US20040229792A1 - Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof - Google Patents

Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof Download PDF

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US20040229792A1
US20040229792A1 US10/768,798 US76879804A US2004229792A1 US 20040229792 A1 US20040229792 A1 US 20040229792A1 US 76879804 A US76879804 A US 76879804A US 2004229792 A1 US2004229792 A1 US 2004229792A1
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Gail Mandel
Simon Halegoua
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NEW YORK SUNY AT STONY BROOK, State University of, Research Foundation of
Icagen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the present invention is in the fields of biotechnology, protein purification and crystallization, x-ray diffraction analysis, three-dimensional computer molecular modeling, and rational drug design (RDD).
  • the invention is directed to isolated peripheral nervous system (PNS) specific sodium channel proteins (SCPs) and encoding nucleic acid, as well as to compounds, compositions and methods for selecting, making and using therapeutic or diagnostic agents having sodium channel modulating activity.
  • PNS peripheral nervous system
  • SCPs specific sodium channel proteins
  • the present invention further provides three-dimensional computer modeling of the PNS SCP, and for RDD, based on the use of x-ray data and/or amino acid sequence data on computer readable media.
  • Voltage-sensitive ion channels are a class of transmembrane proteins that provide a basis for cellular excitability, as the ability to transmit information via ion-generated membrane potentials. In response to changes in membrane potentials, these molecules mediate rapid ion flux through highly selective pores in a nerve cell membrane. If the channel density is high enough, a suitable regenerative depolarization results, termed the action potential.
  • the voltage-sensitive sodium channel is the ion channel most often responsible for generating the action potential in excitable cells.
  • sodium-based action potentials in different excitable tissues look similar (Hille, B., In: Ionic Channels of Excitable Membranes, B. Hille, ed., Sinauer, Sunderland, Mass., (1984), pp. 70-71)
  • recent electophysiological studies indicate that sodium channels in different cells differ in both their structural and functional properties, and many sodium channels with distinct primary structures have now been identified. See, e.g., Mandel, J. Membrane Biol. 125: 193-205 (1992).
  • the type II and type III genes expressed widely in the central nervous system (CNS), are expressed at very low levels in some cells in the PNS (Beckh, S., FEBS Lett. 262:317-322 (1990)).
  • the type II and III mRNAs were barely detectable, by Northern blot analysis, in dorsal root ganglion (DRG), cranial nerves and sciatic nerves.
  • DRG dorsal root ganglion
  • type I mRNA was present in moderately high amounts in DRG and cranial nerve, but in low levels in sciatic nerve.
  • a population of neurons in vertebrate DRG has been identified electrophysiologically that contains, in addition to the more conventional channels, a distinct sodium channel type; this DRG channel has a k D for TTX approximately tenfold higher than the k D of sodium channels in either skeletal muscle or heart (Jones et al., J. Physiol. 389:605-627 (1987)).
  • PC12 pheochromocytoma
  • NGF-treated PC12 cells acquire the ability to generate sodium-based action potentials (Dichter et al., Nature 268:501-504 (1977)).
  • PNS peripheral nervous system
  • SCs PNS sodium channels
  • invention provides peripheral nervous system specific (PNS) sodium channel peptides (SCPs), encoding nucleic acid, vectors, host cells and antibodies, as well as methods of making and using thereof, including recombinant expression, purification, cell-based drug screening, gene therapy, crystallization, X-ray diffraction analysis, as well as computer structure determination and rational drug design utilizing at least one PNS SCP amino acid sequence and/or x-ray diffraction data provided on computer readable media.
  • PNS peripheral nervous system specific
  • SCPs peripheral nervous system specific sodium channel peptides
  • the invention also includes oligonucleotide probes specific for PNS SCP encoding sequences, as well as methods for dectection in a sample, where the probe is labeled.
  • the invention further includes methods for producing a PNS SCP, comprising culturing a host in a culture medium, comprising a PNS SCP nucleic acid; and isolating the PNS SCP from said host or said culture medium.
  • the invention additionally includes an antibody which binds an epitope specific for a PNS SCP, as well as host cells which express the antibody. Diagnostic or therapeutic methods using the antibody are also included in the invention.
  • the invention further includes gene therapy methods and delivery vectors comprising nucleic acid encoding, or complementary to, at least one PNS SCP, and pharmaceutically acceptable compositions thereof.
  • the invention also includes gene therapy by methods that administer an antisense PNS SCP nucleic acid to an animal in amount effective to provide a PNS SC modulating effect, such as an analgesic effect.
  • the present invention further provides methods for purifying and crystallizing a PNS SCP that can be analyzed to obtain x-ray diffraction patterns of sufficiently high resolution to be useful for three-dimensional molecular modeling of the protein.
  • the x-ray diffraction data, atomic coordinates, and/or amino acid sequences provided on computer readable medium are modeled on computer systems, using methods of the invention, to generate secondary, tertiary and/or quaternary structures of a PNS SCP, which structures contribute to their overall three dimensional structure, as well as binding and active sites of the PNS SCP.
  • Molecular modeling methods and computer systems are also provided by the present invention for rational drug design (RDD). These drug design methods use computer modeling programs to find potential ligands or agents that are calculated to bind with sites or domains on the PNS SCP. Potential ligands or agents are then screened for modulating or binding activity. Such screening methods can be selected from assays for at least one biological activity of the protein, as associated with a PNS SCP-related pathology or trauma, according to known sodium channel assays. The resulting ligands provided by methods of the present invention are synthesized and are useful for treating, inhibiting or preventing at least one of PCS SCP-related pathology or trauma in a mammal.
  • RDD rational drug design
  • FIG. 1 depicts a 323 amino acid and corresponding 969 nucleotide sequence of a PNS SCP as amino acids 233-555 of SEQ ID NO:2 and nucleotides 699-1665 of SEQ ID NO:1, as the primary structure of Domain III of the Peripheral Nerve type I (PN1) sodium channel alpha ( ) subunit for both amino acid and DNA sequences.
  • the single amino acid code is used to denote deduced amino acids.
  • YJ1 and YOIC refer to the oligonucleotide primers used to obtain the initial PCR fragment of PN1 cDNA.
  • FIG. 2A-B shows a Northern blot analysis of sodium channel ⁇ subunit mRNA in rat pheochromocytoma (PC12) cells treated with Nerve Growth Factor.
  • the probe used is pRB211 which encodes the highly conserved fourth repeated domain of the rat type II sodium channel. Both type H and PN1 mRNAs are detected with this probe.
  • the probe used contains sequences specific for PN1. The levels of sodium channel mRNA are quantitated with reference to the amount of cyclophilin mRNA, as indicated. Control cells are PC 12 cells grown in the absence of NGF.
  • FIG. 3A-B shows an example of tissue-specific distribution of PN1 mRNA.
  • FIG. 3(A) presents a Northern blot analysis using equal amounts of RNA from tissues. PN1 mRNA is indicated by the dash. 28S refers to the 28S rRNA. The probe contains sequences specific for the PN1 gene. Note the absence of PN1 mRNA in skeletal muscle, cardiac muscle, and the low levels of PN1 mRNA in spinal cord.
  • FIG. 3(B) shows RNAase protection analysis of PN1 mRNA.
  • PN1 refers to the PN1 probe protected by mRNA from the different tissue samples.
  • Actin refers to actin probe sequences protected by the same mRNA.
  • FIG. 4A-F shows localization of PN1 mRNA in Superior Cervical Ganglion (SCG) and Dorsal Root Ganglion (DRG) tissues by in situ hybridization analysis.
  • FIGS. 4A-4B represent neurons hybridized with a PN1-specific antisense RNA probe.
  • FIGS. 4C-4D represent neurons hybridized with the radiolabeled PN1 probe in the presence of non-labeled PN1 competitor DNA.
  • FIGS. 4E-4F represent tissue sections hybridized with an antisense type II probe.
  • FIG. 5 shows a blot analysis comparing Levels of PN1 and brain type I a subunit mRNA in SCG.
  • the pRB11 conserved sodium channel probe detects both type II/IIA and PN1 transcripts.
  • FIG. 6A-B shows a Northern blot analysis which reveals differential expression of PN1 and type I sodium channel mRNAs during postnatal rat development.
  • FIG. 6(A) shows a representative autoradiogram of a Northern blot using radiolabeled antisense pRB211 RNA as probe. Postnatal days 7 (P7) to 42 (P42) are shown.
  • FIG. 6(B) shows a plot of quantitation of the Northern blots showing a decrease in type I mRNA with time after birth.
  • FIG. 7A-D show the deduced primary structure of cloned portion of PN1 a subunit cDNA as a partial 3033 nucleotide (SEQ ID NO:1) sequence and a partial 1011 amino acid (SEQ ID NO:2) sequence.
  • FIG. 8A-D show a comparison of deduced primary amino acid sequences of PN1 (1-988 of SEQ ID NO:2) and brain type II/IIA ⁇ subunit (SEQ ID NO:).
  • FIG. 9A-9D show the entire DNA sequence for a rat PN1 PNS SCP(SEQ ID NO:9).
  • FIG. 10 shows the entire amino sequence for a rat PN1 PNS SCP (SEQ ID NO:10).
  • FIG. 11A-11E shows amino acid sequences for rat PN1 (“RATPN1”) (SEQ ID NO:10) and two expected human PN1 sequences “HULMN1A” (SEQ ID NO:11) “HUMPN1B”(SEQ ID NO:12) HUMPN1C (SEQ ID NO:7) and HUMPN1D (SEQ ID NO:).
  • Alternative sequences include those where “X” is 0, 1, 2, or 3 of the same or different amino acids, which can be optionally selected from Table 1 or Table 2.
  • FIG. 12 shows a computer system suitable for three dimensional structure determination and/or rational drug, design.
  • FIG. 13A-B shows a representative DNA sequence encoding a human PN1 (HUM PN1A) (SEQ ID NO:11)
  • FIG. 14B shows a representative DNA sequence encoding a human PN1 (HUM PN1B) (SEQ ID NO:12)
  • PNS peripheral nervous system specific
  • SCs peripheral nervous system specific sodium channel
  • Certain sodium channels corresponding to PNS SCPs of the invention—are now discovered to be preferentially or selectively expressed in the peripheral nervous system (PNS). These sodium channels modulate peripheral nerve impulse conduction preferentially in the PNS.
  • PNS peripheral nervous system specific
  • SCPs peripheral nervous system specific sodium channel peptides
  • SCPs peripheral nervous system specific sodium channel peptides
  • nucleic acid e.g., nucleic acid
  • vectors e.g., vectors, host cells and antibodies
  • methods of making and using thereof including recombinant expression, purification, cell-based drug screening, gene therapy, crystallization, X-ray diffraction analysis, as well as computer structure determination and rational drug design utilizing at least one PNS SCP amino acid sequence and/or x-ray diffraction data provided on computer readable media.
  • a PNS sodium channel peptide can refer to any subset of a PNS sodium channel (SC) having SC activity, as a fragment, consensus sequence or repeating unit.
  • a PNS SCP of the invention can be prepared by:
  • the SC activity can be screened according to known screening assays for sodium channel activity, in vitro, in situ or in vivo.
  • the minimum peptide sequence to have activity is based on the smallest unit containing or comprising a particular region, domain, consensus sequence, or repeating unit thereof, of at least one PNS SCP.
  • a functional PNS SCP when expressed in a suitable cell, is capable of transporting sodium ions across a lipid bilayer, a cell membrane or a membrane model.
  • the cell In intact cells having sufficient sodium channels, the cell can be capable of generating some form of an action potential, such as in a cell expressing at least one PNS SCP of the present invention.
  • Such transport as measured by suitable SC activity assays, establishes SC activity of one or more PNS SCPs of the invention.
  • a PNS SCP of the invention alternatively includes peptides having a portion of a SC amino acid sequence which substantially corresponds to at least one 20 to 2005 amino acid fragment and/or consensus sequence of a PNS SCP or group of PNS SCPs, wherein the PNS SCP has homology or identity of at least 74-99%, such as 88-99% (or any range or value therein, e.g., 87-99, 88-99, 89-99, 90-99, 91-99, 92-99, 93-99, 94-99, 95-99, 96-99, 97-99, or 98-99%) homology to at least one sequence or consensis sequence of FIGS.
  • a PNS SCP of the invention can maintain SC biological activity. It is preferred that a PNS SCP of the invention is not naturally occurring or is naturally occurring but is in a purified or isolated form which does not occur in nature. Preferably, a PNS SCP of the invention substantially corresponds to an set of domains of PN1, having at least 10 contiguous amino acids of FIGS. 1, 7, 8 , 10 and 11 , or at least 74% homology thereto.
  • a PNS SCP of the invention may comprise at least one domain corresponding to known sodium channel domains, such as rat brain or spinal cord SC domains, such as transmembrane domains, pore lining domains, cytoplasmic domains or extracellular domains, such as IIs6 (e.g., 1-3 to 14-17 (IIs6) 18-23 to 210-214 (cytoplasmic), 229-236 to 254-258 (IIIS1), 268-272 to 293-297 (IIIs2), 300-304 to 321-325 (IIIs3), 326-330 to 347-351 (IIIs4), 368-374 to 389-393 (IIIs5), 474-478 to 500-504 (IIIs6), 553-559 to 577-583 (IVs1), 589-593 to 611-615 (IVs2), 619-623 to 642-646 (IVs3), 654-658 to 678-682 (IVs4), 690-694 to 711-715 (IVs5),
  • IIs6 e.
  • Percent homology or identity can be determined, for example, by comparing sequence information using the GAP computer program version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch ( J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman ( Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • the peptide of the invention corresponds to a SC biologically active portion of SEQ ID NO:2, or variant thereof e.g., as presented in FIG. 11A-D.
  • a PNS SCP of the invention also includes a variant wherein at least one amino acid residue in the peptide has been conservatively replaced, added or deleted by at least one different amino acid.
  • at least one amino acid residue in the peptide has been conservatively replaced, added or deleted by at least one different amino acid.
  • F r a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al., eds, Current Protocols in Molecular Biology, Greene Publishing Assoc., New York, N.Y. (1987, 1992, 1993, 1994, 1995) at ⁇ A. 1.1-A.1.24, and Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), at Appendices C and D.
  • Conservative substitutions of a PNS SCP of the invention includes a variant wherein at least one amino acid residue in the peptide has been conservatively replaced, added or deleted by at least one different amino acid.
  • Such substitutions preferably are made in accordance with the following list as presented in Table 1, which substitutions can be determined by routine experimentation to provide modified structural and functional properties of a synthesized peptide molecule, while maintaining SC biological activity, as determined by known SC activity assays.
  • the term PNS SCP or “substantially corresponding to” includes such substitutions.
  • substitutions of PNS SCPs of the invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue added in its place according to the following Table 2.
  • the types of substitutions which can be made in the protein or peptide molecule of the invention can be based on analysis of the frequencies of amino abid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schultz et al., infra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: TABLE 2 1. Small aliphatic. nonpolar or slightly polar residues: Ala. Ser, Thr (Pro, Gly); 2.
  • Polar, negatively charged residues and their amides Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromatic residues: Phe. Tyr. Trp.
  • alternative substitutions can be made by routine experimentation, to provide alternative PNS SCPs of the invention, e.g., by making one or more conservative substitutions of SC fragments which provide SC activity.
  • at least one substitution, addition or deletion will be evaluated by at least one sodium channel activity screening assay, such as, but not limited to, immunoassays or bioassays, to confirm biological activity, such as, but not limited to, sodium channel activity.
  • Amino acid sequence variants of a PNS SCP of the invention can also be prepared by mutations in the DNA.
  • Such variants include, for example, deletions from, or additions or substitutions of, residues within the amino acid sequence. Any combination of deletion, addition, and substitution can also be made to arrive at the final construct, provided that the final construct possesses some SC activity. Preferably improved SC activity is found over that of the non-variant peptide.
  • mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see, e.g., EP Patent Application Publication No. 75,444; Ausubel, infra; Sambrook, infra).
  • these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding a PNS SCP, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring SC (see, e.g., Ausubel, infra; Sambrook, infra).
  • PNS SCPs can be recombinantly or synthetically produced, or optionally purified, to provide commercially useful amounts of PNS SCPs for use in diagnostic or research applications, according to known method steps (see, e.g., Ausubel, infra, and Sambrook, infra, which references are herein entirely incorporated by reference).
  • the peptide is purified from tissues or cells which naturally produce the peptide.
  • the above-described isolated nucleic acid fragments could be used to expressed the PNS SCP protein in any organism.
  • the samples of the invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts used as the sample.
  • the cells and/or tissue can include, e.g., normal or pathologic animal cells or tissues, such as the peripheral nervous system, and extracts or cell cultures thereof, provided in vivo, in situ or in vitro, as cultured, passaged, non-passaged, transformed, recombinant, or isolated cells and/or tissues.
  • any higher eukaryotic organism can be used as a source of at least one PNS SCI or PNS SCP of the invention, as long as the source organism naturally contains such a peptide.
  • source organism refers to the original organism from which the amino acid sequence of die peptide is derived, regardless of the organism the peptide is expressed in and/or ultimately isolated from.
  • Preferred organisms as sources of at least one PNS SCI or encoding nucleic acid can be any vertebrate animal, such as mammals, birds, bony fish,,electric eels, frogs and toads.
  • the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs).
  • the most preferred source organisms are humans.
  • the present invention relates to an isolated nucleic acid molecule coding for a peptide having an amino acid sequence corresponding to novel PNS SCPs.
  • the isolated nucleic acid molecule comprises a PNS SCP nucleotide sequence with greater than 70% overall identity or homology to. at least a 60 nucleotide sequence present in SEQ ID NO:1 (preferably greater than 80%; more preferably greater than 90%, such as 70-99% any range or value therein).
  • the isolated nucleic acid molecule comprises a PNS SCP nucleotide sequence corresponding to FIGS. 1, 7 or 9 , or encoding at least one domain of FIGS. 1, 7, 8 , 10 and 11 .
  • nucleic acid sequences depicted in SEQ ID NO:1 can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • nucleic acid sequences depicted in SEQ ID NO:1 can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • other DNA sequences which encode substantially the same amino acid sequence of a PNS SCP can be used in the practice of the invention. These include but are not limited to amino acid sequences encoding all or portions of PNS SCP amino acid sequence of FIGS. 1, 8, 10 and 11 , which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • nucleic acid sequence affords an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto.
  • All variations of the nucleotide sequence of the PNS SCP gene and fragments thereof permitted by the genetic code are, therefore, included in this invention. See, e.g., Ausubel, infra, Sambrook, infra.
  • the nucleic acid sequence can comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5′-end and/or the 3′-end of a nucleic acid sequence corresponding to FIGS. 1, 7 or 9 , or encoding at least a portion of FIGS. 1, 8, 10 or 11 , or a variant thereof.
  • Any nucleotide or polynucleotide can be used in this regard, provided that its addition, deletion or substitution does remove the sodium channel activity which is encoded by the nucleotide sequence.
  • the nucleic acid molecule of the invention can, as necessary, have restriction endonuclease recognition sites which do not remove the activity of the encoded PNS SCP.
  • nucleic acid molecules coding for peptides having amino acid sequences corresponding to PNS SCP are provided.
  • the nucleic acid molecule can be isolated from a biological sample containing mammalian nucleic acid, as corresponding to a probe specific for a PNS SC obtained from a higher eukaryotic organism.
  • the nucleic acid molecule can be isolated from a biological sample containing nucleic acid using known techniques, such as but not limited to, primer amplification or cDNA cloning.
  • the nucleic acid molecule can be isolated from a biological sample containing genomic DNA or from a genomic library.
  • suitable biological samples include, but are not limited to, normal or pathologic animal cells or tissues, such as cerebrospinal fluid (CNS), peripheral nervous system (neurons, ganglion) and portions, cells of heart, smooth, skeletal or cardiac muscle, autonomic nervous system, and extracts or cell cultures thereof, provided in vivo, in situ or in vitro, as cultured, passaged, non-passaged, transformed, recombinant, or isolated cells and/or tissues.
  • CNS cerebrospinal fluid
  • peripheral nervous system neurons, ganglion
  • the method of obtaining the biological sample will vary depending upon the nature of the sample.
  • the isolated nucleic acid molecule is also intended to include allelic variations, so long as the sequence encodes a PNS SCP.
  • a PNS SCP allele does not encode the identical amino acid sequence to that found in FIGS. 1, 8, 10 or 11 , or at least domain thereof, it can be, isolated and identified as PNS SCP using the same techniques used herein, and especially nucleic acid amplification techniques to amplify the appropriate gene with primers based on the sequences disclosed herein.
  • Such variations are presented, e.g., in FIG. 11 and in Tables 1 and 2.
  • the cloning of large cDNAs is the same (e.g., PN1 as a PNS SCP of the invention includes overlapping clones of about 13 kDa) but takes more routine experimentation, than smaller cDNAs.
  • One useful method relies on cDNA bacteriophage library screening. (see, e.g., Sambrook, infra, or Ausubel, infra). Probes for the screening are labeled, e.g., with random hexamers and Klenow enzyme (Pharmacia kit).
  • a subcDNA library can be prepared in which a specific PN1 primers are used to prime the reverse transcript reaction in place of oligo dT or random primers.
  • the cDNA sublibrary is then cloned into standard vectors such as lambda zap and screened using conventional techniques. This strategy was used previously (Noda et al. Nature 320:188-192 (1986); Noda et al., Nature 322:826-828 (1986)) to clone the brain type I and II sodium channel cDNAs.
  • the construction of a full-length cDNA is performed by subcloning overlapping fragments into an expression vector (either prokaryotic or eukaryotic). This task is more difficult with large cDNAs because of the paucity of unique restriction sites, but routine restriction, cloning or PCR is used to join the fragments.
  • nucleic Acid Isolated nucleic acid molecules of the present invention are also meant to include those chemically synthesized
  • a nucleic acid molecule with the nucleotide sequence which codes for the expression product of a PNS SCP gene can be designed and, if necessary, divided into appropriate smaller fragments.
  • an oligomer which corresponds to the nucleic acid molecule, or to each of the divided fragments can be synthesized (e.g., of 10-6015 nucleotides or any range or value therein, such as 10-100 nucleotides).
  • Such synthetic oligonucleotides can be prepared, for example, by known techniques (See, e.g., Ausubel, infra, or Sambrook, infra) or by using an automated DNA synthesizer.
  • a labeled oligonucleotide probe be derived synthetically or by cloning. If necessary, the 5′-ends of the oligomers can be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling can be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP can contain high specific activity radioisotopes. Then, the DNA oligomer can be subjected to annealing and ligation with T4 ligase or the like.
  • a Nucleic Acid Probe for the Specific Detection of PNS SCP in another embodiment, relates to a nucleic acid probe of 15-6000 nucleotides for the specific detection of the presence of PNS SCP in a sample comprising the above-described nucleic acid molecules or at least a fragment thereof which binds under stringent conditions to a nucleic acid encoding at least one PNS SCP.
  • the nucleic acid probe can be used to screen an appropriate chromosomal or cDNA library by known hybridization method steps to obtain a PNS SCP encoding nucleic acid molecule of the invention.
  • a chromosomal DNA or cDNA library can be prepared from appropriate cells according to recognized methods in the art (See, e.g., Ausubel, infra; Sambrook, infra).
  • nucleic acid probes having nucleotide sequences which correspond to suitable portions of the amino acid sequence of the PNS SCP.
  • synthesized nucleic acid probes can be used as primers in nucleic acid amplification method steps
  • the invention can thus provide methods for amplification of DNA and/or RNA using heat stable, cross-linked nucleotide primers, which cross linked primers of the invention to provide nucleic acid encoding PNS SCPs according to the invention.
  • RNA or DNA Methods of amplification of RNA or DNA are well known in the art and can be used according to the invention without undue experimentation, based on the teaching and guidance presented herein.
  • the use of nucleic acids encoding portions of PNS SCPs according to the invention, as amplification primers, allows for advantages over known amplification primers, due to the increase in sensitivity, selectivity and/or rate of amplification.
  • RNA amplification processes include, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson et al.; U.S. Pat. No. 5,091,310 to Inn U.S. Pat. No.
  • PCR polymerase chain reaction
  • the hybridization probes of the invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and any other known and suitable labels. After hybridization, the probes can be visualized using known methods.
  • the nucleic acid probes of the invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art (See, e.g., Ausubel, infra; Sambrook, infra). In one embodiment of the above described method, a nucleic acid probe is immobilized on a solid support.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and SEPHAROSE, and acrylic resins, such as polyacrylamide and latex beads.
  • plastics such as polycarbonate
  • complex carbohydrates such as agarose and SEPHAROSE
  • acrylic resins such as polyacrylamide and latex beads.
  • test samples suitable for nucleic acid probing methods of the invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • the present invention relates to methods for detecting the presence of PNS SCP encoding nucleic acid in a sample.
  • Such methods can comprise (a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and (b) detecting the presence of a labeled probe bound to the nucleic acid probe.
  • a labeled probe bound to the nucleic acid probe One skilled in the art can select a suitable, labeled nucleic acid probe according to techniques known in the art as described above. Samples to be tested include, but are not limited to, RNA samples of mammalian tissue.
  • PNS SCP has been found to be expressed in peripheral nerve and dorsal root ganglion cells. Accordingly, PNS SCP probes can be used detect the presence of RNA from PN cells in such a biological sample. Further, altered expression levels of PNS SCP RNA in an individual, as compared to normal levels, can indicate the presence of disease.
  • the PNS SCP probes cart further be used to assay cellular activity in general and specifically in peripheral nervous system tissue.
  • kits for detecting the presence of PNS SCP in a sample comprising at least one container having disposed therein the above-described nucleic acid probe.
  • the kit further comprises other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, allaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin) (See, e.g., Ausubel, infra, Sambrook, infra).
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, TRIS-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
  • nucleic acid probes described in the invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • DNA Constructs Comprising a PNS SCP Nucleic Acid Molecule and Hosts Containing These Constructs.
  • a nucleic acid sequence encoding an PNS SCP of the invention can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Ausubel et al., infra, and are well known in the art.
  • a nucleic acid molecule such as DNA
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as PNS SCPs or Ab fragments in recoverable amounts.
  • the precise nature of the regulatory regions needed for gene expression can vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook, infra and Ausubel infra.
  • the invention accordingly encompasses the expression of an PNS SCP, in either prokaryotic or eukaryotic cells, although eukaryptic expression is preferred.
  • Preferred hosts are bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. It is preferred that the mammalian cell or tissue is of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell can be used.
  • Eukaryotic hosts can include yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture.
  • Preferred eukaryotic hosts can also include, but are not limited to insect cells, mammalian cells either in vivo, or in tissue culture.
  • Preferred mammalian cells include Xenopus oocytes, HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives.
  • Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites.
  • Mammalian cells which can be useful as hosts include cells of fibroblast origin such as, but not limited to, NIH 3T3, VERO or CHO, or cells of lymphoid origin, such as, but not limited to, the hybridoma SP2/O-Ag14 or the murine myeloma P3-X63Ag8, hamster cell lines (e.g., CHO-K1 and progenitors, e.g., CHO-DUXB11) and their derivatives.
  • fibroblast origin such as, but not limited to, NIH 3T3, VERO or CHO
  • lymphoid origin such as, but not limited to, the hybridoma SP2/O-Ag14 or the murine myeloma P3-X63Ag8, hamster cell lines (e.g., CHO-K1 and progenitors, e.g.
  • Neuronally derived cells are preferred for gene therapy of disorders of the nervous system.
  • many possible vector systems are available for the expression of at least one PNS SCP.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals can be derived from viral sources, such as, but not limited to, adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
  • promoters from mammalian expression products such as, but not limited to, actin, collagen, myosin, protein production. See, Ausubel, infra,; Sanbrook, infra.
  • the introduced nucleotide sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors can be employed for this purpose. See, e.g., Ausubel et al., infra, ⁇ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an unglycosylated core protein product.
  • Expression in yeast will produce a glycosylated product.
  • Expression in mammalian cells can be used to ensure “native” glycosylation of the heterologous PNS SCP protein.
  • different vector/host expression systems can effect processing reactions such as proteolytic cleavages to different extents.
  • eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. See, e.g., Ausubel, infra, Sambrook, infra.
  • the DNA construct(s) can be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene molecule(s) results in the production of at least one PNS SCP. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • PNS SCP proteins or fragments of this invention can be obtained by expression from recombinant DNA as described above.
  • a PNS SCP can be purified from biological material. If so desired, the expressed at least one PNS SCP can be isolated and purified in accordance with conventional method steps, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • cells expressing at least one PNS SCP in suitable levels can be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • PNS SCPs can be isolated by the use of specific antibodies, such as, but not limited to, an PNS SCP or SC antibody. Such antibodies can be obtained by known method steps (see, e.g. Colligan, infra; Ausubel, infra.
  • a fist step in the purification of a PNS SCP includes extraction of the PNSSCP fraction from a biological sample, such as peripheral nerve tissue or dorsal root ganglia (DRG), in buffers, with or without solubilizing agents such as urea, formic acid, detergent, or thiocyanate.
  • a second step includes subjecting the solubilized material to ion-exchange chromatography on Mono-Q-or Mono-S columns (Pharmacia LKB Biotechnology, Inc; Piscataway, N.J.).
  • the solubilized material can be separated by any other process wherein molecules can be separated according to charge density, charge distribution and molecular size, for example. Elution of the PNS SCP from the ion-exchange resin are monitored by an immunoassay, such as M-IRMA, on each fraction. Immunoreactive peaks would are then dialyzed, lyophilized, and subjected to molecular sieve, or gel chromatography. In a third step, molecular sieve or gel chromatography is a type of partition chromatography in which separation is based on molecular size. Dextran, polyacrylamide, and agarose gels are commonly used for this type of separation.
  • a fourth step in a purification protocol for a PNS SCP can include analyzing the immunoreactive peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a further gel chromatographic purification step, and staining, such as, for example, silver staining.
  • a fifth step in a purification method can include subjecting the PNS SCP obtained after SDS-PAGE to affinity chromatography, or any other procedure based upon affinity between a substance to be isolated and a molecule to which it can specifically bind.
  • affinity chromatography on SEPHAROSE conjugated to anti-PNS SCP mAbs can be used.
  • Alternative methods, such as reverse-phase HPLC, or any other method characterized by rapid separation with good peak resolution are useful.
  • an Antibody Having Binding Affinity to a PNS SCP Peptide and a Hybridoma Containing the Antibody in another embodiment, the invention relates to an antibody having binding affinity specifically to a PNS SCP peptide as described above or fragment thereof. Those which bind selectively to PNS SCP would be chosen for use in methods which could include, but should not be limited to, the analysis of altered PNS SCP expression in tissue containing PNS SCP.
  • the PNS SCP proteins of the invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
  • the PNS SCP peptide of the invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide would be generated as described herein and used as an immunogen.
  • the antibodies of the invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies.
  • the invention further includes single chain antibodies.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. MAbs can be obtained by methods known to those skilled in the art.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof.
  • a hybridoma producing a mAb of the invention can be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mabs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used.
  • Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse str) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein entirely incorporated by reference.
  • the anti-Id antibody can also be used as an “immunogen” to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id can be epitopically identical to the original mAb which induced the anti-Id.
  • antibodies to the idiotypic determinants of a mAb it is possible to identify other clones expressing antibodies of identical specificity.
  • mAbs generated against a PNS SCP of the invention can be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mabs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a PNS SCP specific epitope. The anti-Id mabs thus have their own idiotypic epitopes, or “idiotopes” structurally similar to the epitope being evaluated.
  • KLH keyhole limpet hemocyanin
  • antibody is also meant to-include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab′) 2 , which are capable of binding antigen.
  • Fab and F(ab′) 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciated that Fab and F(ab′) 2 and other fragments of the antibodies useful in the invention can be used for the detection and/or quantitation of a PNS SCP according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′) 2 fragments).
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • the term “epitope” is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or “antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • an “antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additiolially capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen can have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which can be evoked by other antigens.
  • Immunoassays Antibodies of the invention, directed against a PNS SCP, can be used to detect or diagnose a PNS SC or a PNS SC-related pathologies. Screening methods are provided by the invention can include, e.g., immunoassays employing radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA) methodologies, based on the production of specific antibodies (monoclonal or polyclonal) to a PNS SCP.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbant assay
  • One embodiment for carrying out the diagnostic assay of the invention on a biological sample containing a PNS SCP comprises:
  • the specific concentrations of detectably labeled antibody and PNS SCP, the temperature and time of incubation, as well as other assay conditions can be varied, depending on various factors including the concentration of a PNS SCP in the sample, the nature of the sample, and the like.
  • the binding activity of a given lot of anti-PNS SCP antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like can be added to the assays as is customary or necessary for the particular situation.
  • Detection can be accomplished using any of a variety of assays. For example, by radioactively labeling the PNS SCP-specific antibodies or antibody fragments, it is possible to detect PNS SCP through the use of radioimmune assays.
  • a good description of a radioimmune assay can be found in Colligan, infra, and Ausubel, infra, entirely incorporated by reference herein.
  • the detection of cells which express a PNS SCP can be accomplished by in vivo imaging techniques, in which the labeled antibodies (or fragments thereof) are provided to a subject, and the presence of the PNS SCP is detected without the prior removal of any tissue sample.
  • Such in vivo detection procedures have the advantage of being less invasive than other detection methods, and are, moreover, capable of detecting the presence of PNS SCP in tissue which cannot be easily removed from the patient, such as brain tissue.
  • the type of detection instrument available is a major factor in selecting a given radionuclide.
  • the radionuclide chosen must have a type of decay which is detectable for a given type of instrument
  • any conventional method for visualling diagnostic imaging can be utilized in accordance with this invention.
  • PET positron emission tomography
  • gamma beta
  • MRI magnetic resonance imaging
  • the antibodies useful in the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis.
  • Elements which are particularly useful, as in Magnetic Resonance Imaging (MRI), include 157 Gd, 55 Mn, 162 Dy, and 56 Fe.
  • the antibodies (or fragments thereof) useful in the invention are also particularly suited for use in in vitro immunoassays to detect the presence of a PNS SCP in body tissue, fluids (such as CSF), or cellular extracts.
  • the antibodies (or antibody fragments) can be utilized in liquid phase or, preferably, bound to a solid-phase carrier, as described above.
  • In situ detection can be accomplished by removing a histological specimen from a patient, and providing the combination of labeled antibodies of the invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • an effective amount of a diagnostic reagent is one capable of achieving the desired diagnostic discrimination and will vary depending on such factors as age, condition, sex, the extent of disease of the subject, counter-indications, if any, and other variables to be adjusted by the physician.
  • the amount of such materials which are typically used in a diagnostic test are generally between 0.1 to 5 mg, and preferably between 0.1 to 0.5 mg.
  • the assay of the invention is also ideally suited for the preparation of a kit.
  • a kit can comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay.
  • a container means containing a first- antibody immobilized on a solid phase support can contain a further container means containing a second detectably labeled antibody in solution.
  • Further container means can contain standard solutions comprising serial dilutions of the PNS SCP to be detected.
  • the standard solutions of a PNS SCP can be used to prepare a standard curve with the concentration of PNS SCP plotted on the abscissa and the detection signal on the ordinate.
  • the results obtained from a sample containing a PNS SCP can be interpolated from such a plot to give the concentration of the PNS SCP.
  • Diagnostic Screening and Treatment It is to be understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal that expresses at least one PNS SC.
  • the diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level of PNS SCP based on family history, or a patient in which it is desired to diagnose a PNS SCP-related disease.
  • presymptomatic screening of an individual in need of such screening is now possible using DNA encoding the PNS SCP protein of the invention.
  • the screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of the presence of a missing or aberrant PNS SC gene in individuals, and thus an opinion concerning the likelihood that such individual would develop or has developed a PNS SC-associated disease. This is especially valuable for the identification of carriers of altered or missing PNS SC genes, for example, from individuals with a family history of a PNS SC-related pathology. Early diagnosis is also desired to maximize appropriate timely intervention.
  • a tissue sample would be taken from such individual, and screened for (1) the presence of the “normal” PNS SCP gene; (2) the presence of PNS SCP mRNA and/or (3) the presence of PNS SCP protein.
  • the normal human gene can be characterized based upon, for example, detection of restriction digestion patterns in “normal” versus the patient's DNA, including RFLP analysis, using DNA probes prepared against the PNS SCP sequence (or a functional fragment thereof) taught in the invention.
  • PNS SCP mRNA can be characterized and compared to normal PNS SCP mRNA (a) levels and/or (b) size as found in a human population not at risk of developing PNS SCP-associated disease using similar probes.
  • PNS SCP protein can be (a) detected and/or (b) quantitated using a biological assay for PNS SCP activity or using an immunological assay and PNS SCP antibodies.
  • the immunological assay is preferred for its speed.
  • An (1) aberrant PNS SCP DNA size pattern, and/or (2) aberrant PNS SCP mRNA sizes or levels and/or (3) aberrant PNS SCP protein levels would indicate that the patient is at risk for developing a PNS SCP-associated disease.
  • the screening and diagnostic methods of the invention do not require that the entire PNS SCP DNA coding sequence be used for the probe. Rather, it is only necessary to use a fragment or length of nucleic acid that is sufficient to detect the presence of the PNS SCP gene in a DNA preparation from a normal or affected individual, the absence of such gene, or an altered physical property of such gene (such as a change in electrophoretic migration pattern).
  • Prenatal diagnosis can be performed when desired, using any known method to obtain fetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy.
  • Prenatal chromosome analysis can be used to determine if the portion of the chromosome possessing the normal PNS SCP gene is present in a heterozygous state.
  • PNS SCP Purification and Crystallization Methods.
  • a PNS SCP as a membrane protein is purified in soluble form using detergents (e.g., octyglucosides) or other suitable amphiphillic molecules.
  • the resulting PNS SCP is in sufficient purity and concentration for crystallization.
  • the purified PNS SCP is then isolated and assayed for biological activity and for lack of aggregation (which interferes with crystallization).
  • the purified and cleaved PNS SCP preferably runs as a single band under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges).
  • PAGE polyacrylamide gel electrophoresis
  • the purified PNS SCP is preferably crystallized under varying conditions of at least one of the following: pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands and concentration of purified and cleaved PNS SCP by known methods. See, e.g., Michel, Trends in Biochem Sci. 8:56-59 (1983); Deisenhofer et al. J. Mol. Biol 180:385-398 (1984); Weiss et al. FEBS Lett. 267:268-272 (1990). Blundell, et al. Protein Crystallography Academic Press, London (1976); Oxender et al. eds., Protein Engineering Liss, N.Y.
  • Crystallized protein is also tested for at least one SC activity and differently sized and shaped crystals are further tested for suitability in X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals.
  • the hanging drop method is preferably used to crystallize a purified soluble, PNS SCP protein. See, e.g., Taylor et al., J. Mol. Biol. 226:1287-1290 (1992); Takimoto et al. (1992), infra, CRYSTAL SCREEN, Hampton Research.
  • a mixture of the protein and precipitant can include the following: ⁇ pH (e.g., 4-10); ⁇ buffer type (e.g., tromethamine (TRIZMA), sodium azide, phosphate, sodium, rcacodylate acetates, imidazole, Tris HCl, sodium hepes); ⁇ buffer concentration (e.g., 0.1-100 mM); ⁇ salt type (e.g., sodium azide, calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate; calcium acetate); ⁇ polymer type and concentration: (e.g., polyethylene glycol (PEG) 1-50%, type 6000-10,000); ⁇ other precipitating ligands (salts: potassium, sodium, tartrate, ammonium sulfate, sodium acetate, lithium sulfate, sodium formate, sodium citrate, magnesium formate, sodium phosphate,
  • the above mixtures are used and screened by varying at least one of pH, buffer type; buffer concentration, precipitating salt type or concentration, PEG type, PEG concentration, and cleaved protein concentration. Crystals ranging in size from 0.1-1.5 mm are formed in 1-14 days. These crystals diffract X-rays to at least 10 ⁇ resolution, such as 1.5-10.0 ⁇ , or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 ⁇ or less being preferred for the highest resolution. In addition to diffraction patterns having this highest resolution, lower resolution, such as 25-3.5 ⁇ can further be used.
  • Protein Crystals appear after 1-14 days and continue to grow on subsequent days. Some of the crystals are removed, washed, and assayed for biological activity, which activity is preferred for using in further characterizations. Other washed crystals are preferably run n a stained gel and those that-migrate in the same position as the purified cleaved PNS SCP are preferably used. From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, bipyramidal, rhomboid, and cubic. Initial X-ray analyses are expected to indicate that such crystals diffract at moderately high to high resolution. When fewer crystals are produced in a drop, they can be much larger size, e.g., 0.2-1.5 mm.
  • PNS SCP X-ray Crystallography Methods
  • the crystals so produced for a PNS SCP are X-ray analyzed using a suitable X-ray source. A suitable number of diffraction patterns are obtained.
  • Crystals are preferably stable for at least 10 hrs in the X-ray beam.
  • Frozen crystals e.g., ⁇ 220 to ⁇ 50° C.
  • longer X-ray exposures e.g., 4-72 hrs
  • multiple frames are optionally collected as the crystal is rotated in the X-ray beam, e.g.; for 12-96 hrs.
  • Crystals are preferably analyzed using a synchrotron high energy X-ray source. Using frozen crystals, X-ray diffraction data is collected on crystals that diffract to a resolution of 10-1.5 ⁇ , with lower resolutions also useful, such as 25-10 ⁇ , sufficient to solve the three-dimensional structure of a PNS SCP in considerable detail, as presented herein.
  • An amino acid sequence of a PNS SCP and/or x-ray diffraction data useful for computer molecular modeling of a PNS SCP or a portion thereof, can be “provided” in a variety of mediums to facilitate use thereof.
  • provided refers to a manufacture, which contains a PNS SCP amino acid sequence and/or x-ray diffraction data of the present invention, e.g., the amino sequence provided in FIGS. 1, 8, 10 or 11 , a representative fragment thereof, or an amino acid sequence having at least 80-100% overall identity to a 5-2005 amino acid fragment of an amino acid sequence of FIGS. 11 A-D or a variant thereof.
  • Such a method provides the amino acid sequence and/or x-ray diffraction data in a form which allows a skilled artisan to analyze and molecular model the three dimension structure of a PNS SCP or subdomain thereof.
  • PNS SCP or at least one subdomain thereof, amino acid sequence and/or x-ray diffraction data of the present invention is recorded on computer readable medium.
  • computer readable medium refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as optical discs or CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • “recorded” refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising an amino acid sequence and/or x-ray diffraction data information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon an amino acid sequence and/or x-ray diffraction data of the present invention.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the sequence and x-ray data information of the present invention on computer readable medium.
  • the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • a skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the information of the present invention.
  • the present invention further provides systems; particularly computer-based systems; which contain the sequence and/or diffraction data described herein. Such systems are designed to do molecular modeling and RDD for a PNS SCP or at least one subdomain thereof.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the sequence and/or x-ray diffraction data of the present invention.
  • the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • input means input means
  • output means output means
  • data storage means data storage means
  • the computer-based systems of the present invention comprise a data storage means having stored therein a PNS SCP or fragment sequence and/or x-ray diffraction data of the present invention and the necessary hardware means and software means for supporting and implementing an analysis means.
  • data storage means refers to memory which can store sequence or x-ray diffraction data of the present invention, or a memory access means which can access manufactures having recorded thereon the sequence or x-ray data of the present invention.
  • search means or “analysis means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence or x-ray data stored within the data storage means. Search means are used to identify fragments or regions of a PNS SCP which match a particular target sequence or target motif.
  • search means are used to identify fragments or regions of a PNS SCP which match a particular target sequence or target motif.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting computer analyses that can be adapted for use in the present computer-based systems.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration or electron density map which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzymic active sites, structural subdomains, epitopes, functional domains and signal sequences.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • a variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify structural motifs or electron density maps.
  • a skilled artisan can readily recognize that any one of the publicly available computer modeling programs can be used as the search means for the computer-based systems of the present invention.
  • FIG. 12 provides a block diagram of a computer system 102 that can be used to implement the present invention.
  • the computer system 102 includes a processor 106 connected to a bus 104 .
  • main memory 108 preferably implemented as random access memory, RAM
  • secondary storage memory 110 such as a hard drive 112 and a removable storage medium 114 .
  • the removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc.
  • a removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage medium 114 .
  • the computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114 once inserted in the removable medium storage device 114 .
  • a monitor 120 can be used as connected to the bus 104 to visualize the structure determination data.
  • Amino acid, encoding nucleotide or other sequence and/or x-ray diffraction data of the present invention may be stored in a well known manner in the main memory 108 , any of the secondary storage devices 110 , and/or a removable storage device 116 .
  • Software for accessing and processing the amino acid sequence and/or x-ray diffraction data (such as search tools, comparing tools, etc.) reside in main memory 108 during execution.
  • Three Dimensional Structure Determination One or more computer modeling steps and/or computer algorythms are used to provide a molecular 3-D model of a cleaved PNS SCP, using amino acid sequence data from FIGS. 1, 8, 10 or 11 (or variants thereof) and/or x-ray diffraction data. If only the amino acid sequence is used, for three-dimensional structure determination then a suitable modeling program can be used, e.g., LINUS (Rose et. al. Proteins: Structure, Function and Genetics (June, 1995) and references cited herein.
  • LINUS Rose et. al. Proteins: Structure, Function and Genetics (June, 1995) and references cited herein.
  • the PNS SCP model has no or A1a-substituted (for surface) residues in disallowed regions of the Ramachandran plot, and gives a positive 3D-1D profile (Luthy et al., Nature 356:83-85 (1992)), suggesting that all the residues are in acceptable environments (Kraulis (1991), infra).
  • the dissallowed regions can be corrected by the use of suitable algorythms, such as the RAVE program described herein.
  • Phase determination is optionally used for solving the three-dimensional structure of a cleaved PNS SCP. This structure can then be used for RDD of modulators of PNS SCP neuraminidase, endothelin cathepsin A or other biological activity, e.g., which is relevant to a PNS SCP related pathology.
  • Electron density maps can be calculated using such programs as those from the CCP4 computing package (SERC (UK) Collaborative Computing Project 4, Daresbury Laboratory, UK, 1979). Cycles of two-fold averaging can further be used, such as with the program RAVE (Kleywegt & Jones, Bailey et al., eds., First Map to Final Model, SERC Daresbury Laboratory, UK, pp.59-66 (1994)) and gradual model expansion.
  • RAVE Ket & Jones
  • eds. First Map to Final Model
  • SERC Daresbury Laboratory UK, pp.59-66 (1994)
  • For map visualization and model building a program usch as “O” Jones (1991), infra) can be used.
  • Rigid body and positional refinement can be carried out using a program such as X-PLOR (Brünger (1992), infra), e.g., with the stereochemical parameters of Engh and Huber ( Acta Cryst. A 47:392-400 (1991)). If the model at this stage in the averaged maps still misses residues (e.g., at least 5-10 per subunit), the some or all of the missing residues can be incorporated in the model during additional cycles of positional refinement and model building.
  • the refinement procedure can start using data from lower resolution (e.g., 25-10 ⁇ to 10-3.0 ⁇ and then gradually extended to include data from 12-6 ⁇ to 3.0-1.5 ⁇ ).
  • ⁇ -values for individual atoms can be refined once data between 2.9 and 1.5 ⁇ has been added. Subsequently waters can be gradually added.
  • a program such as ARP (Lamzin and Wilson, Acta Cryst. D 49:129-147 (1993)) can be used to add crystallographic waters and as a tool to check for bad areas in the model. Programs such as PROCHECK (Lackowski et al., J. Appl. Cryst. 26:283-291 (1993)), WHATIF (Vriend, J. Mol. Graph.
  • a program such as DSSP can be used to assign the secondary structure elements (Kabsch and Sander (1983), infra).
  • a program such as SUPPOS from the BIOMOL crystallographic computing package) can be used to for some or all of the least-squares superpositions of various models and parts of models. Solvent accessible surfaces and electrostatic potentials can be calculated using such programs as GRASP (Nicholls et al. (1991), infra).
  • the structure of a PNS SCP can thus be solved with the molecular replacement procedure such as by using X-PLOR (Brünger (1992), infra).
  • a partial search model for the monomer can be constructed using a related protein, such as wheat serine carboxypeptidase structure (Liao et al. (1992), infra).
  • the rotation and translation function can be used to yield two or more orientations and positions for two subunits to form a physiological dimer as determined based on their interactions. Cyclical two-fold density averaging can also be done using the RAVE program and model expansion can also be used to add missing residues for each monomer, resulting in a model with 95-99.9% of the total number residues.
  • the model can be refined in a program such as X-PLOR (Brünger (1992), supra), to a suitable crystallographic R factor .
  • the model data is then saved on computer readable medium for use in further analysis, such as rational drug design.
  • a diagnostic or therapeutic PNS SCP modulating ligand of the present invention can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies.
  • Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
  • any other known diagnostic or therapeutic agent can be used in a method of the invention.
  • the time-dependent nature of modulation of ligand K i values are determined, (e.g., by the method of Henderson ( Biochem. J. 127:321-333 (1972)).
  • the substrate (or blank where appropriate) and enzyme are pre-incubated in buffer. Reactions are initiated by the addition of substrate. Aliquots are removed over a suitable time course and each quenched by addition into the aliquots of suitable quenching solution (e.g. sodium hydroxide in aqueous ethanol).
  • suitable quenching solution e.g. sodium hydroxide in aqueous ethanol.
  • concentration of product is determined, e.g. fluorometrically, using a spectrometer.
  • PROCNLIN from SAS (SAS Institute Inc., Cary, N.C., USA) which performs nonlinear regression using least-square techniques.
  • the iterative method used is optionally the multivariate secant method, similar to the Gauss-Newton method, except that the derivatives in the Taylor series are estimated from the histogram of iterations rather than supplied analytically.
  • a suitable convergence criterion is optionally used, e.g., where there is a change in loss function of less than 10 ⁇ 8 .
  • PNS SCP crystals are soaked for 2 days in 0.01-100 mM ligand and X-ray diffraction data are collected on an area detector and/or an image plate detector (e.g., a Mar image plate detector) using a rotating anode X-ray source. Data are collected to as high a resolution as possible, e.g., 1.5-3.5 ⁇ , and merged with an R-factor on suitable intensities.
  • An atomic model of the inhibitor is built into the difference Fourier map (F inhibitor complex ⁇ F native ).
  • the model can be refined to a solution in a cycle of simulated annealing (Brünger (1987), infra) involving 10-500 cycles of energy refinement, 100-10,000 1-FS steps of room temperature dynamics and/or 10-500 more cycles of energy refinement. Harmonic restraints are also used for the atom refinement, except for atoms within a 10-15 ⁇ radius of the inhibitor. An R-factor is selected for the model for both the r.m.s. deviations from the ideal bond lengths, as well as for the angles, respectively. Direct measurements of enzyme inhibition provide further confirmation that the modeled ligands are modulators of at least one biological activity of a PNS SC.
  • Ligands of a PNS SCP are thus also provided by the present invention. Demonstration of clinically useful levels, e.g., in vivo activity is also important.
  • animal models e.g., rat, mouse, rabbit
  • modulation of a PNS SCP occurs in suitable animal models, using the ligands discovered by molecular modeling and x-ray crystallography.
  • a diagnostic or therapeutic PNS SCP modulating agent or ligand of the present invention can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies, as described herein.
  • Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds r metals, chemiluminescent compounds and bioluminescent compounds.
  • any other known diagnostic or therapeutic agent can be used in a method of the invention.
  • a therapeutic agent used in the invention can have a therapeutic effect on the target cell as a cell or neuron of the peripheral nervous system, the effect selected from, but not limited to: correcting a defective gene or protein, a drug action, a toxic effect, a growth stimulating effect, a growth inhibiting effect, a metabolic effect, a catabolic affect, an anabolic effect, a neurohumoral effect, a cell differentiation stimulatory effect, a cell differentiation inhibitory effect, a neuromodulatory effect, a pluripotent stem cell stimulating effect, and any other known therapeutic effects that modulates at least one SC in a cell of the peripheral nervous system can be provided by a therapeutic agent delivered to a target cell via pharmaceutical administration or via a delivery vector according to the invention.
  • a therapeutic nucleic acid as a therapeutic agent can have, but is not limited to, at least one of the following therapeutic effects on a target cell: inhibiting transcription of a DNA sequence; inhibiting translation of an RNA sequence; inhibiting reverse transcription of an RNA or DNA sequence; inhibiting a post-translational modification of a protein; inducing transcription of a DNA sequence; inducing translation of an RNA sequence; inducing reverse transcription of an RNA or DNA sequence; inducing a post-translational modification of a protein; transcription of the nucleic acid as an RNA; translation of the nucleic acid as a protein or enzyme; and incorporating the nucleic acid into a chromosome of a target cell for constitutive or transient expression of the therapeutic nucleic acid.
  • Therapeutic effects of therapeutic nucleic acids can include, but are not limited to: turning off a defective gene or processing the expression thereof, such as antisense RNA or DNA; inhibiting viral replication or synthesis; gene therapy as expressing a heterologous nucleic acid encoding a therapeutic protein or correcting a defective protein; modifying a defective or underexpression of an RNA such as an hnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or an enzyme that generates a compound as a drug or prodrug in pathological or normal cells expressing the chimeric receptor, and any other known therapeutic effects.
  • a therapeutic nucleic acid of the invention which encodes, or provides the therapeutic effect any known toxin, prodrug or gene drug for delivery to pathogenic nervous cells can also include genes under the control of a tissue specific transcriptional regulatory sequence (MRSs) specific for pathogenic SC containing cells. Such TRSs would further limit the expression of the therapeutic agent in the target cell, according to known methods.
  • MRSs tissue specific transcriptional regulatory sequence
  • Non-limiting examples of such PNS SCP modulating agents or ligands of the present invention and methods thereof include methyl/halophenyl-substituted piperizine compounds, such as lidoflazine (see; e.g., Merck Index Monograph 5311 and U.S. Pat. No. 3,267,104, both entirely incoporated herein by reference).
  • Such compounds were tested and found to inhibit sodium channel activity of at least one PNS SCP of the present invention in cell lines expressing at least one PNS SCP, such as PC12, PK1-4 and other isolated or recombinant cells expressing at least one PNS SCP of the present invention.
  • the present invention provides PNS SCP modulating agents or ligands as methyl/halophenyl-substituted piperizines.
  • the substitutions can include alkyl- and/or halophenyl-substituted piperizines.
  • the present invention further provides a method for modulating the activity of the PNS SCP protein in a cell.
  • agents antagonists or agonists
  • agents which have been identified to inhibit or enhance the activity of PNS SCP can be formulated so that the agent can be contacted with a cell expressing a PNS SCP protein in vivo.
  • the contacting of such a cell with such an agent results in the in vivo modulation of the activity of the PNS SCP proteins. So long as a formulation barrier or toxicity barrier does not exist, agents identified in the assays described above will be effective for in vivo use.
  • the invention in another embodiment, relates to a method of administering PNS SCP or a PNS SCP modulating compound or composition (including PNS SCP antagonists and agonists) to an animal (preferably, a mammal (specifically, a human)) in an amount sufficient to effect an altered level of PNS SCP in the animal.
  • the administered PNS SC or PNS SCP modulating compound or composition could specifically effect PNS SCP associated functions.
  • administration of PNS SC or PNS SCP modulating compound or composition could be used to alter PNS SCP levels in the peripheral nervous system.
  • PNS SCP antagonists can be used to treat pain due to trauma or pathology involving the central or peripheral nervous system, or pathologies related to the abnormally high levels of expression of at least one naturally occurring nervous system specific (NS) sodium channel (SC), where a PNS SCP antagonist also inhibits at least one NS SC, or where the pain is mediated to some extent by PN SC.
  • NS nervous system specific
  • Such pathologies include, but are not limited to; inflammatory diseases, neuropathies (e.g., diabetic neuropathy), dystrophies (e.g., reflex sympathetic dystrophy, post-herpetic neuralgia); trauma (tissue damage by any cause); focal pain by any cause.
  • Inflammatory diseases can include, but are not limited to, chronic inflammatory pathologies and vascular inflammatory pathologies.
  • Chronic inflammatory pathologies include, but are not limited to sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki's pathology.
  • PNS SCP agonists can be used to treat pathologies involving the central or peripheral nervous system, or pathologies related to the abnormally low levels of expression of at least one naturally occurring nervous system specific (NS) sodium channel (SC), where a PNS SCP agonist also enhances or stimulates at least one NS SC.
  • NS nervous system specific
  • Such pathologies include, but are not limited to, neurodegenerative diseases, diseases of the gastrointestinal tract due to dysfunction of the enteric nervous system (e.g., colitis, ileitis, inflammatory bowel syndrome); diseases of the cardiovascular system (e.g., hypertension and congestive heart failure); diseases of the genitourinary tract involving sympathetic and parasympathetic innervation (e.g., benign prostrate hyperplasia, impotence); diseases of the neuromuscular system (e.g., muscular dystrophy, multiple sclerosis, epilepsy).
  • diseases of the gastrointestinal tract due to dysfunction of the enteric nervous system e.g., colitis, ileitis, inflammatory bowel syndrome
  • diseases of the cardiovascular system e.g., hypertension and congestive heart failure
  • diseases of the genitourinary tract involving sympathetic and parasympathetic innervation e.g., benign prostrate hyperplasia, impotence
  • diseases of the neuromuscular system e.g., muscular dys
  • Neurodegenerative diseases can include, but are not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; hyperkinetic movement disorders, such as Huntington's Chorea and senile chorea; hypokinetic movement disorders, such as Parkinson's disease; progressive supranucleo palsy; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia; multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); and systemic disorders Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi-system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); or
  • compositions of the invention for a PNS SC related pathology can be administered by any means that achieve its intended purpose, for example, to treat or prevent a cancer or precancer us condition.
  • prevention involves administration of a Pharmaceutical composition prior to the induction of the disease.
  • suppression involves administration of the composition prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events can be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events.
  • prophylaxis as distinct from “treatment” to encompass both “preventing” and “suppressing”as defined herein.
  • protection is meant to include “prophylaxis.” See, e.g., Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein.
  • the “protection” provided need not be absolute, i.e., the disease need not be totally prevented or eradicated, provided that there is a statistically significant improvement relative to a control population. Protection can be limited to mitigating the severity or rapidity of onset of symptoms of the disease.
  • At least one PNS SC modulating compound or composition of the invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as previously described.
  • administration can be by -various parenteral routes such as subcutaneous, intravenous, intradernal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes.
  • parenteral administration can be by bolus injection or by gradual perfusion over time.
  • An additional mode of using of a diagnostic/pharmaceutical compound or composition of the invention is by topical application.
  • a diagnostic/pharmaceutical compound or composition of the invention can be incorporated into topically applied vehicles such as salves or ointments.
  • a diagnostic/pharmaceutical compound or composition according to the invention for topical applications, it is preferred to administer an effective amount of a diagnostic/pharmaceutical compound or composition according to the invention to target area, e.g., skin surfaces, mucous membranes, and the like, which are adjacent to peripheral neurons which are to be treated.
  • This amount will generally range from about 0.0001 mg to about 1 g of a PNS SC modulating compound per application, depending upon the area to be treated, whether the use is diagnostic, prophylactic or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed.
  • a preferred topical preparation is an ointment, wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base.
  • a typical regimen for treatment or prophylaxis comprises administration of an effective amount over a period of one or several days, up to and including between one week and about six months.
  • the dosage of a diagnostic/pharmaceutical compound or composition of the invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the diagnostic/pharmaceutical effect desired.
  • the ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts.
  • the total dose required-for each treatment can be administered by multiple doses or in a single dose.
  • the diagnostic/pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology.
  • Effective amounts of a diagnostic/pharmaceutical compound or composition of the invention are from about 0.1 ⁇ g to about 100 mg/kg body weight, administered at intervals of 4-72 hours, for a period of 2 hours to 1 year, and/or any range or value therein, such as 0.0001-1.0, 1-10, 10-50 and 50-100, 0.0001-0.001, 0.001-0.01, 0.01-1.0, 1.0-10, 5-10, 10-20, 20-50 and 50-100 mg/kg, at intervals of 1-4, 4-10, 10-16, 16-24, 24-36, 36-48, 48-72 hours, for a period of 1-14, 14-28, or 30-44 days, or 1-24 weeks, or any range or value therein.
  • the recipients of administration of compounds and/or compositions of the invention can be any vertebrate animal, such as mammals, birds, bony fish, frogs and toads.
  • the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs).
  • the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans.
  • a delivery vector of the present invention can be, but is not limited to, a viral vector, a liposome, an anti-PNS SCP or anti-SC antibody, or a SC ligand, one or more of which delivery vectors is associated with a diagnostic or therapeutic agent.
  • the delivery vector can comprise any diagnostic or therapeutic agent which has a therapeutic or diagnostic effect on the target cell.
  • the target cell specificity of the delivery vector is thus provided by use of a target cell specific delivery vector.
  • the delivery vector can also be a recombinant viral vector comprising at least one binding domain selected from the group consisting of an antibody or fragment, a chimeric binding site antibody or fragment, a target cell or specific ligand, a receptor which binds a target cell ligand, an anti-idiotypic antibody, a liposome or other component which is specific for the target cell.
  • a PNS SCP can be already associated with the target cell, or the delivery vector can bind the target cell via a ligand to a target cell receptor or vice versa.
  • the therapeutic or diagnostic agent such as a therapeutic or diagnostic nucleic acid, protein, drug, compound composition and the like, is delivered preferentially to the target cell, e.g., where the nucleic acid is preferably incorporated into the chromosome of the target cell, to the partial or complete exclusion of non-target cells.
  • the invention is thus intended to provide delivery vectors, containing one or more therapeutic and/or diagnostic agents, including vectors suitable for gene therapy.
  • PNS SCP DNA can be provided to the PNS cells of such patient in a manner and amount that permits the expression of the PNS SCP protein provided by such gene, for a time and in a quantity sufficient to treat such patient, such as a suitable delivery vector.
  • a suitable delivery vector Many vector systems are known in die art to provide such delivery to human patients in need of a gene or protein missing from the cell.
  • retrovirus systems can be used, especially modified retrovirus systems and especially herpes simplex virus systems. Such methods are provided for, in, for example, the teachings of Breakefield, et al., The New Biologist 3:203-218 (1991); Huang, Q.
  • the PNS SCP modulating compound or composition is expressed as a recombinant gene in a cell, so that the cells can be transplanted into a mammal, preferably a human in need of gene therapy.
  • a genetic sequence which encodes for all or part of the PNS SCP modulating compound or composition is added into a vector and introduced into a host cell.
  • diseases that can be suitable for gene therapy include, but are not limited to, neurodegenerative diseases or disorders, Alzheimer's, schizophrenia, epilepsy, neoplasms and cancer.
  • the means by which the vector carrying the gene can be introduced into the cell include but is not limited to, microinjection, electroporation, transduction, or transfection using DEAE-Dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art (Sambrook infra, Ausubel, infra).
  • Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. See, generally, Osol et al., eds. Remington's Pharmaceutical Science, 16th Ed., (1980).
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising PNS SC or PNS SCP modulating compound or composition in an amount sufficient to alter PNS SCP associated activity, and a pharmaceutically acceptable diluent, carrier, or excipient
  • concentrations and dosage unit sizes can be readily determined by one skilled in the art (See, e.g., Osol et al. ed., Remington's Pharmaceutical Sciences, 16th Ed., Mack, Easton Pa. (1980) and WO 91/19008).
  • compositions comprising an effective amount of at least one PNS SCP antisense oligonucleotide, in combination with a pharmaceutically acceptable carrier.
  • antisense oligos include, but are not limited to, at least one nucleotide sequence of 12-500 bases in length which is complementary to a DNA sequence of SEQ ID NO:1, or a DNA sequence encoding at least 4 amino acids of SEQ ID NO:2 or FIG. 11A-11E.
  • the PNS SCP nucleic acid can be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • a preferred sterol is cholesterol.
  • the PNS SCP gene therapy nucleic acids and the pharmaceutical compositions of the invention can be administered by any means that achieve their intended purpose.
  • administration can be by parenteral, subcutaneous, intravenous, intramuscular, intra-peritoneal, or transdermal routes.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions within the scope of this invention include all compositions wherein the PNS SCP antisense oligonucleotide is contained in an amount effective to achieve enhanced expression of at least one PNS SCP in a peripheral nervous system neuron or ganglion. While individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art.
  • the PNS SCP nucleic acid can be administered to mammals, e.g. humans, at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated.
  • Suitable formulations for parenteral administration include aqueous solutions of the PNS SCP nucleic acid in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions can be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension can also contain stabilizers.
  • At least one PNS SCP can be coded by DNA constructs which are administered in the form of virions, which are preferably incapable of replicating in vivo (see, for example, Taylor, WO 92/06693).
  • DNA constructs can be administered using herpes-based viruses (Gage et al., U.S. Pat. No. 5,082,670).
  • PNS SCP antisense RNA sequences, PNS SCP ribozymes, and PNS SCP EGS can be coded by RNA constructs which are administered in the form of virions, such as recombinant, replication deficient retroviruses or adenoviruses.
  • retroviral vectois The preparation of retroviral vectois is well known in the art (see; for example, Brown et al., “Retroviral Vectors,” in DNA Cloning: A Practical Approach, Volume 3, IRL Press, Washington, D.C. (1987)).
  • Specificity for gene expression in the peripheral nervous system can be conferred by using appropriate cell-specific regulatory sequences, such as cell-specific enhancers and promoters. Since protein phosphorylation is critical for neuronal regulation (Kennedy, “Second Messengers and Neuronal Function,” in An Introduction to Molecular Neurobiology, Hall, E A, Sinauer Associates, Inc. (1992)), protein kinase promoter sequences can be used to achieve sufficient levels of PNS SCP gene expression.
  • gene therapy can be used to alleviate sodium channel related pathology by inhibiting the inappropriate expression of a particular form of PNS SC.
  • gene therapy can be used to alleviate such pathologies by providing the appropriate expression level of a particular form of PNS SCP.
  • particular PNS SCP nucleic acid sequences can be coded by DNA or RNA constructs which are administered in the form of viruses, as described above.
  • PC12 cells and PKI-4 PC12 subclones were grown as previously described (Mandel et al., 1988).
  • NGF 2.5 S subunit, kindly supplied by Dr. S. Halegoua, SUNY at Stony Brook
  • the PKI-4 PC12 subclone which expresses the cAMP-dependent kinase inhibitor protein (PKI) was also provided by Dr. S. Halegoua (see D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993)).
  • PKI-4 PC12 subclone which expresses the cAMP-dependent kinase inhibitor protein (PKI) was also provided by Dr. S. Halegoua (see D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993)).
  • PCR Amplification Total cellular RNA was isolated, according to the method of Cathala et al. DNA 2:329-335 (1983), from a PC12 subclone (PKI-4) which expresses high levels of the cAM-dependent protein kinase inhibitor protein. Two ⁇ g of total RNA prepared time NGF-treated PKI-4 cells was used to synthesize first strand cDNA using random hexamer primers for the reverse transcriptase reaction. The cDNA then served as template for the PCR amplification, using a pair of degenerate oligonucleotide primers that specified a 400 base pair region within repeat domain III of the sodium: channel ⁇ subunit gene.
  • the 5′ primer (designated YJI:GCG AAGCTT (TC)TIATITT(TC)I(GATC)IAT(ATC)ATGGG (SEQ ID NO:3), underline indicates a HindIII restriction site), corresponded to amino acids FWLIFSIM at positions 1347-1354 in the type II sodium channel gene.
  • the 3′ primer (designated YO1C: GCA GGATCC (AG)TT(AG)AAA(AG)TT(AG)TC(AGT)AT(AGT)AT(AGCT)AC(AGCT)CC (SEQ ID NO:5), underline indicates a BamH1 restriction site) corresponded to amino acids GVIIDNFN at positions 1470-1447 in the type II gene.
  • the amplification reaction mixture consisted of 5% of the cDNA, 1 mM MgCl 2 , 0.2 mM dNTPSs, 0.5 ⁇ M each primer, Taq polymerase (Perkin-Elmer) in a buffer consisting of 0.1 M KCl, 0.1 M-TRIS HCl (pH 8.3) and gelatin (1 mg/ml).
  • the reaction was performed in a Perkin-Elmer thermocycler as follows: 5 cycles of denaturation (94° C., 1 min.), annealing (37° C., 1 min.), and extension (72° C., 1 min) followed by 25 cycles of denaturation (94° C., 1 min.), annealing (50° C., 1 min.) and extension (72° C., 1 min.).
  • the PCR products were excised from a low melt agarose gel (SEAPLAQUE GTG, FMC BIOPRODUCTS) and subcloned into a Bluescript II SK plasmid vector previously restricted with HindIII and BamH1.
  • the clones were screened for cDNA inserts by miniprep (Sambrook et al., infra) and sequenced in both directions by dideoxy chain termination (Sequenase 2.0 kit, UNITED STATES BIOCHEMICAL). Sequence data was compiled and analyzed using GENWORKS software (INTELLIGENETICS, INC., Mountain View, Calif.).
  • RNA blots were cross-linked to the nylon using Stratalinker UV crosslinker (STRATAGENE CORP.) and hybridized to 32 P-UTP-labeled antisense RNA probes generated from the following linearized templates: pPC12-1, pRB211 (Cooperman, infra, 1987), p1B15 (cyclophilin; Danielson et al., DNA 7:261-267 (1988)), and rat brain type 1, which contains 51 bp of intron, 5′ untranslated sequence and 267 bp of coding sequence of the type I sodium channel.
  • Stratalinker UV crosslinker STRATAGENE CORP.
  • RNA probes were transcribed with either T3 (pPC12-1), T7, (pNach1), or SP6 (pRB211, p1B15) RNA polymerase according to the manufacturer's instructions (PROMEGA CORP, Madison, Wis.). The blots were washed once in 2 ⁇ SSC, 0.1% NaDodSO 4 for 15 min. at 68° C., followed by two washes in 0.2 ⁇ SSC, 0.1% NaDodSO 4 for 15 min. at 68° C. Autoradiography with preflashed XAR-5 film (EASTMAN KODAK CO., Rochester, N.Y.) was used for quantitation of mRNA by densitometry.
  • Ribonuclease protections assays were performed by use of a kit (RPA II, AMBION INC., Austin, Tex.). Total RNA was hybridized with 10 4 cpm of antisense RNA probe generated from pPC12-1. To control for differences in the amount of total RNA between samples, we included an antisense RNA probe for ⁇ actin, transcribed from pTRI- ⁇ -actin (AMBION, INC.).
  • Sections were hybridized with antisense digoxigenin-labeled RNA probes transcribed from pPC12-1 or pNach2 (Cooperman et al., Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) according to the manufacturer's instructions for RNA labeling with digoxigenin-UTP (BOEHRINGER MANNHEIM). Unlabeled probes were synthesized by replacing digoxigenin-UTP with rUTP.
  • Each section was covered with ⁇ 100 ⁇ l of hybridization solution containing 20 mM TRIS HCl (pH 8.0), 2.5 mM EDTA, 50% formamide, 0.3 M NaCl, 1 ⁇ Denhardt's, 10% dextran sulfate, 1 mg/ml tRNA, and probe at a concentration of 0.7 ⁇ g/ml. Sections were then covered with PARAFILM coverslips and incubated in a humid chamber overnight at 45° C. After hybridization, sections were washed in 50% formamide, 2 ⁇ SSC at 45° C.
  • cDNAs from an NGF-treated PC12 subclone which preferentially expresses PN1 mRNA (PKI-4 cells) D'Arcangelo et al were amplified by the polymerase chain reaction (PCR), using a pair of degenerate oligonucleotide primers that specify a 400 base pair (bp) region of the sodium channel ⁇ subunit gene (see Methods, FIG. 1). Both primers specified putative membrane-spanning regions within repeat domain III, which are highly conserved among voltage-gated sodium channels.
  • the amplified regions between the primers include the strictly-conserved pore-lining residues, as well as residues which are divergent among the different mammalian ⁇ subunits.
  • Sequence analysis of the PCR products revealed a cDNA, pPC12-1, which encoded a portion of a novel putative sodium channel ⁇ subunit (FIG. 1). Additional cDNAs were further isolated which encapsulated the entire PN1 coding region.
  • pPC12-1 encode part of the PN1 gene
  • the cDNA was used to generate antisense RNA probes for Northern blot analysis of mRNA from control and NGF-treated PC12 cells (FIG. 2B).
  • a duplicate blot (FIG. 2A) was hybridized with an antisense probe pRB211, which encode a highly-conserved region of the sodium channel ⁇ subunit (Cooperman et al., Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) and which cross-hybridizes with the PN1 transcript, and that, as shown by D'Arcangelo et al., J. Cell Biol.
  • a random- and oligo (dT)-primed Lambda ZAP II cDNA library (STRATAGENE, 5.6 ⁇ 10 6 independent clones) was prepared from poly(A)+mRNA isolated from the same PC12 subclone from which pPC12-1 was isolated. Screening 4 ⁇ 10 4 recombinants with a probe generated from pPC12-1 resulted in isolation of 2 additional, overlapping cDNAs which are joined to give a 3033 bp cDNA (FIG. 7). Additional cDNAs were further isolated which encapsulated the entire PN1 coding region.
  • the S4 domain thought to serve as the voltage sensor, exhibits the highly-conserved pattern of a positively-charged residue (lysine or arginine) at every third position; Furthermore, the putative pore-lining segments (IIISS1-IIISS2) contain residues shown to be involved in sodium-selective permeation (Heinemann et al., Nature 356:441-443 (1992)) as well as TTX affinity (Terlaue et al., FEBS Lett. 293:93-96 (1991)).
  • the sodium channel ⁇ subunit undergoes several characteristic post-translational modifications. All sodium channels sequenced to date exhibit a distinctive pattern of asparagine-linked (N-linked) glycosylation sites, which are found almost exclusively in the extracellular loops joining the S5and S6 transmembrane helices.
  • the N-linked glycosylation sites of PN1 are in good agreement with this pattern; three potential extracellular glycosylation sites are located between IIIS5 and IIIS6. Two of the sites are also found in the types I, II and III sodium channels.
  • the ⁇ subunit is phosphorylated by protein kinase C (PKC), and deduced PN1 sequence contains the highly-conserved consensus PKC phosphorylation-site at serine 1506 (FIG. 1). This residue is located in the cytoplasmic loop joining domains III and IV that has been implicated in channel inactivation, and mutational analysis has shown that this serine is required for PKC modulation of channel inactivation (West et al., 1991).
  • PKC protein kinase C
  • the deduced primary structure of PN1 contains all of the hallmark structural and functional domains characteristics a ⁇ subunit the voltage-gated sodium channel.
  • the PN1 gene is expressed preferentially in the PNS.
  • total RNA was isolated from adult rat brain, spinal cord, SCG, DRG, skeletal muscle, and cardiac muscle and subjected to Northern blot analysis. Blots were hybridized with the PN1-specific antisense probe generated from pPC12-1. As shown in FIG. 3A, we found high levels of hybridization to an ⁇ 11 kb transcript in both SCG and DRG. Much lower, but detectable levels hybridization were seen to transcripts in both spinal cord and brain. No detectable hybridization was observed to mRNA from skeletal muscle, cardiac muscle, or liver.
  • RNA protection analyses were also prepared. Total RNA was isolated from the same tissues used in Northern blot analysis, as well as adrenal gland, and hybridized to PN1-specific antisense probe (pPC12-1). mRNA from SCG, DRG, brain, spinal cord, and adrenal gland protected a 343 bp fragment of the PN1 probe (FIG. 4B). The non-protected bases represent oligonucleotide primer and plasmid sequences. The PN1 probe was not protected by mRNA from either skeletal muscle or cardiac muscle.
  • PN1 mRNA levels in both SCG and DRG are approximately 40-fold greater than in spinal cord, adrenal gland and brain.
  • the PN1 gene is expressed in sympathetic and sensory neurons. To determine whether the PN1 gene is expressed in neurons of peripheral ganglia, in situ hybridization was used to examine the cellular distribution of PN1 mRNA in adult rat SCG and DRG. Cryostat sections were hybridized with a PN1 -specific digoxigenin-labeled RNA probe (pPC12-1), which was visualized using an anti-digoxigenin antibody conjugated to alkaline phosphatase. As shown in FIGS. 4A, B the PN1 antisense probe labeled most neuronal cell bodies in both SCG and DRG.
  • PN1 probe stained most neuronal cell bodies in both SCG and DRG, we found that cell-to-cell variability in PN1 mRNA levels differed between the two ganglia.
  • SCG neurons were fairly homogeneous, in that the intensity of reaction product was relatively constant between different cells.
  • DRG neurons were quite heterogeneous in that the staining intensity varied considerably from cell to cell. For example, in FIG. 4B, arrows indicate two DRG neurons of approximately the same diameter which differ markedly in staining intensity.
  • PN2 probe did not stain non-neuronal cells such as satellite cells and Schwann cells. However, it is possible that these cells contain very low levels of PN1 mRNA which are not detectable by this method.
  • SCG neurons also express the type I sodium channel gene.
  • mRNA from SCG contains two distinct sodium channel gene transcripts. As we have demonstrated, the larger, 11 kb transcript encodes the PN1 sodium channel. The smaller transcript, however, has not yet been identified. We hypothesized that this smaller transcript encoded the type I sodium channel, because moderate levels of type I mRNA have been found in other PNS tissues (Beckh, S., FEBS Lett. 262:317-322 (1990)).
  • Northern blots of SCG mRNA isolated from adult rats were hybridized with an antisense probe specific for the type I sodium channel gene (pNach1, see Methods above). As shown in FIG. 5, the type I-specific probe hybridized specifically to the smaller transcript.
  • SCG mRNA protects the type I probe in an RNas protection assay.
  • the putative PN1 ⁇ subunit and type I ⁇ subunit genes are differentially regulated during development.
  • Several studies have shown that the types I, II and III sodium channel genes are differentially regulated during development in both the central and peripheral nervous systems.
  • To determine whether the PN1 and type I genes are also independently regulated during development we measured their relative mRNA levels in SCG isolated from rats of different postnatal ages. To visualize both transcripts simultaneously, Northern blots were hybridized with the conserved sodium channel gene probe pRB211. As shown in FIG. 6A, in SCG removed on postnatal day 7 (P7), the levels of PN1 and type I mRNA are approximately equal.
  • PN1 is the predominant sodium channel gene transcript, with levels of PN1 mRNA several-fold greater than that of type I.
  • PNS SCP-ligand e.g., antagonists and agonists
  • a PNS SCP-ligand e.g., antagonists and agonists
  • An assay for PNS SCP activity in such cells is used to determine the functionality of the PNS SCP protein in the presence of at least one agent which can act as antagonist or agonist, and thus, agents that interfere or enhance the activity of PNS SCP are identified.
  • Two or more cell lines (each expressing a different PNS SCP) are used, as well as optionally using one or more cell lines expressing a CNS specific sodium channel as a control.
  • agents are selected and screened (1) at random; (2) by a rational selection; and or (3) by design using for example, computer modeling techniques.
  • Agent determination methods include Computer Assisted Molecular Design (CAMD)) PNS SCP-agent binding, sophisticated chemical synthesis and testing, targeted screening, peptide combinatorial library technology, antisense technology and/or biological assays, according to known methods. See, e.g., Rapaka et al., eds., Medications Development: Drug Discovery, Databases, and Computer - Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, U.S. Dept.
  • CVD Computer Assisted Molecular Design
  • cell expression libraries, or other cells are used to that have been selected or genetically engineered to express and display a PNS SCP via the use of the PNS SCP nucleic acids of the invention are preferred in such methods, as host cell lines may be chosen which are devoid of related receptors. Rapaka, infra, (1993), at pages 58-65.
  • a PNS SCP agent in the context of the present invention refers to any chemical or biological molecule that associates with a PNS SCP in vitro, in situ or in vivo, and can be, but is not limited to, synthetic, recombinant or naturally derived chemical compounds and compositions, e.g., organic compounds, nucleic acids, peptides, carbohydrates, vitamin derivatives, hormones, neurotransmitters, viruses or receptor binding domains thereof, opsins, rhodopsins, nucleosides, nucleotides, coagulation cascade factors, odorants or pheremones, toxins, growth factors, platelet activating factors, neuroactive peptides, neurohumors, or any biologically active compound, such as drugs or naturally occurring compounds.
  • synthetic, recombinant or naturally derived chemical compounds and compositions e.g., organic compounds, nucleic acids, peptides, carbohydrates, vitamin derivatives, hormones, neurotransmitters, viruses or receptor binding domains thereof
  • agents are selected and screened at random or rationally selected or designed using computer modeling techniques. For random screening, potential agents are selected and assayed for their ability to bind to the PNS SCP, or a fragment thereof. Alternatively, agents may be rationally selected or designed. As used herein, a agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of at least one specific PNS SCP (e.g., as presented in FIG. 11). For example, one skilled in the art can readily adapt currently available procedures to generate agents capable of binding to a specific peptide sequence in order to generate rationally designed compounds, such as chemical compounds, nucleic acids or peptides.
  • a method of screening for an agent that modulates the activity of at least one PNS SCP comprising:
  • Any cell can be used in the above assay so long as it expresses a functional form of PNS SCP protein and the PNS SCP activity can be measured.
  • the preferred expression cells are eukaryotic cells or organisms. Such cells can be modified to contain DNA sequences encoding the PNS SCP protein using routine procedures known in the art Alternatively, one skilled in the art can introduce mRNA encoding the PNS SCP protein directly into the cell.
  • stem cell populations for either neuronal or glial cells can be genetically engineered to express a functional PNS SCP ion channel.
  • Such cells expressing the PNS SCP ion channel can be transplanted to the diseased or injured region of the mammal's neurological system ( Neural Transplantation. A Practical Approach, Donnet & Djorklund, eds., Oxford University Press, New York, N.Y. (1992)).
  • embryonic tissue or fetal neurons can be genetically engineered to express functional PNS SCP ion channel and transplanted to the diseased or injured region of the mammal's limbic system. The feasibility of transplanting fetal dopamine neurons into Parkinsonian patients has been demonstrated. (Lindvall et al., Archives of Neurology 46:615-631 (1989)).
  • RNA polymerase such as SP6 polymerase or T7 polymerase with a capping analog such as M 7 G(5′)ppp(5′)G.
  • RNA e.g., about 50 nl, corresponding to 2-5 ng
  • stage V and stage VI oocytes isolated from Xenopus, and incubated for 3-5 days at 19° C.
  • Oocytes axe tested for sodium channel expression with a two-microelectrode voltage clamp (Trimmer et al, Neuron 3:33-49 1989).
  • cDNAs encoding a voltage-dependent sodium channel is cloned into any one of a number of mammalian expression vectors, and transfected into mammalian cells which do not express endogenous voltage-dependent sodium channels (such as fibroblast cell lines). Transfected clones are selected expressing the cloned, transfected cDNA. Sodium channel expression is measured with a whole cell voltage clamp technique using a patch electrode (D'Arcangelo et al., J. Cell. Biol. 122:915-921 (1993)).
  • PNS SCPs and Cell Lines Useful for Drug Screening Any cell line expressing (Naturally, by induction or due to recombinant expression of a PNS SCP) can be used for drug screening.
  • PC12 cells are mutants deficient in Protein Kinase A (PKA) activity and which express both PN1 and Type II sodium channels.
  • PKA Protein Kinase A
  • A126-1B2 cells are a cell line which express PN1, but are now discovered to does not express Type II sodium channels.
  • PKI-4 is a PC12 cell line transfected with a cDNA encoding a peptide inhibitor of PKA.
  • Each of these cell lines can be used as one source of a PNS SCP of the present invention, or as a cell line itself to use in drug screening.
  • Treatment of PC12 cells with NGF reduces both a PNS SCP (PN1) and type II sodium channels, while NGF induces only PN1 in A 126-182 cells.
  • PKI-4 cells express a PNS SCP (PN1) without NGF treatment (D'Arcangelo et al, J. Cell Biol. 122:915-921 (1993)).
  • heterologous expression systems can also be used in which cell lines (such as Chinese Hamster Ovary cells (CHO)) are stably transfected with a cDNA encoding PN-1. Method steps for transfecting and stably expressing cDNA to form heterologous cell lines, are well known in the art An advantage of using transfected cells is that clones are obtained that express very high levels of a PNS SCP, such as PN-1.
  • cell lines such as Chinese Hamster Ovary cells (CHO)
  • Method steps for transfecting and stably expressing cDNA to form heterologous cell lines are well known in the art
  • An advantage of using transfected cells is that clones are obtained that express very high levels of a PNS SCP, such as PN-1.
  • Labeled ligand binding neurotoxins can be used to characterize PNS sodium channels. For example previous studies have identified at least six distinct neurotoxin binding sites on previously characterized non-PNS-sodium channels (reviewed in Lombert et al., FEB 219(2):355-359 (1987)). Many of these sites are thought to be allosterically coupled to one another (for review, see Strichartz et al., Ann. Rev. Neurosci. 10:237-267 (1987), and references cited therein). In other words, binding of a drug or toxin to a particular neurotoxin site can be sensitive to drug binding at not only that site, but other sites on the channel as well. This is advantageous for a drug screening program in that for a given labeled ligand, the likelihood of identifying agents that preferrentially bind to a PNS SCP is increased.
  • A126-1B2, PC12, PKI-4 or other cells expressing a PNS SCP cells are grown using standard techniques, and optionally treated with NGF for 1-2 days to induce PN-1 expression. Cells are harvested and tested for ion flux activity with alternative potential agents.
  • binding reactions are conducted e.g., at 37° C., then stopped. Samples are quickly filtered with vacuum washed with ice-cold buffer, and bound radioactivity determined by scintillation counting.
  • Ion Flux directly tests the ability of a potential PNS SCP agent to inhibit or enhance the activity of a PNS SCP function, by their ability to inhibit or enhance the influx of ion tracers through a PNS SCP.
  • routine methods can be used to screen compounds for modulating PNS SCP ion channel activity, e.g., ( 14 C) guanidimium ion flux using intact cells expressing at least one PNS SCP. Additionally these methods are well known to be easily modified for use with 22 Na. Similarly, these known method steps could be modified for use with substrate-attached cells or vesicles prepared from broken cells, according to known method steps.
  • guanidinium flux assay For a guanidinium flux assay the methods for 22 Na are modified from those of Reith ( Europ. J. Pharmacol. 188:33-41 (1990) for brain synaptosomes), e.g., as described in Example 2 below. Aliquots of a cell suspension containing heterologous cells expressing at least one PNS SCP are incubated for 10 minutes at 37° C. in the presence of channel openers (typically, 100 ⁇ M veratridine) and test drugs in a total volume of 100 ⁇ M (0.20-0.25 mg protein). Ion flux is initiated by the addition of HEPES/TRIS solution also containing 4mM guanidine HCl (final) and 1000 dpm/nmol ( 14 C) guanidine.
  • channel openers typically, 100 ⁇ M veratridine
  • test drugs in a total volume of 100 ⁇ M (0.20-0.25 mg protein). Ion flux is initiated by the addition of HEPES/TRIS solution also containing 4m
  • the reaction is continued for 30 seconds and is stopped by the addition of ice-cold incubation buffer, followed by rapid filtration under vacuum over Whatman GF/C filter.
  • the filters are washed rapidly with ice-cold incubation buffer and radioactivity determined by scintillation counting. Nonspecific uptake is determined in parallel by the inclusion of 1 mM tetrodotoxin during both preincubation and uptake.
  • methyl/halophenyl substituted compounds such as lidoflazine (see, e.g., Merck Index Monograph 5311 and U.S. Pat. No. 3,267,104, both entirely incoporated herein by reference), were tested and found to inhibit sodium channel activity of at least one PNS SCP of the present invention in cell lines expressing at least one PNS SCP, with a pIC50 of 6.51 for lidoflazine on PKI-4 cells.
  • the present invention provides PNS SCP modulating agents as methyl/halophenyl-substituted piperizines.
  • a human peripheral nervous system cDNA library (as a human DRG library) was used for polymerase chain reaction (PCR) amplification.
  • the PCR used a 5′ primer corresponding to DNA encoding amino acids 604-611 of SEQ ID NO:2, and a corresponding 3′ primer encoding amino acids 723-731 of SEQ ID NO:2.
  • the PCR reaction mixture consisted of 5% of the cDNA, 1 mM MgCl 2 , 0.2 mM dNTPSs, 0.5 mM, each primer, Taq polymerase (Perkin-Elmer) in a buffer consisting of 0.1 M KCl, 0.1 M TRIS HCl (pH 8.3) and gelatin (1 mg/ml).
  • the reaction was performed in a Perkin-Elmer thermocycler as follows: five cycles of denaturations (94° C., 1 min.), annealing (37° C., 1 min), and extension (72° C., 1 min.), followed by 25 cycles of denaturation (94° C., 1 min.), annealing (50° C., 1 min.), and extension (72° C., 1 min.).
  • the resulting PCR products provided a human amplified cDNA which encoded amino acids 646-658 of SEQ ID NO:2, as presented in FIG. 11A-E.
  • PCR primers corresponding to SEQ ID NO:1 are used to isolate clones from the human DRG cDNA library which encompass the entire coding region of one or more human PNS SCPs of the present invention.
  • a 5′ primer includes the sequence 5′TTTGTGCCCCACAGACCCCAG3′ (SEQ ID NO: 13) and a 3′ primer includes the sequence 5′ ACACAAATTCTTGATCTGGAATTGCT3′(SEQ ID NO: 14) or 5′CAACCTC AGACAGAGAG CAATGA 3′ (SEQ ID NO: 15), which are used for nested PCR.
  • PCR is performed to obtain cDNAs encoding a human PNS SCP.
  • Additional PCR is performed by “walking” 5′ or 3′ of the sequence corresponding to the above PCR product. In this way cDNAs encompassing the entire coding region of one or more human PNS SCPs are provided.
  • the resulting additional cDNA clones or PCR products, encoding the entire human PNS SCP, are subcloned into a plasmid vector previously restricted with suitable restriction sites.
  • the clones are screened for cDNA inserts by miniprep (Sambrook et al., infra) and sequenced in both directions by dideoxy chain termination (Sequenase 2.0 kit, United States Biochemical). Sequence data is compiled and analyzed using GeneWorks software (IntelliGenetics, Inc., Mountain View, Calif.).
  • Transcripts of the size of the resulting human PNS SCP are then confirmed to be present in human PNS mRNA or cDNA (encoding a 1970-1990 amino acid sequence of FIG. 11A-E). However, as in Example 1, such transcripts are not expected to be detected in mRNA from brain. This expected result confirms new human members of the sodium channel gene family (termed Human Peripheral Nerve type 1 (HUMPN1A (deposited as ATCC No. ______) and HUMPN1B (Deposited as ATCC No. ______) of FIG. 11A-E, where X is 0, 1, 2 or 3 of the same or different amino acid).
  • HUMPN1A Human Peripheral Nerve type 1
  • HUMPN1B Deposited as ATCC No. ______

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Abstract

Cloning, expression, viral and delivery vectors and hosts which contain nucleic acid coding for at least one peripheral nervous system specific (PNS) sodium channel peptide (SCP), isolated PNS SCP, and compounds and compositions and methods, are provided, for isolating, crystallizing, x-ray analysing molecular modeling, rational drug designing, selecting, making and using therapeutic or diagnostic agents or ligands having at least one peripheral nervous system specific (PNS) sodium channel (SC) modulating activity.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation-in-part of U.S. application. Ser. No. 08/482,401, filed Jun. 7, 1995, which is a continuation-in-part of U.S. application Ser. No. 08/334,029 filed Nov. 2, 1994,both of which disclosures are entirely incorporated herein by reference[0001]
    • STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
  • [0002] The present invention was made with U.S. government support Therefore, the U.S. government has certain rights in the invention.
    • FIELD OF THE INVENTION
  • The present invention is in the fields of biotechnology, protein purification and crystallization, x-ray diffraction analysis, three-dimensional computer molecular modeling, and rational drug design (RDD). The invention is directed to isolated peripheral nervous system (PNS) specific sodium channel proteins (SCPs) and encoding nucleic acid, as well as to compounds, compositions and methods for selecting, making and using therapeutic or diagnostic agents having sodium channel modulating activity. The present invention further provides three-dimensional computer modeling of the PNS SCP, and for RDD, based on the use of x-ray data and/or amino acid sequence data on computer readable media. [0003]
    • BACKGROUND OF THE INVENTION
  • Voltage-sensitive ion channels are a class of transmembrane proteins that provide a basis for cellular excitability, as the ability to transmit information via ion-generated membrane potentials. In response to changes in membrane potentials, these molecules mediate rapid ion flux through highly selective pores in a nerve cell membrane. If the channel density is high enough, a suitable regenerative depolarization results, termed the action potential. [0004]
  • The voltage-sensitive sodium channel is the ion channel most often responsible for generating the action potential in excitable cells. Although sodium-based action potentials in different excitable tissues look similar (Hille, B., [0005] In: Ionic Channels of Excitable Membranes, B. Hille, ed., Sinauer, Sunderland, Mass., (1984), pp. 70-71) recent electophysiological studies indicate that sodium channels in different cells differ in both their structural and functional properties, and many sodium channels with distinct primary structures have now been identified. See, e.g., Mandel, J. Membrane Biol. 125: 193-205 (1992).
  • Functionally distinct sodium channels have been described in a variety of neuronal cell types (Llinas et al., [0006] J. Physiol. 305:197-213 (1980); Kostytk et al., Neuroscience 6:2423-2430(1981); Bossu et al., Neurosci. Lett. 51:241-246 (1984) 1981, Gilly et al., Nature 309:448-450 (1984); French et al, Neurosci. Lett. 56:289-294 (1985); Ikeda et al., J. Neurophysiol. 55:527-539 (1986); Jones et al., J. Physiol. 389:605-627 (1987); Alonso & Llinas, 1989; Gilly et al., J. Neurosci 9:1362-1374 (1989)) and in skeletal muscle (Gonoi et al., J. Neurosci 5:2559-2564 (1985); Weiss et al., Science 233:361-364 (1986)). The kinetics of sodium currents in glia and neurons can also be distinguished (Barres et al., Neuron 2:1375-1388 (1989)).
  • The type II and type III genes, expressed widely in the central nervous system (CNS), are expressed at very low levels in some cells in the PNS (Beckh, S., [0007] FEBS Lett. 262:317-322 (1990)). The type II and III mRNAs were barely detectable, by Northern blot analysis, in dorsal root ganglion (DRG), cranial nerves and sciatic nerves. On the other hand, type I mRNA was present in moderately high amounts in DRG and cranial nerve, but in low levels in sciatic nerve. A comparison of the amount of all three brain mRNAs, relative to total sodium channel mRNA detected with a conserved cDNA probe, suggested the presence of additional, as yet unidentified, sodium channel types in DRG neurons. Consistent with the mRNA studies, immunochemical studies showed that neither type I nor type II sodium channel alpha subunits made up a significant component of the total sodium channels in the superior cervical ganglion or sciatic nerve (Gordon et al., Proc. Natl. Acad. Sci. USA 84:8682-8686 (1987)).
  • A population of neurons in vertebrate DRG has been identified electrophysiologically that contains, in addition to the more conventional channels, a distinct sodium channel type; this DRG channel has a k[0008] D for TTX approximately tenfold higher than the kD of sodium channels in either skeletal muscle or heart (Jones et al., J. Physiol. 389:605-627 (1987)).
  • The localization of different sodium channels to specific regions in the nervous system supports the possibility that cell-specific regulation of this gene family is at the transcriptional level. By analogy with other eukaryotic genes, distinct DNA elements can be present which mediate cell-specific and temporal regulation of individual sodium channel genes. [0009]
  • Studies of sodium channel gene regulation have been facilitated by the use of well-characterized cell lines, such as pheochromocytoma (PC12) cells, a popular cell model for neuronal differentiation (Green et al., [0010] Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976); Halegoua et al., Curr. Top. Microbiol. Immunol. 165:119-170 (1991)). In addition to extending neurites and initiating synthesis of certain neurotransmitters, NGF-treated PC12 cells acquire the ability to generate sodium-based action potentials (Dichter et al., Nature 268:501-504 (1977)). This ability is conferred by an increase in the density of functional sodium channels in the membranes of the NGF-treated cells (Rudy et al., J. Neurosci. 7:1613-1625 (1987); Mandel et al., Proc. Natl. Acad Sci. USA 85:924-928 (1988); O'Lague et al., Proc. Natl. Acad. Sci. USA 77:1701-1705 (1980)). Northern blot analysis revealed that undifferentiated PC12 cells contained a basal level of sodium channel mRNA which increased coincident with the increase in channel activity observed after treatment with NGF (Mandel et al., Proc. Natl. Acad. Sci. USA 85:924-928 (1988)).
  • There is a long standing need to diagnose and/or treat pathologies relating to impaired peripheral nervous system (PNS) nerve conduction associated with PNS injury or in genetic or other disease states, such as those involving lack of or defects in, PNS sodium channels (SCs). In view of the possibility of cell or tissue specific sodium channels, the discovery and use of isolated PNS SCs and encoding nucleic acid would provide an opportunity to diagnose or treat such pathologies by either screening suitable PNS SC modulating drugs or molecules (e.g., analgesics), or by using recombinant PNS SCs for in situ or in vivo gene therapy to replace or supplement PNS SCs in at least one portion of the peripheral nervous system of a mammalian patient suffering from a PNS SC related pathology. [0011]
    • SUMMARY OF THE INVENTION
  • The present invention (hereinafter, “invention”) provides peripheral nervous system specific (PNS) sodium channel peptides (SCPs), encoding nucleic acid, vectors, host cells and antibodies, as well as methods of making and using thereof, including recombinant expression, purification, cell-based drug screening, gene therapy, crystallization, X-ray diffraction analysis, as well as computer structure determination and rational drug design utilizing at least one PNS SCP amino acid sequence and/or x-ray diffraction data provided on computer readable media. [0012]
  • The invention also includes oligonucleotide probes specific for PNS SCP encoding sequences, as well as methods for dectection in a sample, where the probe is labeled. The invention further includes methods for producing a PNS SCP, comprising culturing a host in a culture medium, comprising a PNS SCP nucleic acid; and isolating the PNS SCP from said host or said culture medium. [0013]
  • The invention additionally includes an antibody which binds an epitope specific for a PNS SCP, as well as host cells which express the antibody. Diagnostic or therapeutic methods using the antibody are also included in the invention. [0014]
  • The invention further includes gene therapy methods and delivery vectors comprising nucleic acid encoding, or complementary to, at least one PNS SCP, and pharmaceutically acceptable compositions thereof. [0015]
  • The invention also includes gene therapy by methods that administer an antisense PNS SCP nucleic acid to an animal in amount effective to provide a PNS SC modulating effect, such as an analgesic effect. [0016]
  • The present invention further provides methods for purifying and crystallizing a PNS SCP that can be analyzed to obtain x-ray diffraction patterns of sufficiently high resolution to be useful for three-dimensional molecular modeling of the protein. The x-ray diffraction data, atomic coordinates, and/or amino acid sequences provided on computer readable medium, are modeled on computer systems, using methods of the invention, to generate secondary, tertiary and/or quaternary structures of a PNS SCP, which structures contribute to their overall three dimensional structure, as well as binding and active sites of the PNS SCP. [0017]
  • Molecular modeling methods and computer systems are also provided by the present invention for rational drug design (RDD). These drug design methods use computer modeling programs to find potential ligands or agents that are calculated to bind with sites or domains on the PNS SCP. Potential ligands or agents are then screened for modulating or binding activity. Such screening methods can be selected from assays for at least one biological activity of the protein, as associated with a PNS SCP-related pathology or trauma, according to known sodium channel assays. The resulting ligands provided by methods of the present invention are synthesized and are useful for treating, inhibiting or preventing at least one of PCS SCP-related pathology or trauma in a mammal. [0018]
  • Further objects, features, utilities, embodiments and/or advantages of the present invention will be apparent from the additional description provided herein.[0019]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts a 323 amino acid and corresponding 969 nucleotide sequence of a PNS SCP as amino acids 233-555 of SEQ ID NO:2 and nucleotides 699-1665 of SEQ ID NO:1, as the primary structure of Domain III of the Peripheral Nerve type I (PN1) sodium channel alpha ( ) subunit for both amino acid and DNA sequences. The single amino acid code is used to denote deduced amino acids. YJ1 and YOIC refer to the oligonucleotide primers used to obtain the initial PCR fragment of PN1 cDNA. [0020]
  • FIG. 2A-B shows a Northern blot analysis of sodium channel α subunit mRNA in rat pheochromocytoma (PC12) cells treated with Nerve Growth Factor. In FIG. 2(A), the probe used is pRB211 which encodes the highly conserved fourth repeated domain of the rat type II sodium channel. Both type H and PN1 mRNAs are detected with this probe. In FIG. 2(B), the probe used contains sequences specific for PN1. The levels of sodium channel mRNA are quantitated with reference to the amount of cyclophilin mRNA, as indicated. Control cells are PC 12 cells grown in the absence of NGF. [0021]
  • FIG. 3A-B shows an example of tissue-specific distribution of PN1 mRNA. FIG. 3(A) presents a Northern blot analysis using equal amounts of RNA from tissues. PN1 mRNA is indicated by the dash. 28S refers to the 28S rRNA. The probe contains sequences specific for the PN1 gene. Note the absence of PN1 mRNA in skeletal muscle, cardiac muscle, and the low levels of PN1 mRNA in spinal cord. FIG. 3(B) shows RNAase protection analysis of PN1 mRNA. PN1 refers to the PN1 probe protected by mRNA from the different tissue samples. Actin refers to actin probe sequences protected by the same mRNA. [0022]
  • FIG. 4A-F shows localization of PN1 mRNA in Superior Cervical Ganglion (SCG) and Dorsal Root Ganglion (DRG) tissues by in situ hybridization analysis. FIGS. 4A-4B represent neurons hybridized with a PN1-specific antisense RNA probe. FIGS. 4C-4D represent neurons hybridized with the radiolabeled PN1 probe in the presence of non-labeled PN1 competitor DNA. FIGS. 4E-4F represent tissue sections hybridized with an antisense type II probe. [0023]
  • FIG. 5 shows a blot analysis comparing Levels of PN1 and brain type I a subunit mRNA in SCG. The pRB11 conserved sodium channel probe detects both type II/IIA and PN1 transcripts. [0024]
  • FIG. 6A-B shows a Northern blot analysis which reveals differential expression of PN1 and type I sodium channel mRNAs during postnatal rat development. FIG. 6(A) shows a representative autoradiogram of a Northern blot using radiolabeled antisense pRB211 RNA as probe. Postnatal days 7 (P7) to 42 (P42) are shown. FIG. 6(B) shows a plot of quantitation of the Northern blots showing a decrease in type I mRNA with time after birth. [0025]
  • FIG. 7A-D show the deduced primary structure of cloned portion of PN1 a subunit cDNA as a partial 3033 nucleotide (SEQ ID NO:1) sequence and a partial 1011 amino acid (SEQ ID NO:2) sequence. [0026]
  • FIG. 8A-D show a comparison of deduced primary amino acid sequences of PN1 (1-988 of SEQ ID NO:2) and brain type II/IIA α subunit (SEQ ID NO:). [0027]
  • FIG. 9A-9D show the entire DNA sequence for a rat PN1 PNS SCP(SEQ ID NO:9). [0028]
  • FIG. 10 shows the entire amino sequence for a rat PN1 PNS SCP (SEQ ID NO:10). [0029]
  • FIG. 11A-11E shows amino acid sequences for rat PN1 (“RATPN1”) (SEQ ID NO:10) and two expected human PN1 sequences “HULMN1A” (SEQ ID NO:11) “HUMPN1B”(SEQ ID NO:12) HUMPN1C (SEQ ID NO:7) and HUMPN1D (SEQ ID NO:). Alternative sequences include those where “X” is 0, 1, 2, or 3 of the same or different amino acids, which can be optionally selected from Table 1 or Table 2. [0030]
  • FIG. 12 shows a computer system suitable for three dimensional structure determination and/or rational drug, design. [0031]
  • FIG. 13A-B shows a representative DNA sequence encoding a human PN1 (HUM PN1A) (SEQ ID NO:11) [0032]
  • FIG. 14B shows a representative DNA sequence encoding a human PN1 (HUM PN1B) (SEQ ID NO:12) [0033]
    • DETAILED DESCRIPTION OF THE INVENTION
  • A need exists for modulating the activity of at least one peripheral nervous system specific (PNS) sodium channel (SCs). Such modulation could potentially provide analgesic or diagnostic agents for pain or pathologies associated with nerve conduction in the PNS. [0034]
  • Certain sodium channels—corresponding to PNS SCPs of the invention—are now discovered to be preferentially or selectively expressed in the peripheral nervous system (PNS). These sodium channels modulate peripheral nerve impulse conduction preferentially in the PNS. The present invention provides peripheral nervous system specific (PNS) sodium channel peptides (SCPs), encoding nucleic acid, vectors, host cells and antibodies, as well as methods of making and using thereof, including recombinant expression, purification, cell-based drug screening, gene therapy, crystallization, X-ray diffraction analysis, as well as computer structure determination and rational drug design utilizing at least one PNS SCP amino acid sequence and/or x-ray diffraction data provided on computer readable media. [0035]
  • A PNS sodium channel peptide (PNS SCP) can refer to any subset of a PNS sodium channel (SC) having SC activity, as a fragment, consensus sequence or repeating unit. A PNS SCP of the invention can be prepared by: [0036]
  • (a) recombinant DNA methods; [0037]
  • (b) proteolytic digestion of the intact molecule or a fragment thereof; [0038]
  • (c) chemical peptide synthesis methods well-known in the art; and/or [0039]
  • (d) by any other method capable of producing a PNS SCP and having a conformation similar to an active portion of a PNS SCP and having SC activity. The SC activity can be screened according to known screening assays for sodium channel activity, in vitro, in situ or in vivo. The minimum peptide sequence to have activity is based on the smallest unit containing or comprising a particular region, domain, consensus sequence, or repeating unit thereof, of at least one PNS SCP. [0040]
  • According to the invention, a PNS SCP includes an association of two or more polypeptide domains, such as transmembrane, pore lining domains, or fragments thereof, corresponding to a PNS SCP, such as 1-40 domains or any range or value therein. Transmembrane, cytoplasmic pore lining or other domains of a PNS SCP of the invention may have at least 74% homology, such as 74-100% overall homology or identity, or any range or value therein to one or more corresponding SC domains as described herein (eg., as presented FIGS. 1, 7, [0041] 8, 10 or 11). As would be understood by one of ordinary skill in the art, the above configuration of domains are provided as part of a PNS SCP of the invention, such that a functional PNS SCP, when expressed in a suitable cell, is capable of transporting sodium ions across a lipid bilayer, a cell membrane or a membrane model. In intact cells having sufficient sodium channels, the cell can be capable of generating some form of an action potential, such as in a cell expressing at least one PNS SCP of the present invention. Such transport, as measured by suitable SC activity assays, establishes SC activity of one or more PNS SCPs of the invention.
  • Accordingly, a PNS SCP of the invention alternatively includes peptides having a portion of a SC amino acid sequence which substantially corresponds to at least one 20 to 2005 amino acid fragment and/or consensus sequence of a PNS SCP or group of PNS SCPs, wherein the PNS SCP has homology or identity of at least 74-99%, such as 88-99% (or any range or value therein, e.g., 87-99, 88-99, 89-99, 90-99, 91-99, 92-99, 93-99, 94-99, 95-99, 96-99, 97-99, or 98-99%) homology to at least one sequence or consensis sequence of FIGS. 1, 7, [0042] 8, 10 r 11. In one aspect, such a PNS SCP can maintain SC biological activity. It is preferred that a PNS SCP of the invention is not naturally occurring or is naturally occurring but is in a purified or isolated form which does not occur in nature. Preferably, a PNS SCP of the invention substantially corresponds to an set of domains of PN1, having at least 10 contiguous amino acids of FIGS. 1, 7, 8, 10 and 11, or at least 74% homology thereto.
  • Alternatively or additionally, a PNS SCP of the invention may comprise at least one domain corresponding to known sodium channel domains, such as rat brain or spinal cord SC domains, such as transmembrane domains, pore lining domains, cytoplasmic domains or extracellular domains, such as IIs6 (e.g., 1-3 to 14-17 (IIs6) 18-23 to 210-214 (cytoplasmic), 229-236 to 254-258 (IIIS1), 268-272 to 293-297 (IIIs2), 300-304 to 321-325 (IIIs3), 326-330 to 347-351 (IIIs4), 368-374 to 389-393 (IIIs5), 474-478 to 500-504 (IIIs6), 553-559 to 577-583 (IVs1), 589-593 to 611-615 (IVs2), 619-623 to 642-646 (IVs3), 654-658 to 678-682 (IVs4), 690-694 to 711-715 (IVs5), 779-783 to 801-805 (IVs6), 348-352 to 368-372, 501-505 to 550-554, 233-555, 676-678 to 689-693, 554-557 to 941-945, or any range or value therein, corresponding to SEQ ID NO:2 as presented in FIG. 7A-7D, or variants thereof as presented substitutions in Table 1 or Table 2, having 74-100% overall homology or any range or value therein. At least one of such domains are present in the PNS SCPs presented in FIG. 11A-E, or fragments thereof, as non-limiting examples. Alternative domains are also encoded by DNA which hybridizes under stringent conditions to at least 30 contiguous nucleotides of FIGS. 1, 7, [0043] 9, 13 or 14, or having codons substituted therefor which encode the same amino acid as a particular codon. Additionally, phosphorylation (e.g., PKA and PKC) domains, as would be recognized by the those skilled in the art are also considered when providing a PNS SCP or encoding nucleic acid according to the invention.
  • Percent homology or identity can be determined, for example, by comparing sequence information using the GAP computer program version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch ([0044] J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. In a preferred embodiment, the peptide of the invention corresponds to a SC biologically active portion of SEQ ID NO:2, or variant thereof e.g., as presented in FIG. 11A-D.
  • Thus, one of ordinary skill in the art, given the teachings and guidance presented in the present specification, will know how to add, delete or substitute other amino acid residues in other positions of a SC to obtain a PNS SCP, including substituted, deletional or additional variants, e.g. with a substitution as presented in Tables 1 or 2 below. [0045]
  • A PNS SCP of the invention also includes a variant wherein at least one amino acid residue in the peptide has been conservatively replaced, added or deleted by at least one different amino acid. For a detailed description of protein chemistry and structure, See, e.g., Schulz, et al., [0046] Principles of Protein Structure, Springer-Verlag, N.Y., 1978, and Creighton, T. E., Proteins, Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. F r a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al., eds, Current Protocols in Molecular Biology, Greene Publishing Assoc., New York, N.Y. (1987, 1992, 1993, 1994, 1995) at §§ A. 1.1-A.1.24, and Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), at Appendices C and D.
  • Conservative substitutions of a PNS SCP of the invention includes a variant wherein at least one amino acid residue in the peptide has been conservatively replaced, added or deleted by at least one different amino acid. Such substitutions preferably are made in accordance with the following list as presented in Table 1, which substitutions can be determined by routine experimentation to provide modified structural and functional properties of a synthesized peptide molecule, while maintaining SC biological activity, as determined by known SC activity assays. In the context of the invention, the term PNS SCP or “substantially corresponding to” includes such substitutions. [0047]
    TABLE 1
    Original Exemplary
    Residue Substitution
    Ala Gly; Ser
    Arg Lys
    Asn Gln; His
    Asp Glu
    Cys Ser
    Gln Asn
    Glu Asp
    Gly Ala; Pro
    His Asn; Gln
    Ile Leu; Val
    Leu Ile; Val
    Lys Arg; Gln; Glu
    Met Leu; Tyr; Ile
    Phe Met; Leu; Tyr
    Ser Thr
    Thr Ser
    Trp Tyr
    Tyr Trp; Phe
    Val Ile; Leu
  • Alternatively, another group of substitutions of PNS SCPs of the invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue added in its place according to the following Table 2. The types of substitutions which can be made in the protein or peptide molecule of the invention can be based on analysis of the frequencies of amino abid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schultz et al., infra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: [0048]
    TABLE 2
    1. Small aliphatic. nonpolar or slightly polar residues:
    Ala. Ser, Thr (Pro, Gly);
    2. Polar, negatively charged residues and their amides:
    Asp, Asn, Glu, Gln;
    3. Polar, positively charged residues:
    His, Arg, Lys;
    4. Large aliphatic, nonpolar residues:
    Met, Leu, Ile, Val (Cys); and
    5. Large aromatic residues: Phe. Tyr. Trp.
  • Most deletions and additions, and substitutions according to the invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. “Characteristics” is defined in a non-inclusive manner to define both changes in secondary structure, e.g. α-helix or β-sheet, as well as changes in physiological activity, e.g. in receptor binding assays. [0049]
  • Accordingly, based on the above examples of specific substitutions, alternative substitutions can be made by routine experimentation, to provide alternative PNS SCPs of the invention, e.g., by making one or more conservative substitutions of SC fragments which provide SC activity. However, when the exact effect of the substitution, deletion, or addition is to be confirmed, one skilled in the art will appreciate that the effect of at least one substitution, addition or deletion will be evaluated by at least one sodium channel activity screening assay, such as, but not limited to, immunoassays or bioassays, to confirm biological activity, such as, but not limited to, sodium channel activity. [0050]
  • Amino acid sequence variants of a PNS SCP of the invention can also be prepared by mutations in the DNA. Such variants include, for example, deletions from, or additions or substitutions of, residues within the amino acid sequence. Any combination of deletion, addition, and substitution can also be made to arrive at the final construct, provided that the final construct possesses some SC activity. Preferably improved SC activity is found over that of the non-variant peptide. Obviously, mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see, e.g., EP Patent Application Publication No. 75,444; Ausubel, infra; Sambrook, infra). At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding a PNS SCP, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variants typically exhibit the same qualitative biological activity as the naturally occurring SC (see, e.g., Ausubel, infra; Sambrook, infra). [0051]
  • Once a PNS sodium channel structure or characteristics have been determined, PNS SCPs can be recombinantly or synthetically produced, or optionally purified, to provide commercially useful amounts of PNS SCPs for use in diagnostic or research applications, according to known method steps (see, e.g., Ausubel, infra, and Sambrook, infra, which references are herein entirely incorporated by reference). [0052]
  • A variety of methodologies known in the art can be utilized to obtain an isolated PNS SCP of the invention. In one embodiment, the peptide is purified from tissues or cells which naturally produce the peptide. Alternatively the above-described isolated nucleic acid fragments could be used to expressed the PNS SCP protein in any organism. The samples of the invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts used as the sample. [0053]
  • The cells and/or tissue can include, e.g., normal or pathologic animal cells or tissues, such as the peripheral nervous system, and extracts or cell cultures thereof, provided in vivo, in situ or in vitro, as cultured, passaged, non-passaged, transformed, recombinant, or isolated cells and/or tissues. [0054]
  • Any higher eukaryotic organism can be used as a source of at least one PNS SCI or PNS SCP of the invention, as long as the source organism naturally contains such a peptide. As used herein, “source organism” refers to the original organism from which the amino acid sequence of die peptide is derived, regardless of the organism the peptide is expressed in and/or ultimately isolated from. Preferred organisms as sources of at least one PNS SCI or encoding nucleic acid can be any vertebrate animal, such as mammals, birds, bony fish,,electric eels, frogs and toads. Among mammals, the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs). The most preferred source organisms are humans. [0055]
  • One skilled in the art can readily follow known methods for isolating proteins in order to obtain the peptide free of natural contaminants. These include, but are not limited to: immunochromotography, size-exclusion chromatography, HPLC, ion-exchange chromatography, and immunoaffinity chromatography. See, e.g., Ausubel, infra; Sambrook, infra; Colligan, infra. [0056]
  • Isolated Nucleic Acid Molecules Coding for PNS SCP Peptides In one embodiment, the present invention relates to an isolated nucleic acid molecule coding for a peptide having an amino acid sequence corresponding to novel PNS SCPs. In one preferred embodiment, the isolated nucleic acid molecule comprises a PNS SCP nucleotide sequence with greater than 70% overall identity or homology to. at least a 60 nucleotide sequence present in SEQ ID NO:1 (preferably greater than 80%; more preferably greater than 90%, such as 70-99% any range or value therein). In another preferred embodiment, the isolated nucleic acid molecule comprises a PNS SCP nucleotide sequence corresponding to FIGS. 1, 7 or [0057] 9, or encoding at least one domain of FIGS. 1, 7, 8, 10 and 11.
  • Also included within the scope of this invention are the functional equivalents of the herein-described isolated nucleic acid molecules and derivatives thereof. For example, as presented above for PNS SCP amino acid sequences, the nucleic acid sequences depicted in SEQ ID NO:1 can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence of a PNS SCP can be used in the practice of the invention. These include but are not limited to amino acid sequences encoding all or portions of PNS SCP amino acid sequence of FIGS. 1, 8, [0058] 10 and 11, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the PNS SCP gene and fragments thereof permitted by the genetic code are, therefore, included in this invention. See, e.g., Ausubel, infra, Sambrook, infra. [0059]
  • In addition, the nucleic acid sequence can comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5′-end and/or the 3′-end of a nucleic acid sequence corresponding to FIGS. 1, 7 or [0060] 9, or encoding at least a portion of FIGS. 1, 8, 10 or 11, or a variant thereof. Any nucleotide or polynucleotide can be used in this regard, provided that its addition, deletion or substitution does remove the sodium channel activity which is encoded by the nucleotide sequence. Moreover, the nucleic acid molecule of the invention can, as necessary, have restriction endonuclease recognition sites which do not remove the activity of the encoded PNS SCP.
  • Further, it is possible to delete codons or to substitute one or more codons by codons other than degenerate codons to produce a structurally modified peptide, but one which has substantially the same utility or activity of the peptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two peptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code. See, e.g., Ausubel, infra; Sambrook, infra. [0061]
  • Isolation of Nucleic Acid In another aspect of the present invention, isolated nucleic acid molecules coding for peptides having amino acid sequences corresponding to PNS SCP are provided. In particular, the nucleic acid molecule can be isolated from a biological sample containing mammalian nucleic acid, as corresponding to a probe specific for a PNS SC obtained from a higher eukaryotic organism. [0062]
  • The nucleic acid molecule can be isolated from a biological sample containing nucleic acid using known techniques, such as but not limited to, primer amplification or cDNA cloning. [0063]
  • The nucleic acid molecule can be isolated from a biological sample containing genomic DNA or from a genomic library. Suitable biological samples include, but are not limited to, normal or pathologic animal cells or tissues, such as cerebrospinal fluid (CNS), peripheral nervous system (neurons, ganglion) and portions, cells of heart, smooth, skeletal or cardiac muscle, autonomic nervous system, and extracts or cell cultures thereof, provided in vivo, in situ or in vitro, as cultured, passaged, non-passaged, transformed, recombinant, or isolated cells and/or tissues. The method of obtaining the biological sample will vary depending upon the nature of the sample. [0064]
  • One skilled in the art will realize that a mammalian genome can be subject to slight allelic variations between individuals. Therefore, the isolated nucleic acid molecule is also intended to include allelic variations, so long as the sequence encodes a PNS SCP. When a PNS SCP allele does not encode the identical amino acid sequence to that found in FIGS. 1, 8, [0065] 10 or 11, or at least domain thereof, it can be, isolated and identified as PNS SCP using the same techniques used herein, and especially nucleic acid amplification techniques to amplify the appropriate gene with primers based on the sequences disclosed herein. Such variations are presented, e.g., in FIG. 11 and in Tables 1 and 2.
  • The cloning of large cDNAs is the same (e.g., PN1 as a PNS SCP of the invention includes overlapping clones of about 13 kDa) but takes more routine experimentation, than smaller cDNAs. One useful method relies on cDNA bacteriophage library screening. (see, e.g., Sambrook, infra, or Ausubel, infra). Probes for the screening are labeled, e.g., with random hexamers and Klenow enzyme (Pharmacia kit). If 5′ cDNAs are not obtained with these approaches, a subcDNA library can be prepared in which a specific PN1 primers are used to prime the reverse transcript reaction in place of oligo dT or random primers. The cDNA sublibrary is then cloned into standard vectors such as lambda zap and screened using conventional techniques. This strategy was used previously (Noda et al. [0066] Nature 320:188-192 (1986); Noda et al., Nature 322:826-828 (1986)) to clone the brain type I and II sodium channel cDNAs. The construction of a full-length cDNA is performed by subcloning overlapping fragments into an expression vector (either prokaryotic or eukaryotic). This task is more difficult with large cDNAs because of the paucity of unique restriction sites, but routine restriction, cloning or PCR is used to join the fragments.
  • Synthesis of Nucleic Acid Isolated nucleic acid molecules of the present invention are also meant to include those chemically synthesized For example, a nucleic acid molecule with the nucleotide sequence which codes for the expression product of a PNS SCP gene can be designed and, if necessary, divided into appropriate smaller fragments. [0067]
  • Then an oligomer which corresponds to the nucleic acid molecule, or to each of the divided fragments, can be synthesized (e.g., of 10-6015 nucleotides or any range or value therein, such as 10-100 nucleotides). Such synthetic oligonucleotides can be prepared, for example, by known techniques (See, e.g., Ausubel, infra, or Sambrook, infra) or by using an automated DNA synthesizer. [0068]
  • A labeled oligonucleotide probe be derived synthetically or by cloning. If necessary, the 5′-ends of the oligomers can be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling can be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP can contain high specific activity radioisotopes. Then, the DNA oligomer can be subjected to annealing and ligation with T4 ligase or the like. [0069]
  • A Nucleic Acid Probe for the Specific Detection of PNS SCP In another embodiment, the present invention relates to a nucleic acid probe of 15-6000 nucleotides for the specific detection of the presence of PNS SCP in a sample comprising the above-described nucleic acid molecules or at least a fragment thereof which binds under stringent conditions to a nucleic acid encoding at least one PNS SCP. [0070]
  • The nucleic acid probe can be used to screen an appropriate chromosomal or cDNA library by known hybridization method steps to obtain a PNS SCP encoding nucleic acid molecule of the invention. A chromosomal DNA or cDNA library can be prepared from appropriate cells according to recognized methods in the art (See, e.g., Ausubel, infra; Sambrook, infra). [0071]
  • In the alternative, organic chemical synthesis is carried out in order to obtain nucleic acid probes having nucleotide sequences which correspond to suitable portions of the amino acid sequence of the PNS SCP. Thus, the synthesized nucleic acid probes can be used as primers in nucleic acid amplification method steps [0072]
  • The invention can thus provide methods for amplification of DNA and/or RNA using heat stable, cross-linked nucleotide primers, which cross linked primers of the invention to provide nucleic acid encoding PNS SCPs according to the invention. [0073]
  • Methods of amplification of RNA or DNA are well known in the art and can be used according to the invention without undue experimentation, based on the teaching and guidance presented herein. According to the invention, the use of nucleic acids encoding portions of PNS SCPs according to the invention, as amplification primers, allows for advantages over known amplification primers, due to the increase in sensitivity, selectivity and/or rate of amplification. [0074]
  • Known methods of DNA or RNA amplification include, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson et al.; U.S. Pat. No. 5,091,310 to Inn U.S. Pat. No. 5,066,584 to Gyllensten et al.; U.S. Pat. No. 4,889,818 to Gelfand et al; U.S. Pat. No. 4,994,370 to Silver et al.; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No. 4,656,134 to Ringold; U.S. Pat. No. 5,340,728 to Grosz et al.; U.S. Pat. No. 5,322,770 to Gelfand et al.; U.S. Pat. No. 5,338,671 to Scalice et al.; PCT WO 92/06200 to Cetus Corp.; PCT WO 94/14978 to Strack et al., which patent disclosures are entirely incorporated herein by reference) and RNA mediated amplification which uses antisense RNA to the target sequence as a template for double stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al., with the tradeneame NASBA), the entire contents of which patents and references are herein entirely incorporated by reference. Reviews of the PCR are provided by Mullis ([0075] Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis et al. (Meth Enzyrol. 155:335-350 (1987)). One skilled in the art can readily design such probes based on the sequence disclosed herein using methods such as computer alignment and sequence analysis known in the art. See, e.g., Ausubel, infra, Sambrook, infra.
  • The hybridization probes of the invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and any other known and suitable labels. After hybridization, the probes can be visualized using known methods. The nucleic acid probes of the invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art (See, e.g., Ausubel, infra; Sambrook, infra). In one embodiment of the above described method, a nucleic acid probe is immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and SEPHAROSE, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art (See, e.g., Ausubel, infra; Sambrook, infra). [0076]
  • The test samples suitable for nucleic acid probing methods of the invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized. [0077]
  • Methods for Detecting The Presence of PNS SCP Encoding Nucleic Acid in a Biological Sample. In another embodiment, the present invention relates to methods for detecting the presence of PNS SCP encoding nucleic acid in a sample. Such methods can comprise (a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and (b) detecting the presence of a labeled probe bound to the nucleic acid probe. One skilled in the art can select a suitable, labeled nucleic acid probe according to techniques known in the art as described above. Samples to be tested include, but are not limited to, RNA samples of mammalian tissue. [0078]
  • PNS SCP has been found to be expressed in peripheral nerve and dorsal root ganglion cells. Accordingly, PNS SCP probes can be used detect the presence of RNA from PN cells in such a biological sample. Further, altered expression levels of PNS SCP RNA in an individual, as compared to normal levels, can indicate the presence of disease. The PNS SCP probes cart further be used to assay cellular activity in general and specifically in peripheral nervous system tissue. [0079]
  • A Kit for Detecting the Presence of PNS SCP in a Sample. In another embodiment, the present invention relates to a kit for detecting the presence of PNS SCP in a sample comprising at least one container having disposed therein the above-described nucleic acid probe. In a preferred embodiment, the kit further comprises other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, allaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin) (See, e.g., Ausubel, infra, Sambrook, infra). [0080]
  • A compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, TRIS-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like. [0081]
  • One skilled in the art will readily recognize that the nucleic acid probes described in the invention can readily be incorporated into one of the established kit formats which are well known in the art. [0082]
  • DNA Constructs Comprising a PNS SCP Nucleic Acid Molecule and Hosts Containing These Constructs. A nucleic acid sequence encoding an PNS SCP of the invention can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Ausubel et al., infra, and are well known in the art. [0083]
  • A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as PNS SCPs or Ab fragments in recoverable amounts. The precise nature of the regulatory regions needed for gene expression can vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook, infra and Ausubel infra. [0084]
  • The invention accordingly encompasses the expression of an PNS SCP, in either prokaryotic or eukaryotic cells, although eukaryptic expression is preferred. [0085]
  • Preferred hosts are bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. It is preferred that the mammalian cell or tissue is of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell can be used. [0086]
  • Eukaryotic hosts can include yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture. Preferred eukaryotic hosts can also include, but are not limited to insect cells, mammalian cells either in vivo, or in tissue culture. Preferred mammalian cells include [0087] Xenopus oocytes, HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives.
  • Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Mammalian cells which can be useful as hosts include cells of fibroblast origin such as, but not limited to, NIH 3T3, VERO or CHO, or cells of lymphoid origin, such as, but not limited to, the hybridoma SP2/O-Ag14 or the murine myeloma P3-X63Ag8, hamster cell lines (e.g., CHO-K1 and progenitors, e.g., CHO-DUXB11) and their derivatives. One preferred type of mammalian cells are cells which are intended to replace the function of the genetically deficient cells in vivo. Neuronally derived cells are preferred for gene therapy of disorders of the nervous system. For a mammalian cell host, many possible vector systems are available for the expression of at least one PNS SCP. A wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals can be derived from viral sources, such as, but not limited to, adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, promoters from mammalian expression products, such as, but not limited to, actin, collagen, myosin, protein production. See, Ausubel, infra,; Sanbrook, infra. [0088]
  • When live insects are to be used, silk moth caterpillars and baculoviral vectors are presently preferred hosts for large scale PNS SCP production according to the invention. Production of PNS SCPs in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express at least one PNS SCP by methods known to those skilled in the related arts. See Ausubel et al, eds. [0089] Current Protocols in Molecular Biology, Wiley Interscience, §§16.8-16.11 (1987, 1992, 1993, 1994).
  • In a preferred embodiment, the introduced nucleotide sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be employed for this purpose. See, e.g., Ausubel et al., infra, §§ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. [0090]
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure “native” glycosylation of the heterologous PNS SCP protein. Furthermore, different vector/host expression systems can effect processing reactions such as proteolytic cleavages to different extents. [0091]
  • As discussed above, expression of PNS SCP in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. See, e.g., Ausubel, infra, Sambrook, infra. [0092]
  • Once the vector or nucleic acid molecule containing the construct(s) has been prepared for expression, the DNA construct(s) can be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene molecule(s) results in the production of at least one PNS SCP. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like). [0093]
  • Isolation of PNS SCP. The PNS SCP proteins or fragments of this invention can be obtained by expression from recombinant DNA as described above. Alternatively, a PNS SCP can be purified from biological material. If so desired, the expressed at least one PNS SCP can be isolated and purified in accordance with conventional method steps, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. For example, cells expressing at least one PNS SCP in suitable levels can be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation. Alternatively, PNS SCPs can be isolated by the use of specific antibodies, such as, but not limited to, an PNS SCP or SC antibody. Such antibodies can be obtained by known method steps (see, e.g. Colligan, infra; Ausubel, infra. [0094]
  • For purposes of the invention, one method of purification which is illustrative, without being limiting, consists of the following steps. A fist step in the purification of a PNS SCP includes extraction of the PNSSCP fraction from a biological sample, such as peripheral nerve tissue or dorsal root ganglia (DRG), in buffers, with or without solubilizing agents such as urea, formic acid, detergent, or thiocyanate. A second step includes subjecting the solubilized material to ion-exchange chromatography on Mono-Q-or Mono-S columns (Pharmacia LKB Biotechnology, Inc; Piscataway, N.J.). Similarly, the solubilized material can be separated by any other process wherein molecules can be separated according to charge density, charge distribution and molecular size, for example. Elution of the PNS SCP from the ion-exchange resin are monitored by an immunoassay, such as M-IRMA, on each fraction. Immunoreactive peaks would are then dialyzed, lyophilized, and subjected to molecular sieve, or gel chromatography. In a third step, molecular sieve or gel chromatography is a type of partition chromatography in which separation is based on molecular size. Dextran, polyacrylamide, and agarose gels are commonly used for this type of separation. One useful gel for the invention is SEPHAROSE 12 (Pharmacia LKB Biotechnology, Inc.). However, other methods, known to those of skill in the art can be used to effectively separate molecules based on size. A fourth step in a purification protocol for a PNS SCP can include analyzing the immunoreactive peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a further gel chromatographic purification step, and staining, such as, for example, silver staining. A fifth step in a purification method can include subjecting the PNS SCP obtained after SDS-PAGE to affinity chromatography, or any other procedure based upon affinity between a substance to be isolated and a molecule to which it can specifically bind. For further purification of a PNS SCP, affinity chromatography on SEPHAROSE conjugated to anti-PNS SCP mAbs (specific mABs generated against substantially pure PNS SCP) can be used. Alternative methods, such as reverse-phase HPLC, or any other method characterized by rapid separation with good peak resolution are useful. [0095]
  • It will be appreciated that other purification steps can be substituted for the preferred method described above. Those of skill in the art will be able to devise alternate purification schemes without undue experimentation. [0096]
  • An Antibody Having Binding Affinity to a PNS SCP Peptide and a Hybridoma Containing the Antibody. In another embodiment, the invention relates to an antibody having binding affinity specifically to a PNS SCP peptide as described above or fragment thereof. Those which bind selectively to PNS SCP would be chosen for use in methods which could include, but should not be limited to, the analysis of altered PNS SCP expression in tissue containing PNS SCP. [0097]
  • The PNS SCP proteins of the invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction. [0098]
  • The PNS SCP peptide of the invention can be used to produce antibodies or hybridomas. One skilled in the art will recognize that if an antibody is desired, such a peptide would be generated as described herein and used as an immunogen. [0099]
  • The antibodies of the invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. [0100]
  • The term “antibody” is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. MAbs can be obtained by methods known to those skilled in the art. See, e.g., Kohler and Milstein, [0101] Nature 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988); Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which references are incorporated entirely herein by reference. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a mAb of the invention can be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mabs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, [0102] Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al, European Patent Application 125023; Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171 496; Morrison et al., European Patent Application 173 494; Neuberger et al, PCT Application WO 86/01533; Kudo et al., European Patent Application 184 187; Morrison et al., European Patent Application 173 494, Sahagan et al., J. Immunol. 137:1066-1074.(1986); Robinson et al., International Patent Publication No. PCT/US86/02269; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al, Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al, Science 240:1041-1043 (1988); and Harlow, infra. These references are entirely incorporated herein by reference.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse str) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein entirely incorporated by reference. [0103]
  • The anti-Id antibody can also be used as an “immunogen” to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id can be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. [0104]
  • Accordingly, mAbs generated against a PNS SCP of the invention can be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mabs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a PNS SCP specific epitope. The anti-Id mabs thus have their own idiotypic epitopes, or “idiotopes” structurally similar to the epitope being evaluated. [0105]
  • The term “antibody” is also meant to-include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab′)[0106] 2, which are capable of binding antigen. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies useful in the invention can be used for the detection and/or quantitation of a PNS SCP according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term “epitope” is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or “antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • An “antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additiolially capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen can have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which can be evoked by other antigens. [0107]
  • Immunoassays. Antibodies of the invention, directed against a PNS SCP, can be used to detect or diagnose a PNS SC or a PNS SC-related pathologies. Screening methods are provided by the invention can include, e.g., immunoassays employing radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA) methodologies, based on the production of specific antibodies (monoclonal or polyclonal) to a PNS SCP. For these assays, biological samples are obtained by, nerve biopsy, or other peripheral nervous system tissue sampling. For example, in one form of RIA, the substance under test is mixed with diluted antiserum in the presence of radiolabeled antigen. In this method, the concentration of the test substance will be inversely proportional to the amount of labeled antigen bound to the, specific antibody and directly related to the amount of free labeled antigen. Other suitable screening methods will be readily apparent to those of skill in the art. [0108]
  • Furthermore, one skilled in the art can readily adapt currently available procedures, as well as the techniques, methods and kits disclosed above with regard to antibodies, to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., “Application of Synthetic Peptides: Antisense Peptides”, In: [0109] Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992), and Kaspczak et al., Biochemistry 28:9230-8 (1989).
  • One embodiment for carrying out the diagnostic assay of the invention on a biological sample containing a PNS SCP, comprises: [0110]
  • (a) contacting a detectably labeled PNS SCP-specific antibody with a solid support to effect immobilization of said PNS SCP-specific antibody or a fragment thereof; [0111]
  • (b) contacting a sample suspected of containing a PNS SCP with said solid support; [0112]
  • (c) incubating said detectably labeled PNS SCP-specific antibody with said support for a time sufficient to allow the immobilized PNS SCP-specific antibody to bind to the PNS SCP; [0113]
  • (d) separating the solid phase support from the incubation mixture obtained in step (c); and [0114]
  • (e) detecting the bound label and thereby detecting and quantifying PNS SCP. [0115]
  • The specific concentrations of detectably labeled antibody and PNS SCP, the temperature and time of incubation, as well as other assay conditions can be varied, depending on various factors including the concentration of a PNS SCP in the sample, the nature of the sample, and the like. The binding activity of a given lot of anti-PNS SCP antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like can be added to the assays as is customary or necessary for the particular situation. [0116]
  • Detection can be accomplished using any of a variety of assays. For example, by radioactively labeling the PNS SCP-specific antibodies or antibody fragments, it is possible to detect PNS SCP through the use of radioimmune assays. A good description of a radioimmune assay can be found in Colligan, infra, and Ausubel, infra, entirely incorporated by reference herein. Preferably, the detection of cells which express a PNS SCP can be accomplished by in vivo imaging techniques, in which the labeled antibodies (or fragments thereof) are provided to a subject, and the presence of the PNS SCP is detected without the prior removal of any tissue sample. Such in vivo detection procedures have the advantage of being less invasive than other detection methods, and are, moreover, capable of detecting the presence of PNS SCP in tissue which cannot be easily removed from the patient, such as brain tissue. [0117]
  • There are many different in vivo labels and methods of labeling known to those of ordinary skill in the art Examples of the types of labels which can be used in the invention include radioactive isotopes and paramagnetic isotopes. Those of ordinary skill in the art will know of other suitable labels for binding to the antibodies used in the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the antibodies can be done using standard techniques common to those of ordinary skill in the art. [0118]
  • For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given radionuclide. The radionuclide chosen must have a type of decay which is detectable for a given type of instrument In general, any conventional method for visualling diagnostic imaging can be utilized in accordance with this invention. For example, positron emission tomography (PET), gamma, beta, and magnetic resonance imaging (MRI) detectors can be used to visualize diagnostic imagining. [0119]
  • The antibodies useful in the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis. Elements which are particularly useful, as in Magnetic Resonance Imaging (MRI), include [0120] 157Gd, 55Mn, 162Dy, and 56Fe.
  • The antibodies (or fragments thereof) useful in the invention are also particularly suited for use in in vitro immunoassays to detect the presence of a PNS SCP in body tissue, fluids (such as CSF), or cellular extracts. In such immunoassays, the antibodies (or antibody fragments) can be utilized in liquid phase or, preferably, bound to a solid-phase carrier, as described above. [0121]
  • In situ detection can be accomplished by removing a histological specimen from a patient, and providing the combination of labeled antibodies of the invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a PNS SCP, but also the distribution of a PNS SCP on the examined tissue. Using the invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. [0122]
  • As used herein, an effective amount of a diagnostic reagent (such as an antibody or antibody fragment) is one capable of achieving the desired diagnostic discrimination and will vary depending on such factors as age, condition, sex, the extent of disease of the subject, counter-indications, if any, and other variables to be adjusted by the physician. The amount of such materials which are typically used in a diagnostic test are generally between 0.1 to 5 mg, and preferably between 0.1 to 0.5 mg. [0123]
  • The assay of the invention is also ideally suited for the preparation of a kit. Such a kit can comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay. [0124]
  • For example, there can be a container means containing a first- antibody immobilized on a solid phase support, and a further container means containing a second detectably labeled antibody in solution. Further container means can contain standard solutions comprising serial dilutions of the PNS SCP to be detected. The standard solutions of a PNS SCP can be used to prepare a standard curve with the concentration of PNS SCP plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample containing a PNS SCP can be interpolated from such a plot to give the concentration of the PNS SCP. [0125]
  • Diagnostic Screening and Treatment It is to be understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal that expresses at least one PNS SC. The diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level of PNS SCP based on family history, or a patient in which it is desired to diagnose a PNS SCP-related disease. [0126]
  • According to the invention, presymptomatic screening of an individual in need of such screening is now possible using DNA encoding the PNS SCP protein of the invention. The screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of the presence of a missing or aberrant PNS SC gene in individuals, and thus an opinion concerning the likelihood that such individual would develop or has developed a PNS SC-associated disease. This is especially valuable for the identification of carriers of altered or missing PNS SC genes, for example, from individuals with a family history of a PNS SC-related pathology. Early diagnosis is also desired to maximize appropriate timely intervention. [0127]
  • In one preferred embodiment of the method of screening, a tissue sample would be taken from such individual, and screened for (1) the presence of the “normal” PNS SCP gene; (2) the presence of PNS SCP mRNA and/or (3) the presence of PNS SCP protein. The normal human gene can be characterized based upon, for example, detection of restriction digestion patterns in “normal” versus the patient's DNA, including RFLP analysis, using DNA probes prepared against the PNS SCP sequence (or a functional fragment thereof) taught in the invention. Similarly, PNS SCP mRNA can be characterized and compared to normal PNS SCP mRNA (a) levels and/or (b) size as found in a human population not at risk of developing PNS SCP-associated disease using similar probes. Lastly, PNS SCP protein can be (a) detected and/or (b) quantitated using a biological assay for PNS SCP activity or using an immunological assay and PNS SCP antibodies. When assaying PNS SCP protein, the immunological assay is preferred for its speed. An (1) aberrant PNS SCP DNA size pattern, and/or (2) aberrant PNS SCP mRNA sizes or levels and/or (3) aberrant PNS SCP protein levels would indicate that the patient is at risk for developing a PNS SCP-associated disease. [0128]
  • The screening and diagnostic methods of the invention do not require that the entire PNS SCP DNA coding sequence be used for the probe. Rather, it is only necessary to use a fragment or length of nucleic acid that is sufficient to detect the presence of the PNS SCP gene in a DNA preparation from a normal or affected individual, the absence of such gene, or an altered physical property of such gene (such as a change in electrophoretic migration pattern). [0129]
  • Prenatal diagnosis can be performed when desired, using any known method to obtain fetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy. Prenatal chromosome analysis can be used to determine if the portion of the chromosome possessing the normal PNS SCP gene is present in a heterozygous state. [0130]
  • Overview of PNS SCP Purification and Crystallization Methods. In general, a PNS SCP as a membrane protein, is purified in soluble form using detergents (e.g., octyglucosides) or other suitable amphiphillic molecules. The resulting PNS SCP is in sufficient purity and concentration for crystallization. The purified PNS SCP is then isolated and assayed for biological activity and for lack of aggregation (which interferes with crystallization). The purified and cleaved PNS SCP preferably runs as a single band under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges). The purified PNS SCP is preferably crystallized under varying conditions of at least one of the following: pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands and concentration of purified and cleaved PNS SCP by known methods. See, e.g., Michel, [0131] Trends in Biochem Sci. 8:56-59 (1983); Deisenhofer et al. J. Mol. Biol 180:385-398 (1984); Weiss et al. FEBS Lett. 267:268-272 (1990). Blundell, et al. Protein Crystallography Academic Press, London (1976); Oxender et al. eds., Protein Engineering Liss, N.Y. (1986); McPherson; The Preparation and Analysis of Protein Crystals Wiley, N.Y. (1982); or the methods provided in a commercial kit, such as CRYSTAL SCREEN (Hampton Research, Riverside, Calif.). The crystallized protein is also tested for at least one SC activity and differently sized and shaped crystals are further tested for suitability in X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals. See, e.g., Blundell., infra, Oxender, infra, McPherson, infra; Wyckoff et al, eds., Diffraction Methods for Biological Macromolecules, Vols. 114-115: Methods in Enzymology, Orlando, Fla. Academic Press (1985).
  • Protein Crystallization Methods. The hanging drop method is preferably used to crystallize a purified soluble, PNS SCP protein. See, e.g., Taylor et al., [0132] J. Mol. Biol. 226:1287-1290 (1992); Takimoto et al. (1992), infra, CRYSTAL SCREEN, Hampton Research. A mixture of the protein and precipitant can include the following:  pH (e.g., 4-10);  buffer type (e.g., tromethamine (TRIZMA), sodium azide, phosphate, sodium, rcacodylate acetates, imidazole, Tris HCl, sodium hepes);  buffer concentration (e.g., 0.1-100 mM);  salt type (e.g., sodium azide, calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate; calcium acetate);  polymer type and concentration: (e.g., polyethylene glycol (PEG) 1-50%, type 6000-10,000);  other precipitating ligands (salts: potassium, sodium, tartrate, ammonium sulfate, sodium acetate, lithium sulfate, sodium formate, sodium citrate, magnesium formate, sodium phosphate, potassium phosphage; organics: 2-propanol; non-volatile: 2-methyl-2,4-pentanediol); and  concentration of purified PNS SCP (e.g., 0.1-100 mg/ml, with added amphiphillic molecules (detergents such as octylgluosides)). See, e.g., CRYSTAL SCREEN, Hampton Research.
  • The above mixtures are used and screened by varying at least one of pH, buffer type; buffer concentration, precipitating salt type or concentration, PEG type, PEG concentration, and cleaved protein concentration. Crystals ranging in size from 0.1-1.5 mm are formed in 1-14 days. These crystals diffract X-rays to at least 10 Å resolution, such as 1.5-10.0 Å, or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 Å or less being preferred for the highest resolution. In addition to diffraction patterns having this highest resolution, lower resolution, such as 25-3.5 Å can further be used. [0133]
  • Protein Crystals. Crystals appear after 1-14 days and continue to grow on subsequent days. Some of the crystals are removed, washed, and assayed for biological activity, which activity is preferred for using in further characterizations. Other washed crystals are preferably run n a stained gel and those that-migrate in the same position as the purified cleaved PNS SCP are preferably used. From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, bipyramidal, rhomboid, and cubic. Initial X-ray analyses are expected to indicate that such crystals diffract at moderately high to high resolution. When fewer crystals are produced in a drop, they can be much larger size, e.g., 0.2-1.5 mm. [0134]
  • PNS SCP X-ray Crystallography Methods The crystals so produced for a PNS SCP are X-ray analyzed using a suitable X-ray source. A suitable number of diffraction patterns are obtained. Crystals are preferably stable for at least 10 hrs in the X-ray beam. Frozen crystals (e.g., −220 to −50° C.) are optionally used for longer X-ray exposures (e.g., 4-72 hrs), the crystals being relatively more stable to the X-rays in the frozen state. To collect the maximum number of useful reflections, multiple frames are optionally collected as the crystal is rotated in the X-ray beam, e.g.; for 12-96 hrs. Larger crystals (>0.2 mm) are preferred, to increase the resolution of the X-ray diffraction. Crystals are preferably analyzed using a synchrotron high energy X-ray source. Using frozen crystals, X-ray diffraction data is collected on crystals that diffract to a resolution of 10-1.5 Å, with lower resolutions also useful, such as 25-10 Å, sufficient to solve the three-dimensional structure of a PNS SCP in considerable detail, as presented herein. [0135]
  • Computer Related Embodiments. An amino acid sequence of a PNS SCP and/or x-ray diffraction data, useful for computer molecular modeling of a PNS SCP or a portion thereof, can be “provided” in a variety of mediums to facilitate use thereof. As used herein, provided refers to a manufacture, which contains a PNS SCP amino acid sequence and/or x-ray diffraction data of the present invention, e.g., the amino sequence provided in FIGS. 1, 8, [0136] 10 or 11, a representative fragment thereof, or an amino acid sequence having at least 80-100% overall identity to a 5-2005 amino acid fragment of an amino acid sequence of FIGS. 11A-D or a variant thereof. Such a method provides the amino acid sequence and/or x-ray diffraction data in a form which allows a skilled artisan to analyze and molecular model the three dimension structure of a PNS SCP or subdomain thereof.
  • In one application of this embodiment, PNS SCP, or at least one subdomain thereof, amino acid sequence and/or x-ray diffraction data of the present invention is recorded on computer readable medium. As used herein, “computer readable medium” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a n amino acid sequence and/or x-ray diffraction data of the present invention. [0137]
  • As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising an amino acid sequence and/or x-ray diffraction data information of the present invention. [0138]
  • A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon an amino acid sequence and/or x-ray diffraction data of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the sequence and x-ray data information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the information of the present invention. [0139]
  • By providing the PNS SCP sequence and/or x-ray diffraction data on computer readable medium, a skilled artisan can routinely access the sequence and x-ray diffraction data to model a PNS SCP, a subdomain thereof, or a ligand thereof. Computer algorythms are publicly and commercially available which allow a skilled artisan to access this data provided in a computer readable medium and analyze it for molecular modeling and/or RDD. [0140]
  • The present invention further provides systems; particularly computer-based systems; which contain the sequence and/or diffraction data described herein. Such systems are designed to do molecular modeling and RDD for a PNS SCP or at least one subdomain thereof. [0141]
  • As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the sequence and/or x-ray diffraction data of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate which of the currently available computer-based system are suitable for use in the present invention. [0142]
  • As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a PNS SCP or fragment sequence and/or x-ray diffraction data of the present invention and the necessary hardware means and software means for supporting and implementing an analysis means. As used herein, “data storage means” refers to memory which can store sequence or x-ray diffraction data of the present invention, or a memory access means which can access manufactures having recorded thereon the sequence or x-ray data of the present invention. [0143]
  • As used herein, “search means” or “analysis means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence or x-ray data stored within the data storage means. Search means are used to identify fragments or regions of a PNS SCP which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting computer analyses that can be adapted for use in the present computer-based systems. [0144]
  • As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration or electron density map which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzymic active sites, structural subdomains, epitopes, functional domains and signal sequences. A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. [0145]
  • A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify structural motifs or electron density maps. A skilled artisan can readily recognize that any one of the publicly available computer modeling programs can be used as the search means for the computer-based systems of the present invention. [0146]
  • One application of this embodiment is provided in FIG. 12. FIG. 12 provides a block diagram of a [0147] computer system 102 that can be used to implement the present invention. The computer system 102 includes a processor 106 connected to a bus 104. Also connected to the bus 104 are a main memory 108 (preferably implemented as random access memory, RAM) and a variety of secondary storage memory 110, such as a hard drive 112 and a removable storage medium 114. The removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage medium 114. The computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114 once inserted in the removable medium storage device 114. A monitor 120 can be used as connected to the bus 104 to visualize the structure determination data.
  • Amino acid, encoding nucleotide or other sequence and/or x-ray diffraction data of the present invention may be stored in a well known manner in the [0148] main memory 108, any of the secondary storage devices 110, and/or a removable storage device 116. Software for accessing and processing the amino acid sequence and/or x-ray diffraction data (such as search tools, comparing tools, etc.) reside in main memory 108 during execution.
  • Three Dimensional Structure Determination One or more computer modeling steps and/or computer algorythms are used to provide a molecular 3-D model of a cleaved PNS SCP, using amino acid sequence data from FIGS. 1, 8, [0149] 10 or 11 (or variants thereof) and/or x-ray diffraction data. If only the amino acid sequence is used, for three-dimensional structure determination then a suitable modeling program can be used, e.g., LINUS (Rose et. al. Proteins: Structure, Function and Genetics (June, 1995) and references cited herein. It is preferred that the PNS SCP model has no or A1a-substituted (for surface) residues in disallowed regions of the Ramachandran plot, and gives a positive 3D-1D profile (Luthy et al., Nature 356:83-85 (1992)), suggesting that all the residues are in acceptable environments (Kraulis (1991), infra). Alternatively, the dissallowed regions can be corrected by the use of suitable algorythms, such as the RAVE program described herein. Phase determination is optionally used for solving the three-dimensional structure of a cleaved PNS SCP. This structure can then be used for RDD of modulators of PNS SCP neuraminidase, endothelin cathepsin A or other biological activity, e.g., which is relevant to a PNS SCP related pathology.
  • Density Modification and Map Interpretation. Electron density maps can be calculated using such programs as those from the CCP4 computing package (SERC (UK) Collaborative Computing Project 4, Daresbury Laboratory, UK, 1979). Cycles of two-fold averaging can further be used, such as with the program RAVE (Kleywegt & Jones, Bailey et al., eds., [0150] First Map to Final Model, SERC Daresbury Laboratory, UK, pp.59-66 (1994)) and gradual model expansion. For map visualization and model building a program usch as “O” (Jones (1991), infra) can be used.
  • Refinement and Model Validation. Rigid body and positional refinement can be carried out using a program such as X-PLOR (Brünger (1992), infra), e.g., with the stereochemical parameters of Engh and Huber ([0151] Acta Cryst. A47:392-400 (1991)). If the model at this stage in the averaged maps still misses residues (e.g., at least 5-10 per subunit), the some or all of the missing residues can be incorporated in the model during additional cycles of positional refinement and model building. The refinement procedure can start using data from lower resolution (e.g., 25-10 Å to 10-3.0 Å and then gradually extended to include data from 12-6 Å to 3.0-1.5 Å). β-values for individual atoms can be refined once data between 2.9 and 1.5 Å has been added. Subsequently waters can be gradually added. A program such as ARP (Lamzin and Wilson, Acta Cryst. D49:129-147 (1993)) can be used to add crystallographic waters and as a tool to check for bad areas in the model. Programs such as PROCHECK (Lackowski et al., J. Appl. Cryst. 26:283-291 (1993)), WHATIF (Vriend, J. Mol. Graph. 8:52-56 (1990)) and PROFILE 3D (Lüthy et al., Nature 356:83-85 (1992)), as well as the geometrical analysis generated by X-PLOR can be been used to check the structure for errors. For the final refinement cycle; 20-5% of the weakest data can be rejected using a IFobsI/σ cutoff and anisotropic scaling between Fobs and Fcalc applied after careful assessment of the quality and completeness of the data
  • Structure Analysis. A program such as DSSP can be used to assign the secondary structure elements (Kabsch and Sander (1983), infra). A program such as SUPPOS (from the BIOMOL crystallographic computing package) can be used to for some or all of the least-squares superpositions of various models and parts of models. Solvent accessible surfaces and electrostatic potentials can be calculated using such programs as GRASP (Nicholls et al. (1991), infra). [0152]
  • Structure Determination. The structure of a PNS SCP can thus be solved with the molecular replacement procedure such as by using X-PLOR (Brünger (1992), infra). A partial search model for the monomer can be constructed using a related protein, such as wheat serine carboxypeptidase structure (Liao et al. (1992), infra). The rotation and translation function can be used to yield two or more orientations and positions for two subunits to form a physiological dimer as determined based on their interactions. Cyclical two-fold density averaging can also be done using the RAVE program and model expansion can also be used to add missing residues for each monomer, resulting in a model with 95-99.9% of the total number residues. The model can be refined in a program such as X-PLOR (Brünger (1992), supra), to a suitable crystallographic R[0153] factor. The model data is then saved on computer readable medium for use in further analysis, such as rational drug design.
  • Rational Design of Drugs that Interact with the PNS SCP. The determination of the three dimensional structure of a cleaved PNS SCP, as described herein, provides a basis for the design of new and specific ligands for the diagnosis and/or treatment of at least one PNS SCP-related pathology. Several approaches can be taken for the use of the crystal structure of a PNS SCP in the rational design of ligands of this protein. A computer-assisted, manual examination of the active site structure is optionally done. The use of software such as GRID (Goodford, [0154] J. Med. Chem. 28:849-857 (1985)) a program that determines probable interaction sites between probes with various functional group characteristics and the enzyme surface—is used to analyze the active site to determine structures of inhibiting compounds. The program calculations, with suitable inhibiting groups on molecules (e.g. protonated primary amines) as the probe, are used to identify potential hotspots around accessible positions at suitable energy contour levels. Suitable ligands, as inhibiting or stimulating modulating compounds or compositions, are then tested for modulating activities of at least one PNS SCP.
  • A diagnostic or therapeutic PNS SCP modulating ligand of the present invention can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. Alternatively, any other known diagnostic or therapeutic agent can be used in a method of the invention. [0155]
  • After preliminary experiments are done to determine the K[0156] m of the substrate with each enzyme activity of a PNS SCP, the time-dependent nature of modulation of ligand Ki values are determined, (e.g., by the method of Henderson (Biochem. J. 127:321-333 (1972)). For example, the substrate (or blank where appropriate) and enzyme are pre-incubated in buffer. Reactions are initiated by the addition of substrate. Aliquots are removed over a suitable time course and each quenched by addition into the aliquots of suitable quenching solution (e.g. sodium hydroxide in aqueous ethanol). The concentration of product is determined, e.g. fluorometrically, using a spectrometer. Plots of fluorescence against time can be close to linear over the assay period, and are used to obtain values for the initial velocity in the presence (Vi) or absence (Vo) of ligand. Error is present in both axes in a Henderson plot, making it inappropriate for standard regression analysis (Leatherbarrow, Trends Biochem. Sci. 15:455-458 (1990)). Therefore, Ki values is obtained from the data by fitting to a modified version of the Henderson equation for competitive inhibition:
  • Qr 2+(E t −Q−I t)r−E t=0
  • where (using the notation of Henderson ([0157] Biochem. J. 127:321-333 (1972)): Q = K i ( A t + K a K a )   and   r = V o V i
    Figure US20040229792A1-20041118-M00001
  • This equation is solved for the positive root with the constraint that [0158]
  • Q=K i((A t +K a)/K a)
  • using PROCNLIN from SAS (SAS Institute Inc., Cary, N.C., USA) which performs nonlinear regression using least-square techniques. The iterative method used is optionally the multivariate secant method, similar to the Gauss-Newton method, except that the derivatives in the Taylor series are estimated from the histogram of iterations rather than supplied analytically. A suitable convergence criterion is optionally used, e.g., where there is a change in loss function of less than 10[0159] −8.
  • Once modulating ligands are found and isolated or synthesized, crystallographic studies of the compounds complexed to a PNS SCP are performed. As a non-limiting example, PNS SCP crystals are soaked for 2 days in 0.01-100 mM ligand and X-ray diffraction data are collected on an area detector and/or an image plate detector (e.g., a Mar image plate detector) using a rotating anode X-ray source. Data are collected to as high a resolution as possible, e.g., 1.5-3.5 Å, and merged with an R-factor on suitable intensities. An atomic model of the inhibitor is built into the difference Fourier map (F[0160] inhibitor complex−Fnative). The model can be refined to a solution in a cycle of simulated annealing (Brünger (1987), infra) involving 10-500 cycles of energy refinement, 100-10,000 1-FS steps of room temperature dynamics and/or 10-500 more cycles of energy refinement. Harmonic restraints are also used for the atom refinement, except for atoms within a 10-15 Å radius of the inhibitor. An R-factor is selected for the model for both the r.m.s. deviations from the ideal bond lengths, as well as for the angles, respectively. Direct measurements of enzyme inhibition provide further confirmation that the modeled ligands are modulators of at least one biological activity of a PNS SC.
  • Ligands of a PNS SCP, based on the crystal structure of this enzyme, are thus also provided by the present invention. Demonstration of clinically useful levels, e.g., in vivo activity is also important. In evaluating PNS SCP inhibitors for biological activity in animal models (e.g., rat, mouse, rabbit) using various oral and parenteral routes of administration are evaluated Using this approach, it is expected that modulation of a PNS SCP occurs in suitable animal models, using the ligands discovered by molecular modeling and x-ray crystallography. [0161]
  • Diagnostic and/or Therapeutic Agents. A diagnostic or therapeutic PNS SCP modulating agent or ligand of the present invention can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies, as described herein. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds r metals, chemiluminescent compounds and bioluminescent compounds. Alternatively, any other known diagnostic or therapeutic agent can be used in a method of the invention. [0162]
  • A therapeutic agent used in the invention can have a therapeutic effect on the target cell as a cell or neuron of the peripheral nervous system, the effect selected from, but not limited to: correcting a defective gene or protein, a drug action, a toxic effect, a growth stimulating effect, a growth inhibiting effect, a metabolic effect, a catabolic affect, an anabolic effect, a neurohumoral effect, a cell differentiation stimulatory effect, a cell differentiation inhibitory effect, a neuromodulatory effect, a pluripotent stem cell stimulating effect, and any other known therapeutic effects that modulates at least one SC in a cell of the peripheral nervous system can be provided by a therapeutic agent delivered to a target cell via pharmaceutical administration or via a delivery vector according to the invention. [0163]
  • A therapeutic nucleic acid as a therapeutic agent can have, but is not limited to, at least one of the following therapeutic effects on a target cell: inhibiting transcription of a DNA sequence; inhibiting translation of an RNA sequence; inhibiting reverse transcription of an RNA or DNA sequence; inhibiting a post-translational modification of a protein; inducing transcription of a DNA sequence; inducing translation of an RNA sequence; inducing reverse transcription of an RNA or DNA sequence; inducing a post-translational modification of a protein; transcription of the nucleic acid as an RNA; translation of the nucleic acid as a protein or enzyme; and incorporating the nucleic acid into a chromosome of a target cell for constitutive or transient expression of the therapeutic nucleic acid. [0164]
  • Therapeutic effects of therapeutic nucleic acids can include, but are not limited to: turning off a defective gene or processing the expression thereof, such as antisense RNA or DNA; inhibiting viral replication or synthesis; gene therapy as expressing a heterologous nucleic acid encoding a therapeutic protein or correcting a defective protein; modifying a defective or underexpression of an RNA such as an hnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or an enzyme that generates a compound as a drug or prodrug in pathological or normal cells expressing the chimeric receptor, and any other known therapeutic effects. [0165]
  • A therapeutic nucleic acid of the invention which encodes, or provides the therapeutic effect any known toxin, prodrug or gene drug for delivery to pathogenic nervous cells can also include genes under the control of a tissue specific transcriptional regulatory sequence (MRSs) specific for pathogenic SC containing cells. Such TRSs would further limit the expression of the therapeutic agent in the target cell, according to known methods. [0166]
  • Non-limiting examples of such PNS SCP modulating agents or ligands of the present invention and methods thereof include methyl/halophenyl-substituted piperizine compounds, such as lidoflazine (see; e.g., Merck Index Monograph 5311 and U.S. Pat. No. 3,267,104, both entirely incoporated herein by reference). Such compounds were tested and found to inhibit sodium channel activity of at least one PNS SCP of the present invention in cell lines expressing at least one PNS SCP, such as PC12, PK1-4 and other isolated or recombinant cells expressing at least one PNS SCP of the present invention. Accordingly, the present invention provides PNS SCP modulating agents or ligands as methyl/halophenyl-substituted piperizines. The substitutions can include alkyl- and/or halophenyl-substituted piperizines. [0167]
  • Pharmaceutical/Diagnostic Administration. Using PNS SCP modulating compounds or compositions (including antagonists and agonists as described above) the present invention further provides a method for modulating the activity of the PNS SCP protein in a cell. In general, agents (antagonists or agonists) which have been identified to inhibit or enhance the activity of PNS SCP can be formulated so that the agent can be contacted with a cell expressing a PNS SCP protein in vivo. The contacting of such a cell with such an agent results in the in vivo modulation of the activity of the PNS SCP proteins. So long as a formulation barrier or toxicity barrier does not exist, agents identified in the assays described above will be effective for in vivo use. [0168]
  • In another embodiment, the invention relates to a method of administering PNS SCP or a PNS SCP modulating compound or composition (including PNS SCP antagonists and agonists) to an animal (preferably, a mammal (specifically, a human)) in an amount sufficient to effect an altered level of PNS SCP in the animal. The administered PNS SC or PNS SCP modulating compound or composition could specifically effect PNS SCP associated functions. Further, since PNS SCP is expressed in -peripheral nervous system tissue, administration of PNS SC or PNS SCP modulating compound or composition could be used to alter PNS SCP levels in the peripheral nervous system. [0169]
  • PNS SCP antagonists can be used to treat pain due to trauma or pathology involving the central or peripheral nervous system, or pathologies related to the abnormally high levels of expression of at least one naturally occurring nervous system specific (NS) sodium channel (SC), where a PNS SCP antagonist also inhibits at least one NS SC, or where the pain is mediated to some extent by PN SC. Such pathologies, include, but are not limited to; inflammatory diseases, neuropathies (e.g., diabetic neuropathy), dystrophies (e.g., reflex sympathetic dystrophy, post-herpetic neuralgia); trauma (tissue damage by any cause); focal pain by any cause. [0170]
  • Inflammatory diseases can include, but are not limited to, chronic inflammatory pathologies and vascular inflammatory pathologies. Chronic inflammatory pathologies include, but are not limited to sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki's pathology. [0171]
  • PNS SCP agonists can be used to treat pathologies involving the central or peripheral nervous system, or pathologies related to the abnormally low levels of expression of at least one naturally occurring nervous system specific (NS) sodium channel (SC), where a PNS SCP agonist also enhances or stimulates at least one NS SC. Such pathologies, include, but are not limited to, neurodegenerative diseases, diseases of the gastrointestinal tract due to dysfunction of the enteric nervous system (e.g., colitis, ileitis, inflammatory bowel syndrome); diseases of the cardiovascular system (e.g., hypertension and congestive heart failure); diseases of the genitourinary tract involving sympathetic and parasympathetic innervation (e.g., benign prostrate hyperplasia, impotence); diseases of the neuromuscular system (e.g., muscular dystrophy, multiple sclerosis, epilepsy). [0172]
  • Neurodegenerative diseases can include, but are not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; hyperkinetic movement disorders, such as Huntington's Chorea and senile chorea; hypokinetic movement disorders, such as Parkinson's disease; progressive supranucleo palsy; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia; multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); and systemic disorders Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi-system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); or any subset thereof. [0173]
  • Pharmaceutical/diagnostic administration of diagnostic/pharmaceutical compound or composition of the invention, for a PNS SC related pathology can be administered by any means that achieve its intended purpose, for example, to treat or prevent a cancer or precancer us condition. [0174]
  • The term “protection”, as in “protection from infection or disease”, as used herein, encompasses “prevention,” “suppression” or “treatment.” “Prevention” involves administration of a Pharmaceutical composition prior to the induction of the disease. “Suppression” involves administration of the composition prior to the clinical appearance of the disease. “Treatment” involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events can be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term “prophylaxis” as distinct from “treatment” to encompass both “preventing” and “suppressing”as defined herein. The term “protection,” as used herein, is meant to include “prophylaxis.” See, e.g., Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein. The “protection” provided need not be absolute, i.e., the disease need not be totally prevented or eradicated, provided that there is a statistically significant improvement relative to a control population. Protection can be limited to mitigating the severity or rapidity of onset of symptoms of the disease. [0175]
  • At least one PNS SC modulating compound or composition of the invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as previously described. [0176]
  • For example, administration can be by -various parenteral routes such as subcutaneous, intravenous, intradernal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes. Alternatively, or concurrently, administration can be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time. [0177]
  • An additional mode of using of a diagnostic/pharmaceutical compound or composition of the invention is by topical application. A diagnostic/pharmaceutical compound or composition of the invention can be incorporated into topically applied vehicles such as salves or ointments. [0178]
  • For topical applications, it is preferred to administer an effective amount of a diagnostic/pharmaceutical compound or composition according to the invention to target area, e.g., skin surfaces, mucous membranes, and the like, which are adjacent to peripheral neurons which are to be treated. This amount will generally range from about 0.0001 mg to about 1 g of a PNS SC modulating compound per application, depending upon the area to be treated, whether the use is diagnostic, prophylactic or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed. A preferred topical preparation is an ointment, wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base. [0179]
  • A typical regimen for treatment or prophylaxis comprises administration of an effective amount over a period of one or several days, up to and including between one week and about six months. [0180]
  • It is understood that the dosage of a diagnostic/pharmaceutical compound or composition of the invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the diagnostic/pharmaceutical effect desired. The ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts. See, e.g., Berkow et al., eds., [0181] The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osol et al., eds., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992), which references are entirely incorporated herein by reference.
  • The total dose required-for each treatment can be administered by multiple doses or in a single dose. The diagnostic/pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology. [0182]
  • Effective amounts of a diagnostic/pharmaceutical compound or composition of the invention are from about 0.1 μg to about 100 mg/kg body weight, administered at intervals of 4-72 hours, for a period of 2 hours to 1 year, and/or any range or value therein, such as 0.0001-1.0, 1-10, 10-50 and 50-100, 0.0001-0.001, 0.001-0.01, 0.01-1.0, 1.0-10, 5-10, 10-20, 20-50 and 50-100 mg/kg, at intervals of 1-4, 4-10, 10-16, 16-24, 24-36, 36-48, 48-72 hours, for a period of 1-14, 14-28, or 30-44 days, or 1-24 weeks, or any range or value therein. [0183]
  • The recipients of administration of compounds and/or compositions of the invention can be any vertebrate animal, such as mammals, birds, bony fish, frogs and toads. Among mammals, the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs). Among birds, the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans. [0184]
  • Gene Therapy. A delivery vector of the present invention can be, but is not limited to, a viral vector, a liposome, an anti-PNS SCP or anti-SC antibody, or a SC ligand, one or more of which delivery vectors is associated with a diagnostic or therapeutic agent. [0185]
  • The delivery vector can comprise any diagnostic or therapeutic agent which has a therapeutic or diagnostic effect on the target cell. The target cell specificity of the delivery vector is thus provided by use of a target cell specific delivery vector. [0186]
  • The delivery vector can also be a recombinant viral vector comprising at least one binding domain selected from the group consisting of an antibody or fragment, a chimeric binding site antibody or fragment, a target cell or specific ligand, a receptor which binds a target cell ligand, an anti-idiotypic antibody, a liposome or other component which is specific for the target cell. A PNS SCP can be already associated with the target cell, or the delivery vector can bind the target cell via a ligand to a target cell receptor or vice versa. [0187]
  • Thus, the therapeutic or diagnostic agent, such as a therapeutic or diagnostic nucleic acid, protein, drug, compound composition and the like, is delivered preferentially to the target cell, e.g., where the nucleic acid is preferably incorporated into the chromosome of the target cell, to the partial or complete exclusion of non-target cells. [0188]
  • The invention is thus intended to provide delivery vectors, containing one or more therapeutic and/or diagnostic agents, including vectors suitable for gene therapy. [0189]
  • In a method of treating a PNS SCP-associated disease in a patient in need of such treatment, functional PNS SCP DNA can be provided to the PNS cells of such patient in a manner and amount that permits the expression of the PNS SCP protein provided by such gene, for a time and in a quantity sufficient to treat such patient, such as a suitable delivery vector. Many vector systems are known in die art to provide such delivery to human patients in need of a gene or protein missing from the cell. For example, retrovirus systems can be used, especially modified retrovirus systems and especially herpes simplex virus systems. Such methods are provided for, in, for example, the teachings of Breakefield, et al., [0190] The New Biologist 3:203-218 (1991); Huang, Q. et al, Experimental Neurology 115:303-316 (1992), WO93/03743 and WO90/09441. Delivery of a DNA sequence encoding a functional PNS SCP protein will effectively replace the missing or mutated PNS SCP gene of the invention.
  • In another embodiment of this invention, the PNS SCP modulating compound or composition is expressed as a recombinant gene in a cell, so that the cells can be transplanted into a mammal, preferably a human in need of gene therapy. To provide gene therapy to an individual, a genetic sequence which encodes for all or part of the PNS SCP modulating compound or composition is added into a vector and introduced into a host cell. Examples of diseases that can be suitable for gene therapy include, but are not limited to, neurodegenerative diseases or disorders, Alzheimer's, schizophrenia, epilepsy, neoplasms and cancer. Examples of vectors that can be used in gene therapy include, but are not limited to, defective retroviral, adenoviral, or other viral vectors (Mulligan, R. C., [0191] Science 260:926-932 (1993)). See Anderson, Gene Therapy, 246 J. Amer. Med. Assn. 2737 (1980); Friedmann, Progress toward human gene therapy, 244 Science 1275 (1989); Anderson, 256 Science 808 (1992); human gene therapy protocols published in Human Gene Therapy, Mary Ann Liebert Publishers, N.Y. (1990-1994); Bank et al., 565 Ann. N.Y. Acad. Sci. 37 (1989); LTR-Vectors (U.S. Pat. No. 4,405,712); Ausubel, infra, §§ 9.10-9.17; Jon A. Wolff., ed, Gene Therapeutics: methods and applications of direct gene transfer, Birkhäuser, Boston (1994).
  • The means by which the vector carrying the gene can be introduced into the cell include but is not limited to, microinjection, electroporation, transduction, or transfection using DEAE-Dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art (Sambrook infra, Ausubel, infra). [0192]
  • Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. See, generally, Osol et al., eds. [0193] Remington's Pharmaceutical Science, 16th Ed., (1980).
  • In another embodiment, the invention relates to a pharmaceutical composition comprising PNS SC or PNS SCP modulating compound or composition in an amount sufficient to alter PNS SCP associated activity, and a pharmaceutically acceptable diluent, carrier, or excipient Appropriate concentrations and dosage unit sizes can be readily determined by one skilled in the art (See, e.g., Osol et al. ed., Remington's Pharmaceutical Sciences, 16th Ed., Mack, Easton Pa. (1980) and WO 91/19008). [0194]
  • Included as well in the invention are pharmaceutical compositions comprising an effective amount of at least one PNS SCP antisense oligonucleotide, in combination with a pharmaceutically acceptable carrier. Such antisense oligos include, but are not limited to, at least one nucleotide sequence of 12-500 bases in length which is complementary to a DNA sequence of SEQ ID NO:1, or a DNA sequence encoding at least 4 amino acids of SEQ ID NO:2 or FIG. 11A-11E. [0195]
  • Alternatively, the PNS SCP nucleic acid can be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid. A preferred sterol is cholesterol. [0196]
  • The PNS SCP gene therapy nucleic acids and the pharmaceutical compositions of the invention can be administered by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intra-peritoneal, or transdermal routes. The dosage administered will be dependent upon the age, health, and weight of the recipient kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. [0197]
  • Compositions within the scope of this invention include all compositions wherein the PNS SCP antisense oligonucleotide is contained in an amount effective to achieve enhanced expression of at least one PNS SCP in a peripheral nervous system neuron or ganglion. While individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art. Typically, the PNS SCP nucleic acid can be administered to mammals, e.g. humans, at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated. [0198]
  • Suitable formulations for parenteral administration include aqueous solutions of the PNS SCP nucleic acid in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension can also contain stabilizers. [0199]
  • Alternatively, at least one PNS SCP can be coded by DNA constructs which are administered in the form of virions, which are preferably incapable of replicating in vivo (see, for example, Taylor, WO 92/06693). For example, such DNA constructs can be administered using herpes-based viruses (Gage et al., U.S. Pat. No. 5,082,670). Alternatively, PNS SCP antisense RNA sequences, PNS SCP ribozymes, and PNS SCP EGS can be coded by RNA constructs which are administered in the form of virions, such as recombinant, replication deficient retroviruses or adenoviruses. The preparation of retroviral vectois is well known in the art (see; for example, Brown et al., “Retroviral Vectors,” in [0200] DNA Cloning: A Practical Approach, Volume 3, IRL Press, Washington, D.C. (1987)).
  • Specificity for gene expression in the peripheral nervous system can be conferred by using appropriate cell-specific regulatory sequences, such as cell-specific enhancers and promoters. Since protein phosphorylation is critical for neuronal regulation (Kennedy, “Second Messengers and Neuronal Function,” in [0201] An Introduction to Molecular Neurobiology, Hall, E A, Sinauer Associates, Inc. (1992)), protein kinase promoter sequences can be used to achieve sufficient levels of PNS SCP gene expression.
  • Thus, gene therapy can be used to alleviate sodium channel related pathology by inhibiting the inappropriate expression of a particular form of PNS SC. Moreover, gene therapy can be used to alleviate such pathologies by providing the appropriate expression level of a particular form of PNS SCP. In this case, particular PNS SCP nucleic acid sequences can be coded by DNA or RNA constructs which are administered in the form of viruses, as described above. [0202]
  • Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the invention, unless specified. [0203]
    • EXAMPLE 1 Cloning and Sequencing of a PNS SC Encoding Nucleic Acid
  • Materials and Methods [0204]
  • Cell Culture. PC12 cells and PKI-4 PC12 subclones were grown as previously described (Mandel et al., 1988). NGF (2.5 S subunit, kindly supplied by Dr. S. Halegoua, SUNY at Stony Brook), was added to the culture medium at final concentration of 110 ng/ml. The PKI-4 PC12 subclone which expresses the cAMP-dependent kinase inhibitor protein (PKI) was also provided by Dr. S. Halegoua (see D'Arcangelo et al., [0205] J. Cell Biol. 122:915-921 (1993)).
  • PCR Amplification. Total cellular RNA was isolated, according to the method of Cathala et al. [0206] DNA 2:329-335 (1983), from a PC12 subclone (PKI-4) which expresses high levels of the cAM-dependent protein kinase inhibitor protein. Two μg of total RNA prepared time NGF-treated PKI-4 cells was used to synthesize first strand cDNA using random hexamer primers for the reverse transcriptase reaction. The cDNA then served as template for the PCR amplification, using a pair of degenerate oligonucleotide primers that specified a 400 base pair region within repeat domain III of the sodium: channel α subunit gene. The 5′ primer (designated YJI:GCGAAGCTT(TC)TIATITT(TC)I(GATC)IAT(ATC)ATGGG (SEQ ID NO:3), underline indicates a HindIII restriction site), corresponded to amino acids FWLIFSIM at positions 1347-1354 in the type II sodium channel gene. The 3′ primer (designated YO1C: GCAGGATCC(AG)TT(AG)AAA(AG)TT(AG)TC(AGT)AT(AGT)AT(AGCT)AC(AGCT)CC (SEQ ID NO:5), underline indicates a BamH1 restriction site) corresponded to amino acids GVIIDNFN at positions 1470-1447 in the type II gene. The amplification reaction mixture consisted of 5% of the cDNA, 1 mM MgCl2, 0.2 mM dNTPSs, 0.5 μM each primer, Taq polymerase (Perkin-Elmer) in a buffer consisting of 0.1 M KCl, 0.1 M-TRIS HCl (pH 8.3) and gelatin (1 mg/ml). The reaction was performed in a Perkin-Elmer thermocycler as follows: 5 cycles of denaturation (94° C., 1 min.), annealing (37° C., 1 min.), and extension (72° C., 1 min) followed by 25 cycles of denaturation (94° C., 1 min.), annealing (50° C., 1 min.) and extension (72° C., 1 min.). The PCR products were excised from a low melt agarose gel (SEAPLAQUE GTG, FMC BIOPRODUCTS) and subcloned into a Bluescript II SK plasmid vector previously restricted with HindIII and BamH1. The clones were screened for cDNA inserts by miniprep (Sambrook et al., infra) and sequenced in both directions by dideoxy chain termination (Sequenase 2.0 kit, UNITED STATES BIOCHEMICAL). Sequence data was compiled and analyzed using GENWORKS software (INTELLIGENETICS, INC., Mountain View, Calif.).
  • cDNA Library Construction and Screening. Poly(A)+ mRNA from the PKI-4 PC12 subclone was purified (mRNA purification kit, PHARMACIA) and used to construct a random- and oligo (dT)-primed Lambda ZAP II cDNA library (STRATAGENE CORP., La Jolla, Calif.). The library consisted of 5.6×10[0207] 6 independent clones prior to amplification. Screening of approximately 4×106 recombinants using the cloned PCR product pPC12-1 labeled by random primers (PHARMACIA kit) resulted in isolation of 5 cDNAs ranging in size from 1-3 kb. Sequence analysis and comparison to published sequences established that the two of the cDNAs together encoded 3033 bp of the novel sodium channel α subunit, PN1.
  • Northern blot analysis and ribonuclease protection assays. Total cellular RNA was isolated from adult Sprague-Dawley rat brain, spinal cord, superior cervical ganglion, dorsal root ganglion, skeletal muscle, cardiac muscle, and adrenal gland using the standard method of Chirgwin, [0208] Biochemistry 18:5294-5299 (1979). RNA was electrophoresed and transferred to nylon membrane as previously described (Cooperman et al, Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) (DURALON-UV; STRATAGENE CORP.). RNA blots were cross-linked to the nylon using Stratalinker UV crosslinker (STRATAGENE CORP.) and hybridized to 32P-UTP-labeled antisense RNA probes generated from the following linearized templates: pPC12-1, pRB211 (Cooperman, infra, 1987), p1B15 (cyclophilin; Danielson et al., DNA 7:261-267 (1988)), and rat brain type 1, which contains 51 bp of intron, 5′ untranslated sequence and 267 bp of coding sequence of the type I sodium channel. RNA probes were transcribed with either T3 (pPC12-1), T7, (pNach1), or SP6 (pRB211, p1B15) RNA polymerase according to the manufacturer's instructions (PROMEGA CORP, Madison, Wis.). The blots were washed once in 2×SSC, 0.1% NaDodSO4 for 15 min. at 68° C., followed by two washes in 0.2×SSC, 0.1% NaDodSO4 for 15 min. at 68° C. Autoradiography with preflashed XAR-5 film (EASTMAN KODAK CO., Rochester, N.Y.) was used for quantitation of mRNA by densitometry.
  • Ribonuclease protections assays were performed by use of a kit (RPA II, AMBION INC., Austin, Tex.). Total RNA was hybridized with 10[0209] 4 cpm of antisense RNA probe generated from pPC12-1. To control for differences in the amount of total RNA between samples, we included an antisense RNA probe for β actin, transcribed from pTRI-β-actin (AMBION, INC.).
  • In situ hybridization. Tissue preparation and hybridization were performed using a modification of the procedure described by Yokouchi et al., [0210] Develop. 113:431-444 (1991). SCG and DRG were dissected from adult Sprague-Dawley rats and fixed in 4% paraformaldehyde (in 0.1 M PBS) for 2-6 hrs. at 4° C. The tissue was then rinsed≈5 min. in 0.1M PBS (pH 7.3), cryoprotected in 30% sucrose (in 0.1 M PBS) for 2 hrs. at 4° C. and embedded in O.C.T. (TISSUE-TEK). Cryostat sections (14 μM) were collected on SUPERFROST/Plus slides (FISHER SCIENTIFIC), dried≈2 hrs. at room temp., and then stored at −80° C.
  • Immediately before prehybridization, sections were brought to room temp. and rehydrated in 0.1M PBS (pH 7.3) containing 0.3% Triton X-100 for 5 min. Sections were then treated with 0.2 N HCl for 20 min., washed in 0.1 M PBS for 5 min., and digested with proteinase K (5 μg/ml in 0.1 M PBS) for 40 min. at 37° C. Sections were then postfixed with 4% paraformaldehyde (in 0.1 M PBS), rinsed with 0.1 M PBS containing 0.1 M glycine for 15 min., and equilibrated in 50% formamide, 2×SSC for 1 hr. (room temp.). [0211]
  • Sections were hybridized with antisense digoxigenin-labeled RNA probes transcribed from pPC12-1 or pNach2 (Cooperman et al., [0212] Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) according to the manufacturer's instructions for RNA labeling with digoxigenin-UTP (BOEHRINGER MANNHEIM). Unlabeled probes were synthesized by replacing digoxigenin-UTP with rUTP. Each section was covered with ≈100 μl of hybridization solution containing 20 mM TRIS HCl (pH 8.0), 2.5 mM EDTA, 50% formamide, 0.3 M NaCl, 1× Denhardt's, 10% dextran sulfate, 1 mg/ml tRNA, and probe at a concentration of 0.7 μg/ml. Sections were then covered with PARAFILM coverslips and incubated in a humid chamber overnight at 45° C. After hybridization, sections were washed in 50% formamide, 2×SSC at 45° C. for 1 hr., followed by RNase digestion in 0.5M NaCl, 10 mM TRIS HCl (pH 8.0), and 20 μg/ml RNase A (BOEHRINGER MANNHEIM). Sections were subsequently washed at 45° C. in 50% formarnide, 2×SSC for 1 hr., and 50% formamide, 1×SSC for 1 hr.
  • Immunological detection was performed using a kit ([0213] GENIUS 3 KIT, BOEHRINGER MANNHEIM), according to the manufacturer's instructions. In most experiments, the sections were incubated in the color solution for ≈3-5 hrs. at room temp. Sections were then coverslipped with AQUA-MOUNT (Lerner Laboratories) and stored in the dark.
  • Densitometry. Levels of sodium channel mRNA were determined by densitometric analysis of the autoradiograms using Bio Image software (Millipore Corp., Ann Arbor, Mich.). Levels of RNA were normalized to the quantitated levels of cyclophilin mRNA. [0214]
  • Results [0215]
  • Isolation of a cDNA expressed preferentially in peripheral nerve. D'Arcangelo et al., [0216] J. Cell Biol. 122:915-921 (1993) showed previously that NGF treatment of PC12 cells increase the level of an ≈11 kb sodium channel gene transcript which did not hybridize to probes specific for any of the known sodium channel genes. A transcript identical in size was also detected in mRNA from adult rat sympathetic and sensory ganglia, but not in mRNA from brain. These results suggested that the transcript encoded a new member of the sodium channel gene family (termed Peripheral Nerve type 1 (PN1)).
  • To confirm the identity of the PN1 gene, cDNAs from an NGF-treated PC12 subclone which preferentially expresses PN1 mRNA (PKI-4 cells) D'Arcangelo et al were amplified by the polymerase chain reaction (PCR), using a pair of degenerate oligonucleotide primers that specify a 400 base pair (bp) region of the sodium channel α subunit gene (see Methods, FIG. 1). Both primers specified putative membrane-spanning regions within repeat domain III, which are highly conserved among voltage-gated sodium channels. The amplified regions between the primers include the strictly-conserved pore-lining residues, as well as residues which are divergent among the different mammalian α subunits. Sequence analysis of the PCR products revealed a cDNA, pPC12-1, which encoded a portion of a novel putative sodium channel α subunit (FIG. 1). Additional cDNAs were further isolated which encapsulated the entire PN1 coding region. [0217]
  • To determine whether pPC12-1 encode part of the PN1 gene, the cDNA was used to generate antisense RNA probes for Northern blot analysis of mRNA from control and NGF-treated PC12 cells (FIG. 2B). For comparison, a duplicate blot (FIG. 2A) was hybridized with an antisense probe pRB211, which encode a highly-conserved region of the sodium channel α subunit (Cooperman et al., [0218] Proc. Nat'l Acad. Sci. USA 84:8721 (1987)) and which cross-hybridizes with the PN1 transcript, and that, as shown by D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993), levels of the detected transcript should increase rapidly and transiently following NGF treatment (maximal=5 hrs). Comparison of FIGS. 2A and 2B shows that pPC12-1 fulfilled both of these criteria. Also, consistent with D'Arcangelo et al., J. Cell Biol. 122:915-921 (1993), we found that NGF induction of the transcript detected by pPC12-1 is independent of cAMP-dependent protein kinase activity.
  • To isolate additional cDNAs encoding PN1, a random- and oligo (dT)-primed Lambda ZAP II cDNA library (STRATAGENE, 5.6×10[0219] 6 independent clones) was prepared from poly(A)+mRNA isolated from the same PC12 subclone from which pPC12-1 was isolated. Screening 4×104 recombinants with a probe generated from pPC12-1 resulted in isolation of 2 additional, overlapping cDNAs which are joined to give a 3033 bp cDNA (FIG. 7). Additional cDNAs were further isolated which encapsulated the entire PN1 coding region.
  • Analysis of the deduced primary structure of PN1. As shown in FIG. 8, the deduced primary structure of PN1 encodes repeat domain II of the sodium channel α subunit gene. Comparison with the [0220] type 11 sodium channel shows that the PN1 sequence contains all of the structural motifs characteristic of voltage-gated sodium channels, including six putative transmembrane domains (IIIS1-IIIS6). The S4 domain, thought to serve as the voltage sensor, exhibits the highly-conserved pattern of a positively-charged residue (lysine or arginine) at every third position; Furthermore, the putative pore-lining segments (IIISS1-IIISS2) contain residues shown to be involved in sodium-selective permeation (Heinemann et al., Nature 356:441-443 (1992)) as well as TTX affinity (Terlaue et al., FEBS Lett. 293:93-96 (1991)).
  • In addition to such highly-conserved structural features, the sodium channel αsubunit undergoes several characteristic post-translational modifications. All sodium channels sequenced to date exhibit a distinctive pattern of asparagine-linked (N-linked) glycosylation sites, which are found almost exclusively in the extracellular loops joining the S5and S6 transmembrane helices. The N-linked glycosylation sites of PN1 are in good agreement with this pattern; three potential extracellular glycosylation sites are located between IIIS5 and IIIS6. Two of the sites are also found in the types I, II and III sodium channels. [0221]
  • The α subunit is phosphorylated by protein kinase C (PKC), and deduced PN1 sequence contains the highly-conserved consensus PKC phosphorylation-site at serine[0222] 1506 (FIG. 1). This residue is located in the cytoplasmic loop joining domains III and IV that has been implicated in channel inactivation, and mutational analysis has shown that this serine is required for PKC modulation of channel inactivation (West et al., 1991).
  • The entire DNA (FIG. 9A-D) and amino acid (FIG. 10) sequences were determined. The rat PN1 amino acid sequence was compared with new human sequences (FIG. 11A-E) presented in Example 2. [0223]
  • In sum, the deduced primary structure of PN1 contains all of the hallmark structural and functional domains characteristics a α subunit the voltage-gated sodium channel. [0224]
  • The PN1 gene is expressed preferentially in the PNS. To determine whether the PN1 gene was expressed preferentially in the PNS, total RNA was isolated from adult rat brain, spinal cord, SCG, DRG, skeletal muscle, and cardiac muscle and subjected to Northern blot analysis. Blots were hybridized with the PN1-specific antisense probe generated from pPC12-1. As shown in FIG. 3A, we found high levels of hybridization to an ≈11 kb transcript in both SCG and DRG. Much lower, but detectable levels hybridization were seen to transcripts in both spinal cord and brain. No detectable hybridization was observed to mRNA from skeletal muscle, cardiac muscle, or liver. [0225]
  • Ribonuclease (RNase) protection analyses were also prepared. Total RNA was isolated from the same tissues used in Northern blot analysis, as well as adrenal gland, and hybridized to PN1-specific antisense probe (pPC12-1). mRNA from SCG, DRG, brain, spinal cord, and adrenal gland protected a 343 bp fragment of the PN1 probe (FIG. 4B). The non-protected bases represent oligonucleotide primer and plasmid sequences. The PN1 probe was not protected by mRNA from either skeletal muscle or cardiac muscle. [0226]
  • To determine the relative amounts of PN1 mRNA in the various tissues, autoradiographs from three separate RNase protection experiments were analyzed by densitometry. To control for small differences in the amount of total RNA between samples, we included a probe for a β actin. PN1 mRNA levels in both SCG and DRG are approximately 40-fold greater than in spinal cord, adrenal gland and brain. [0227]
  • The PN1 gene is expressed in sympathetic and sensory neurons. To determine whether the PN1 gene is expressed in neurons of peripheral ganglia, in situ hybridization was used to examine the cellular distribution of PN1 mRNA in adult rat SCG and DRG. Cryostat sections were hybridized with a PN1 -specific digoxigenin-labeled RNA probe (pPC12-1), which was visualized using an anti-digoxigenin antibody conjugated to alkaline phosphatase. As shown in FIGS. 4A, B the PN1 antisense probe labeled most neuronal cell bodies in both SCG and DRG. To confirm that the hybridization signal was due to binding of the probe specifically to PN mRNA, we performed two different negative controls: (1) Sections were hybridized-with the digoxigenin-labeled probe in the presence of a 100-fold excess of unlabeled PN1 antisense probe. (2) Previous experiments have shown that SCG and DRG contain extremely low levels of type II sodium channel mRNA (Beckh, S., [0228] FEBS Lett 262:317-322 (1990)). Therefore, we also hybridized sections with a type II-specific antisense probe. As shown, in FIG. 4C-F, both of these control experiments greatly reduced the hybridization signal. Also, consistent with the results of Northern blot and RNase protection analyses, we found that hybridization of the labeled PN1 probe to sections of adult rat cerebral cortex yielded no detectable staining.
  • Although the PN1 probe stained most neuronal cell bodies in both SCG and DRG, we found that cell-to-cell variability in PN1 mRNA levels differed between the two ganglia. SCG neurons were fairly homogeneous, in that the intensity of reaction product was relatively constant between different cells. DRG neurons, however, were quite heterogeneous in that the staining intensity varied considerably from cell to cell. For example, in FIG. 4B, arrows indicate two DRG neurons of approximately the same diameter which differ markedly in staining intensity. [0229]
  • Finally, we found that the PN2 probe did not stain non-neuronal cells such as satellite cells and Schwann cells. However, it is possible that these cells contain very low levels of PN1 mRNA which are not detectable by this method. [0230]
  • SCG neurons also express the type I sodium channel gene. Earlier Northern blot analysis has shown that mRNA from SCG contains two distinct sodium channel gene transcripts. As we have demonstrated, the larger, 11 kb transcript encodes the PN1 sodium channel. The smaller transcript, however, has not yet been identified. We hypothesized that this smaller transcript encoded the type I sodium channel, because moderate levels of type I mRNA have been found in other PNS tissues (Beckh, S., [0231] FEBS Lett. 262:317-322 (1990)). To test this hypothesis, Northern blots of SCG mRNA isolated from adult rats were hybridized with an antisense probe specific for the type I sodium channel gene (pNach1, see Methods above). As shown in FIG. 5, the type I-specific probe hybridized specifically to the smaller transcript. Furthermore, we have found that SCG mRNA protects the type I probe in an RNas protection assay.
  • The putative PN1α subunit and type Iα subunit genes are differentially regulated during development. Several studies have shown that the types I, II and III sodium channel genes are differentially regulated during development in both the central and peripheral nervous systems. To determine whether the PN1 and type I genes are also independently regulated during development, we measured their relative mRNA levels in SCG isolated from rats of different postnatal ages. To visualize both transcripts simultaneously, Northern blots were hybridized with the conserved sodium channel gene probe pRB211. As shown in FIG. 6A, in SCG removed on postnatal day 7 (P7), the levels of PN1 and type I mRNA are approximately equal. However, by P14, their relative abundance has shifted such that level of PN1 mRNA exceeds that of type I by ≈*-fold. This increase in ratio of PN1 to type I mRNA levels continues for at least the next four postnatal weeks. By P42, PN1 is the predominant sodium channel gene transcript, with levels of PN1 mRNA several-fold greater than that of type I. [0232]
  • To quantitate the development changes in mRNA levels, autoradiographs from three separate experiments were analyzed by densitometry. To control for differences in the amount of total RNA between lanes, blots were subsequently hybridizing blots with a probe for the internal control cyclophilin. As shown in FIG. 6B, in which percent maximum mRNA is plotted versus postnatal age, the shift in relative abundance of the two transcripts in largely due to a developmental decrease in level of type I sodium channel mRNA. From P7 to P42, the level of type I mRNA decreases by approximately 80%. [0233]
    • EXAMPLE 2 Drug Screening for PN-1 Antagonists
  • The ability of a PNS SCP-ligand (e.g., antagonists and agonists) to inhibit or enhance the activity of a PNS SCP is be evaluated with cells expressing at least one PNS SCP. An assay for PNS SCP activity in such cells is used to determine the functionality of the PNS SCP protein in the presence of at least one agent which can act as antagonist or agonist, and thus, agents that interfere or enhance the activity of PNS SCP are identified. Two or more cell lines (each expressing a different PNS SCP) are used, as well as optionally using one or more cell lines expressing a CNS specific sodium channel as a control. [0234]
  • These agents are selected and screened (1) at random; (2) by a rational selection; and or (3) by design using for example, computer modeling techniques. [0235]
  • There are numerous variations of assays which can be used by a skilled artisan without the need for undue experimentation in order to isolate, modulating agents or ligands of a PNS SCP. Agent determination methods include Computer Assisted Molecular Design (CAMD)) PNS SCP-agent binding, sophisticated chemical synthesis and testing, targeted screening, peptide combinatorial library technology, antisense technology and/or biological assays, according to known methods. See, e.g., Rapaka et al., eds., [0236] Medications Development: Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, U.S. Dept. of Health and Human Services, Rockville, Md. (1993); Langone, Methods in Enzymology, Volume 203, Molecular Design and Modeling:Concepts and Applications, Part B, Antibodies and Antigens, Nucleic Acids, Polysaccharides and Drugs, Section III, pp 587-702, Academic Press, New York (1991)).
  • Alternatively, cell expression libraries, or other cells are used to that have been selected or genetically engineered to express and display a PNS SCP via the use of the PNS SCP nucleic acids of the invention are preferred in such methods, as host cell lines may be chosen which are devoid of related receptors. Rapaka, infra, (1993), at pages 58-65. [0237]
  • A PNS SCP agent in the context of the present invention refers to any chemical or biological molecule that associates with a PNS SCP in vitro, in situ or in vivo, and can be, but is not limited to, synthetic, recombinant or naturally derived chemical compounds and compositions, e.g., organic compounds, nucleic acids, peptides, carbohydrates, vitamin derivatives, hormones, neurotransmitters, viruses or receptor binding domains thereof, opsins, rhodopsins, nucleosides, nucleotides, coagulation cascade factors, odorants or pheremones, toxins, growth factors, platelet activating factors, neuroactive peptides, neurohumors, or any biologically active compound, such as drugs or naturally occurring compounds. [0238]
  • The agents are selected and screened at random or rationally selected or designed using computer modeling techniques. For random screening, potential agents are selected and assayed for their ability to bind to the PNS SCP, or a fragment thereof. Alternatively, agents may be rationally selected or designed. As used herein, a agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of at least one specific PNS SCP (e.g., as presented in FIG. 11). For example, one skilled in the art can readily adapt currently available procedures to generate agents capable of binding to a specific peptide sequence in order to generate rationally designed compounds, such as chemical compounds, nucleic acids or peptides. See, e.g., Rapaka, infra, (1993); Hurby et al., “Application of Synthetic Peptides: Antisense Peptides,” in [0239] Synthetic Peptides: A User's Guide, W.H. Freeman, New York (1992), pp. 289-307; and Kaspczak et al., Biochemistry 28:9230-2938 (1989).
  • A method of screening for an agent that modulates the activity of at least one PNS SCP comprising: [0240]
  • (a) incubating at least one cell line expressing at least one PNS SCP with an agent to be tested; and [0241]
  • (b) assaying the at least one cell for the activity of the at least one PNS SCP protein by measuring the agents effect on PNS SCP binding or PNS SCP activity preferably the or assay distinguishes the agent's effect on alternative PNS SCP and determines that the agent has little or no effect on CNS sodium channels, or has relatively less effect on CNS sodium channels. [0242]
  • Any cell can be used in the above assay so long as it expresses a functional form of PNS SCP protein and the PNS SCP activity can be measured. The preferred expression cells are eukaryotic cells or organisms. Such cells can be modified to contain DNA sequences encoding the PNS SCP protein using routine procedures known in the art Alternatively, one skilled in the art can introduce mRNA encoding the PNS SCP protein directly into the cell. [0243]
  • In an alternative embodiment stem cell populations for either neuronal or glial cells can be genetically engineered to express a functional PNS SCP ion channel. Such cells expressing the PNS SCP ion channel, can be transplanted to the diseased or injured region of the mammal's neurological system ([0244] Neural Transplantation. A Practical Approach, Donnet & Djorklund, eds., Oxford University Press, New York, N.Y. (1992)). In another embodiment, embryonic tissue or fetal neurons can be genetically engineered to express functional PNS SCP ion channel and transplanted to the diseased or injured region of the mammal's limbic system. The feasibility of transplanting fetal dopamine neurons into Parkinsonian patients has been demonstrated. (Lindvall et al., Archives of Neurology 46:615-631 (1989)).
  • At least two types of approaches are currently used to express voltage-dependent sodium channel clones in order to generate functional channel proteins. In one approach, mRNA encoding the cloned cDNA is expressed in [0245] Xenopus oocytes. The sodium channel cDNA is cloned into a bacterial expression vector such as the pGEM recombinant plasmid (Melton, et al., 1984). Transcription of the cloned cDNA is carried out using an RNA polymerase such as SP6 polymerase or T7 polymerase with a capping analog such as M7G(5′)ppp(5′)G. The resulting RNA (e.g., about 50 nl, corresponding to 2-5 ng) is injected into stage V and stage VI oocytes isolated from Xenopus, and incubated for 3-5 days at 19° C. Oocytes axe tested for sodium channel expression with a two-microelectrode voltage clamp (Trimmer et al, Neuron 3:33-49 1989).
  • In an alternative approach, cDNAs encoding a voltage-dependent sodium channel is cloned into any one of a number of mammalian expression vectors, and transfected into mammalian cells which do not express endogenous voltage-dependent sodium channels (such as fibroblast cell lines). Transfected clones are selected expressing the cloned, transfected cDNA. Sodium channel expression is measured with a whole cell voltage clamp technique using a patch electrode (D'Arcangelo et al., [0246] J. Cell. Biol. 122:915-921 (1993)).
  • Sources of PNS SCPs and Cell Lines Useful for Drug Screening. Any cell line expressing (Naturally, by induction or due to recombinant expression of a PNS SCP) can be used for drug screening. As a non-limiting example, PC12 cells are mutants deficient in Protein Kinase A (PKA) activity and which express both PN1 and Type II sodium channels. A126-1B2 cells are a cell line which express PN1, but are now discovered to does not express Type II sodium channels. PKI-4 is a PC12 cell line transfected with a cDNA encoding a peptide inhibitor of PKA. Each of these cell lines can be used as one source of a PNS SCP of the present invention, or as a cell line itself to use in drug screening. Treatment of PC12 cells with NGF reduces both a PNS SCP (PN1) and type II sodium channels, while NGF induces only PN1 in A 126-182 cells. PKI-4 cells express a PNS SCP (PN1) without NGF treatment (D'Arcangelo et al, [0247] J. Cell Biol. 122:915-921 (1993)).
  • Additionally or alternatively, heterologous expression systems can also be used in which cell lines (such as Chinese Hamster Ovary cells (CHO)) are stably transfected with a cDNA encoding PN-1. Method steps for transfecting and stably expressing cDNA to form heterologous cell lines, are well known in the art An advantage of using transfected cells is that clones are obtained that express very high levels of a PNS SCP, such as PN-1. [0248]
  • To screen for PNS SCP modulators, as antagonists or agonists, drugs are examined for their ability to: [0249]
  • (a) inhibit or enhance the binding of radioligands to a PNS SCP (labeled ligand binding reaction), and/or [0250]
  • (b) to inhibit or enhance ion flux through the channel of the PNS SCP in a cell line that expresses a PNS SCP. [0251]
  • Labeled ligand binding neurotoxins can be used to characterize PNS sodium channels. For example previous studies have identified at least six distinct neurotoxin binding sites on previously characterized non-PNS-sodium channels (reviewed in Lombert et al., [0252] FEB 219(2):355-359 (1987)). Many of these sites are thought to be allosterically coupled to one another (for review, see Strichartz et al., Ann. Rev. Neurosci. 10:237-267 (1987), and references cited therein). In other words, binding of a drug or toxin to a particular neurotoxin site can be sensitive to drug binding at not only that site, but other sites on the channel as well. This is advantageous for a drug screening program in that for a given labeled ligand, the likelihood of identifying agents that preferrentially bind to a PNS SCP is increased.
  • The techniques described herein for measuring labeled ligand binding to a PNS SCP of the invention in intact cells (e.g., PC12 PKI or PNS SCP expressing heterologous cell lines) in suspension are similar to those described previously for radioligand binding to other sodium channels in brain synaptosomal preparations (see, e.g., Catterall et al, [0253] J. Biol. Chem. 256(17):8922-8927 (1981)). However, it is well recognized by those skilled in the art that these techniques are routinely modified for the use of substrate-attached cells or broken cell preparations, based on the teaching and guidance presented herein.
  • A126-1B2, PC12, PKI-4 or other cells expressing a PNS SCP cells are grown using standard techniques, and optionally treated with NGF for 1-2 days to induce PN-1 expression. Cells are harvested and tested for ion flux activity with alternative potential agents. [0254]
  • For both radioligands, binding reactions are conducted e.g., at 37° C., then stopped. Samples are quickly filtered with vacuum washed with ice-cold buffer, and bound radioactivity determined by scintillation counting. [0255]
  • Ion Flux directly tests the ability of a potential PNS SCP agent to inhibit or enhance the activity of a PNS SCP function, by their ability to inhibit or enhance the influx of ion tracers through a PNS SCP. [0256]
  • Most previous sodium channel studies have employed [0257] 22Na as a tracer (for example, see Catterall et al. J. Biol. Chem. 256(17):8922-8927 (1981)). However, the high toxicity of 2Na can be a disadvantage for its use in high-throughput drug screening. A less toxic alternative is (14C) guanidimium ion, influx of which has been shown to be a reliable indicator of sodium channel opening (Reith, Europ. J. Pharmacol. 188:33-41 (1990)). Accordingly, routine methods can be used to screen compounds for modulating PNS SCP ion channel activity, e.g., (14C) guanidimium ion flux using intact cells expressing at least one PNS SCP. Additionally these methods are well known to be easily modified for use with 22Na. Similarly, these known method steps could be modified for use with substrate-attached cells or vesicles prepared from broken cells, according to known method steps.
  • For a guanidinium flux assay the methods for [0258] 22Na are modified from those of Reith (Europ. J. Pharmacol. 188:33-41 (1990) for brain synaptosomes), e.g., as described in Example 2 below. Aliquots of a cell suspension containing heterologous cells expressing at least one PNS SCP are incubated for 10 minutes at 37° C. in the presence of channel openers (typically, 100 μM veratridine) and test drugs in a total volume of 100 μM (0.20-0.25 mg protein). Ion flux is initiated by the addition of HEPES/TRIS solution also containing 4mM guanidine HCl (final) and 1000 dpm/nmol (14C) guanidine. The reaction is continued for 30 seconds and is stopped by the addition of ice-cold incubation buffer, followed by rapid filtration under vacuum over Whatman GF/C filter. The filters are washed rapidly with ice-cold incubation buffer and radioactivity determined by scintillation counting. Nonspecific uptake is determined in parallel by the inclusion of 1 mM tetrodotoxin during both preincubation and uptake.
  • Using the guanidinium flux assay several methyl/halophenyl substituted compounds, such as lidoflazine (see, e.g., Merck Index Monograph 5311 and U.S. Pat. No. 3,267,104, both entirely incoporated herein by reference), were tested and found to inhibit sodium channel activity of at least one PNS SCP of the present invention in cell lines expressing at least one PNS SCP, with a pIC50 of 6.51 for lidoflazine on PKI-4 cells. Accordingly, the present invention provides PNS SCP modulating agents as methyl/halophenyl-substituted piperizines. [0259]
    • EXAMPLE 3 Identification of Human PNS SCP Sequence from a Human Peripheral Nervous System cDNA Library
  • Similar to the procedures provided in Example 1, a human peripheral nervous system cDNA library (as a human DRG library) was used for polymerase chain reaction (PCR) amplification. The PCR used a 5′ primer corresponding to DNA encoding amino acids 604-611 of SEQ ID NO:2, and a corresponding 3′ primer encoding amino acids 723-731 of SEQ ID NO:2. [0260]
  • The PCR reaction mixture consisted of 5% of the cDNA, 1 mM MgCl[0261] 2, 0.2 mM dNTPSs, 0.5 mM, each primer, Taq polymerase (Perkin-Elmer) in a buffer consisting of 0.1 M KCl, 0.1 M TRIS HCl (pH 8.3) and gelatin (1 mg/ml). The reaction was performed in a Perkin-Elmer thermocycler as follows: five cycles of denaturations (94° C., 1 min.), annealing (37° C., 1 min), and extension (72° C., 1 min.), followed by 25 cycles of denaturation (94° C., 1 min.), annealing (50° C., 1 min.), and extension (72° C., 1 min.).
  • The resulting PCR products provided a human amplified cDNA which encoded amino acids 646-658 of SEQ ID NO:2, as presented in FIG. 11A-E. [0262]
    • EXAMPLE 4 Cloning and Sequencing of Human PN-1 Sequence from Human Dorsal Root Ganglion cDNA Library
  • As in Examples 1 and 3 above, additional PCR primers corresponding to SEQ ID NO:1 are used to isolate clones from the human DRG cDNA library which encompass the entire coding region of one or more human PNS SCPs of the present invention. A 5′ primer includes the sequence 5′TTTGTGCCCCACAGACCCCAG3′ (SEQ ID NO: 13) and a 3′ primer includes the sequence 5′ ACACAAATTCTTGATCTGGAATTGCT3′(SEQ ID NO: 14) or 5′[0263] CAACCTC AGACAGAGAG CAATGA 3′ (SEQ ID NO: 15), which are used for nested PCR. According to Examples 1 and 3 above, PCR is performed to obtain cDNAs encoding a human PNS SCP.
  • Additional PCR is performed by “walking” 5′ or 3′ of the sequence corresponding to the above PCR product. In this way cDNAs encompassing the entire coding region of one or more human PNS SCPs are provided. [0264]
  • The resulting additional cDNA clones or PCR products, encoding the entire human PNS SCP, are subcloned into a plasmid vector previously restricted with suitable restriction sites. The clones are screened for cDNA inserts by miniprep (Sambrook et al., infra) and sequenced in both directions by dideoxy chain termination (Sequenase 2.0 kit, United States Biochemical). Sequence data is compiled and analyzed using GeneWorks software (IntelliGenetics, Inc., Mountain View, Calif.). The expected alternative amino acid sequences for a human PN1 sequence or presented in FIG. 11A-D and as SEQ ID NOS:7, 11 and 12, where Xaa represents 0, 1, 2 or 3 amino acids. [0265]
  • Transcripts of the size of the resulting human PNS SCP are then confirmed to be present in human PNS mRNA or cDNA (encoding a 1970-1990 amino acid sequence of FIG. 11A-E). However, as in Example 1, such transcripts are not expected to be detected in mRNA from brain. This expected result confirms new human members of the sodium channel gene family (termed Human Peripheral Nerve type 1 (HUMPN1A (deposited as ATCC No. ______) and HUMPN1B (Deposited as ATCC No. ______) of FIG. 11A-E, where X is 0, 1, 2 or 3 of the same or different amino acid). [0266]
  • Complete DNA and amino acid sequences of novel human PN1s are then confirmed and are expected to contain all of the structural and functional domain characteristics of an α subunit of a mammalian voltage-gated sodium channel. [0267]
  • All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology f the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. [0268]
  • 1 19 3033 base pairs nucleic acid both both DNA (genomic) CDS 1..3033 1 AGG AAC CTT GTG GTC CTG AAC CTG TTT CTG GCT CTT TTG CTG AGT TCC 48 Arg Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser 1 5 10 15 TTT AGT TCT GAC AAT CTT ACA GCA ATT GAG GAA GAC ACC GAT GCA AAC 96 Phe Ser Ser Asp Asn Leu Thr Ala Ile Glu Glu Asp Thr Asp Ala Asn 20 25 30 AAC CTC CAG ATC GCA GTG GCC AGA ATT AAG AGG GGA ATC AAT TAC GTG 144 Asn Leu Gln Ile Ala Val Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val 35 40 45 AAA CAG ACC CTG CGT GAA TTC ATT CTA AAA TCA TTT TCC AAA AAG CCA 192 Lys Gln Thr Leu Arg Glu Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro 50 55 60 AAG GGC TCC AAG GAC ACA AAA CGA ACA GCA GAT CCC AAC AAC AAG AAA 240 Lys Gly Ser Lys Asp Thr Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys 65 70 75 80 GAA AAC TAT ATT TCA AAC CGT ACC CTT GCG GAG ATG AGC AAG GAT CAC 288 Glu Asn Tyr Ile Ser Asn Arg Thr Leu Ala Glu Met Ser Lys Asp His 85 90 95 AAT TTC CTC AAA GAA AAG GAT AGG ATC AGT GGT TAT GGC AGC AGT CTA 336 Asn Phe Leu Lys Glu Lys Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu 100 105 110 GAC AAA AGC TTT ATG GAT GAA AAT GAT TAC CAG TCC TTT ATC CAT AAC 384 Asp Lys Ser Phe Met Asp Glu Asn Asp Tyr Gln Ser Phe Ile His Asn 115 120 125 CCC AGC CTC ACA GTG ACA GTG CCA ATT GCA CCT GGG GAG TCT GAT TTG 432 Pro Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu 130 135 140 GAG ATT ATG AAC ACA GAA GAG CTT AGC AGT GAC TCA GAC AGT GAC TAC 480 Glu Ile Met Asn Thr Glu Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr 145 150 155 160 AGC AAA GAG AAA CGG AAC CGA TCA AGC TCT TCT GAG TGC AGC ACT GTT 528 Ser Lys Glu Lys Arg Asn Arg Ser Ser Ser Ser Glu Cys Ser Thr Val 165 170 175 GAC AAC CCT CTG CCA GGA GAA GAG GAG GCT GAA GCA GAG CCC GTA AAC 576 Asp Asn Pro Leu Pro Gly Glu Glu Glu Ala Glu Ala Glu Pro Val Asn 180 185 190 GCA GAT GAG CCT GAA GCC TGC TTT ACA GAT GGT TGT GTG AGG AGA TTT 624 Ala Asp Glu Pro Glu Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe 195 200 205 CCA TGC TGC CAA GTT AAT GTA GAC TCT GGG AAA GGG AAA GTT TGG TGG 672 Pro Cys Cys Gln Val Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp 210 215 220 ACC ATC AGG AAG ACG TGC TAC AGG ATA GTT GAA CAC AGC TGG TTT GAA 720 Thr Ile Arg Lys Thr Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu 225 230 235 240 AGC TTC ATC GTT CTC ATG ATC CTG CTC AGC AGT GGA GCT CTG GCT TTT 768 Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe 245 250 255 GAA GAT ATC TAT ATT GAA AAG AAA AAG ACC ATT AAG ATT ATC CTG GAG 816 Glu Asp Ile Tyr Ile Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu 260 265 270 TAT GCT GAC AAG ATA TTC ACC TAC ATC TTC ATT CTG GAA ATG CTT CTA 864 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 275 280 285 AAA TGG GTC GCA TAT GGG TAT AAA ACA TAT TTC ACT AAT GCC TGG TGT 912 Lys Trp Val Ala Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys 290 295 300 TGG CTG GAC TTC TTA ATT GTT GAT GTG TCT CTA GTT ACT TTA GTA GCC 960 Trp Leu Asp Phe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala 305 310 315 320 AAC ACT CTT GGC TAC TCA GAC CTT GGC CCC ATT AAA TCT CTA CGG ACA 1008 Asn Thr Leu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr 325 330 335 CTG AGG GCC CTA AGA CCC CTA AGA GCC TTG TCT AGA TTT GAA GGA ATG 1056 Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met 340 345 350 AGG GTA GTG GTC AAC GCA CTC ATA GGA GCA ATC CCT TCC ATC ATG AAC 1104 Arg Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn 355 360 365 GTG CTT CTC GTG TGC CTT ATA TTC TGG CTA ATA TTT AGC ATC ATG GGA 1152 Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly 370 375 380 GTC AAT CTG TTT GCT GGC AAG TTC TAT GAG TGT GTC AAC ACC ACC GAT 1200 Val Asn Leu Phe Ala Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp 385 390 395 400 GGG TCA CGA TTT CCT ACA TCT CAA GTT GCA AAC CGT TCT GAG TGT TTT 1248 Gly Ser Arg Phe Pro Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe 405 410 415 GCC CTG ATG AAC GTT AGT GGA AAT GTG CGA TGG AAA AAC CTG AAA GTA 1296 Ala Leu Met Asn Val Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val 420 425 430 AAC TTC GAC AAC GTT GGG CTT GGT TAC CTG TCG CTG CTT CAA GTT GCA 1344 Asn Phe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 435 440 445 ACA TTC AAG GGC TGG ATG GAT ATT ATG TAT GCA GCA GTT GAC TCT GTT 1392 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val 450 455 460 AAT GTA AAT GAA CAG CCG AAA TAC GAA TAC AGT CTC TAC ATG TAC ATT 1440 Asn Val Asn Glu Gln Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile 465 470 475 480 TAC TTT GTC ATC TTC ATC ATC TTC GGC TCA TTC TTC ACG TTG AAC CTG 1488 Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 485 490 495 TTC ATT GGT GTC ATC ATA GAT AAT TTC AAC CAA CAG AAA AAA AAG CTT 1536 Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu 500 505 510 GGA GGT CAA GAT ATC TTT ATG ACA GAA GAA CAG AAG AAA TAC TAT AAT 1584 Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 515 520 525 GCA ATG AAG AAG CTT GGG TCC AAA AAA CCA CAA AAA CCA ATT CCA AGG 1632 Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg 530 535 540 CCA GGG AAC AAA TTC CAA GGA TGT ATA TTT GAC TTA GTG ACA AAC CAA 1680 Pro Gly Asn Lys Phe Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln 545 550 555 560 GCT TTT GAT ATC ACC ATC ATG GTT CTT ATA TGC CTC AAC ATG GTA ACC 1728 Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu Asn Met Val Thr 565 570 575 ATG ATG GTA GAA AAA GAG GGG CAA ACT GAG TAC ATG GAT TAT GTT TTA 1776 Met Met Val Glu Lys Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu 580 585 590 CAC TGG ATC AAC ATG GTC TTC ATT ATC CTG TTC ACT GGG GAG TGT GTG 1824 His Trp Ile Asn Met Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val 595 600 605 CTG AAG CTA ATC TCC CTC AGA CAT TAC TAC TTC ACT GTG GGT TGG AAC 1872 Leu Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn 610 615 620 ATT TTG TAT TTT GTG GTA GTG ATC CTC TCC ATT GTA GGA ATG TTT CTC 1920 Ile Leu Tyr Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 625 630 635 640 GCT GAG ATG ATA GAG AAG TAT TTC GTG TCC CCT ACC CTG TTC CGA GTC 1968 Ala Glu Met Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val 645 650 655 ATC CGC CTG GCC AGG ATT GGA CGA ATC CTA CGC CTG ATC AAA GGC GCC 2016 Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 660 665 670 AAG GGG ATC CGC ACT CTG CTC TTT GCT TTG ATG ATG TCC CTT CCT GCG 2064 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 675 680 685 CTG TTC AAC ATC GGC CTC CTG CTT TTC CTG GTC ATG TTC ATC TAC GCC 2112 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala 690 695 700 ATC TTT GGG ATG TCC AAC TTT GCC TAC GTT AAA AAG GAG GCT GGA ATT 2160 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile 705 710 715 720 AAT GAC ATG TTC AAC TTT GAG ACT TTT GGC AAC AGC ATG ATC TGC TTG 2208 Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu 725 730 735 TTC CAA ATC ACC ACC TCT GCC GGC TGG GAC GGA CTG CTG GCC CCC ATC 2256 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile 740 745 750 CTC AAC AGC GCA CCT CCC GAC TGT GAC CCT AAA AAA GTT CAC CCA GGA 2304 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro Lys Lys Val His Pro Gly 755 760 765 AGT TCA GTG GAA GGG GAC TGT GGG AAC CCA TCC GTG GGG ATT TTT TAC 2352 Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Tyr 770 775 780 TTT GTC AGC TAC ATC ATC ATA TCC TTC CTG GTG GTG GTG AAC ATG TAC 2400 Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr 785 790 795 800 ATC GCT GTC ATC CTG GAG AAC TTC AGC GTC GCC ACC GAA GAG AGC ACT 2448 Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Thr 805 810 815 GAG CCT CTG AGT GAG GAC GAC TTT GAG ATG TTC TAC GAG GTC TGG GAG 2496 Glu Pro Leu Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu 820 825 830 AAG TTC GAC CCT GAC GCC ACT CAG TTC ATA GAG TTC TGC AAG CTC TCT 2544 Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser 835 840 845 GAC TTT GCA GCT GCC CTG GAT CCT CCC CTC CTC ATC GCA AAG CCA AAC 2592 Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn 850 855 860 AAA GTC CAG CTC ATT GCC ATG GAC CTG CCC ATG GTG AGT GGA GAC CGC 2640 Lys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg 865 870 875 880 ATC CAC TGC CTG GAC ATC TTG TTT GCT TTT ACA AAG CGG GTC CTG GGT 2688 Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly 885 890 895 GAG GGT GGA GAG ATG GAT TCT CTT CGT TCA CAG ATG GAA GAA AGG TTC 2736 Glu Gly Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe 900 905 910 ATG TCA GCC AAT CCT TCT AAA GTG TCC TAT GAA CCC ATC ACG ACC ACA 2784 Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr 915 920 925 CTG AAG AGA AAA CAA GAG GAG GTG TCC GCG ACT ATC ATT CAG CGT GCT 2832 Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala 930 935 940 TAC AGA CGG TAT CGC CTC AGA CAA CAC GTC AAG AAT ATA TCG AGT ATA 2880 Tyr Arg Arg Tyr Arg Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile 945 950 955 960 TAC ATA AAA GAT GGA GAC AGG GAT GAT GAT TTG CCC AAT AAA GAA GAT 2928 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp 965 970 975 ACA GTT TTT GAT AAC GTG AAC GAG AAC TCA AGT CCG GAA AAG ACA GAT 2976 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp 980 985 990 GTA ACT GCC TCA ACC ATC TCG CCA CCT TCC TAT GAC AGT GTC ACA AAG 3024 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys 995 1000 1005 CCA GAT CAA 3033 Pro Asp Gln 1010 1011 amino acids amino acid linear protein 2 Arg Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser 1 5 10 15 Phe Ser Ser Asp Asn Leu Thr Ala Ile Glu Glu Asp Thr Asp Ala Asn 20 25 30 Asn Leu Gln Ile Ala Val Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val 35 40 45 Lys Gln Thr Leu Arg Glu Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro 50 55 60 Lys Gly Ser Lys Asp Thr Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys 65 70 75 80 Glu Asn Tyr Ile Ser Asn Arg Thr Leu Ala Glu Met Ser Lys Asp His 85 90 95 Asn Phe Leu Lys Glu Lys Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu 100 105 110 Asp Lys Ser Phe Met Asp Glu Asn Asp Tyr Gln Ser Phe Ile His Asn 115 120 125 Pro Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu 130 135 140 Glu Ile Met Asn Thr Glu Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr 145 150 155 160 Ser Lys Glu Lys Arg Asn Arg Ser Ser Ser Ser Glu Cys Ser Thr Val 165 170 175 Asp Asn Pro Leu Pro Gly Glu Glu Glu Ala Glu Ala Glu Pro Val Asn 180 185 190 Ala Asp Glu Pro Glu Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe 195 200 205 Pro Cys Cys Gln Val Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp 210 215 220 Thr Ile Arg Lys Thr Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu 225 230 235 240 Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe 245 250 255 Glu Asp Ile Tyr Ile Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu 260 265 270 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 275 280 285 Lys Trp Val Ala Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys 290 295 300 Trp Leu Asp Phe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala 305 310 315 320 Asn Thr Leu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr 325 330 335 Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met 340 345 350 Arg Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn 355 360 365 Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly 370 375 380 Val Asn Leu Phe Ala Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp 385 390 395 400 Gly Ser Arg Phe Pro Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe 405 410 415 Ala Leu Met Asn Val Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val 420 425 430 Asn Phe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 435 440 445 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val 450 455 460 Asn Val Asn Glu Gln Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile 465 470 475 480 Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 485 490 495 Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu 500 505 510 Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 515 520 525 Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg 530 535 540 Pro Gly Asn Lys Phe Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln 545 550 555 560 Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu Asn Met Val Thr 565 570 575 Met Met Val Glu Lys Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu 580 585 590 His Trp Ile Asn Met Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val 595 600 605 Leu Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn 610 615 620 Ile Leu Tyr Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 625 630 635 640 Ala Glu Met Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val 645 650 655 Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 660 665 670 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 675 680 685 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala 690 695 700 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile 705 710 715 720 Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu 725 730 735 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile 740 745 750 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro Lys Lys Val His Pro Gly 755 760 765 Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Tyr 770 775 780 Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr 785 790 795 800 Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Thr 805 810 815 Glu Pro Leu Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu 820 825 830 Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser 835 840 845 Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn 850 855 860 Lys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg 865 870 875 880 Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly 885 890 895 Glu Gly Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe 900 905 910 Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr 915 920 925 Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala 930 935 940 Tyr Arg Arg Tyr Arg Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile 945 950 955 960 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp 965 970 975 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp 980 985 990 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys 995 1000 1005 Pro Asp Gln 1010 29 base pairs nucleic acid single linear cDNA misc_feature 12 /note= “Base is Inosine” misc_feature 15 /note= “Base is Inosine” misc_feature 19 /note= “Base is Inosine” misc_feature 21 /note= “Base is Inosine” 3 GCGAAGCTTY TNATNTTYNN NATHATGGG 29 8 amino acids amino acid single linear peptide 4 Phe Trp Leu Ile Phe Ser Ile Met 1 5 34 base pairs nucleic acid single linear cDNA 5 GCAGGATCCR TTRAAARTTR TCDATDATNA CNCC 34 8 amino acids amino acid single linear peptide 6 Gly Val Ile Ile Asp Asn Phe Asn 1 5 2005 amino acids amino acid single linear peptide 7 Met Ala Arg Ser Val Leu Val Pro Pro Gly Pro Asp Ser Phe Arg Phe 1 5 10 15 Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Gln Arg Ile Ala Glu Glu 20 25 30 Lys Ala Lys Arg Pro Lys Gln Glu Arg Lys Asp Glu Asp Asp Glu Asn 35 40 45 Gly Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Ser Leu Pro Phe 50 55 60 Ile Tyr Gly Asp Ile Pro Pro Glu Met Val Ser Glu Pro Leu Glu Asp 65 70 75 80 Leu Asp Pro Tyr Tyr Ile Asn Lys Lys Thr Phe Ile Val Leu Asn Lys 85 90 95 Gly Lys Ala Ile Ser Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu 100 105 110 Thr Pro Phe Asn Pro Ile Arg Lys Leu Ala Ile Lys Ile Leu Val His 115 120 125 Ser Leu Phe Asn Val Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Val 130 135 140 Phe Met Thr Met Ser Asn Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr 145 150 155 160 Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Leu Ala 165 170 175 Arg Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asn Pro Trp Asn 180 185 190 Trp Leu Asp Phe Thr Val Ile Thr Phe Ala Tyr Val Thr Glu Phe Val 195 200 205 Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala 210 215 220 Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala 225 230 235 240 Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val 245 250 255 Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly 260 265 270 Asn Leu Arg Asn Lys Cys Leu Gln Trp Pro Pro Asp Asn Ser Thr Phe 275 280 285 Glu Ile Asn Ile Thr Ser Phe Phe Asn Asn Ser Leu Asp Trp Asn Gly 290 295 300 Thr Ala Phe Asn Arg Thr Val Asn Met Phe Asn Trp Asp Glu Tyr Ile 305 310 315 320 Glu Asp Lys Ser His Phe Tyr Phe Leu Glu Gly Gln Asn Asp Ala Leu 325 330 335 Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly Tyr Ile 340 345 350 Cys Val Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp 355 360 365 Thr Phe Ser Trp Ala Phe Leu Ser Leu Phe Arg Leu Met Thr Gln Asp 370 375 380 Phe Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr 385 390 395 400 Tyr Met Ile Phe Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu 405 410 415 Ile Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu Gln Asn 420 425 430 Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe Gln Gln 435 440 445 Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln Ala Ala Ala 450 455 460 Ala Ala Ala Ser Ala Glu Ser Arg Asp Phe Ser Gly Ala Gly Gly Ile 465 470 475 480 Gly Val Phe Ser Glu Ser Ser Ser Val Ala Ser Lys Leu Ser Ser Lys 485 490 495 Ser Glu Lys Glu Leu Lys Asn Arg Arg Lys Lys Lys Lys Gln Lys Glu 500 505 510 Gln Ala Gly Glu Glu Glu Lys Glu Asp Ala Val Arg Lys Ser Ala Ser 515 520 525 Glu Asp Ser Ile Arg Lys Lys Gly Phe Gln Phe Ser Leu Glu Gly Ser 530 535 540 Arg Leu Thr Tyr Glu Lys Arg Phe Ser Ser Pro His Gln Ser Leu Leu 545 550 555 560 Ser Ile Arg Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser Arg Ala Ser 565 570 575 Leu Phe Asn Phe Lys Gly Arg Val Lys Asp Ile Gly Ser Glu Asn Asp 580 585 590 Phe Ala Asp Asp Glu His Ser Thr Phe Glu Asp Asn Asp Ser Arg Arg 595 600 605 Asp Ser Leu Phe Val Pro His Arg His Gly Glu Arg Arg Pro Ser Asn 610 615 620 Val Ser Gln Ala Ser Arg Ala Ser Arg Gly Ile Pro Thr Leu Pro Met 625 630 635 640 Asn Gly Lys Met His Ser Ala Val Asp Cys Asn Gly Val Val Ser Leu 645 650 655 Val Gly Gly Pro Ser Ala Leu Thr Ser Pro Val Gly Gln Leu Leu Pro 660 665 670 Glu Gly Thr Thr Thr Glu Thr Glu Ile Arg Lys Arg Arg Ser Ser Ser 675 680 685 Tyr His Val Ser Met Asp Leu Leu Glu Asp Pro Ser Arg Gln Arg Ala 690 695 700 Met Ser Met Ala Ser Ile Leu Thr Asn Thr Met Glu Glu Leu Glu Glu 705 710 715 720 Ser Arg Gln Lys Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn Met Cys 725 730 735 Leu Ile Trp Asp Cys Cys Lys Pro Trp Leu Lys Val Lys His Val Val 740 745 750 Asn Leu Val Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr Ile Cys 755 760 765 Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His Tyr Pro Met Thr 770 775 780 Glu Gln Phe Ser Ser Val Leu Ser Val Gly Asn Leu Val Phe Thr Gly 785 790 795 800 Ile Phe Thr Ala Glu Met Phe Leu Lys Ile Ile Ala Met Asp Pro Tyr 805 810 815 Tyr Tyr Phe Gln Glu Gly Trp Asn Ile Phe Asp Gly Phe Ile Val Ser 820 825 830 Leu Ser Leu Met Glu Leu Gly Leu Ala Asn Val Glu Gly Leu Ser Val 835 840 845 Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser Trp 850 855 860 Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly Ala 865 870 875 880 Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe Ala 885 890 895 Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys Val Cys 900 905 910 Lys Ile Ser Asn Asp Cys Glu Leu Pro Arg Trp His Met His His Phe 915 920 925 Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu Trp Ile 930 935 940 Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Thr Met Cys Leu 945 950 955 960 Thr Val Phe Met Met Val Met Val Ile Gly Asn Leu Val Val Leu Asn 965 970 975 Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu Ala 980 985 990 Ala Thr Asp Asp Asp Asn Glu Met Asn Asn Leu Gln Ile Ala Val Gly 995 1000 1005 Arg Met Gln Lys Gly Ile Asp Phe Val Lys Arg Lys Ile Arg Glu Phe 1010 1015 1020 Ile Gln Lys Ala Phe Val Arg Lys Gln Lys Ala Leu Asp Glu Ile Lys 1025 1030 1035 1040 Pro Leu Glu Asp Leu Asn Asn Lys Lys Asp Ser Cys Ile Ser Asn His 1045 1050 1055 Thr Thr Ile Glu Ile Gly Lys Asp Leu Asn Tyr Leu Lys Asp Gly Asn 1060 1065 1070 Gly Thr Thr Ser Gly Ile Gly Ser Ser Val Glu Lys Tyr Val Val Asp 1075 1080 1085 Glu Ser Asp Tyr Met Ser Phe Ile Asn Asn Pro Ser Leu Thr Val Thr 1090 1095 1100 Val Pro Ile Ala Leu Gly Glu Ser Asp Phe Glu Asn Leu Asn Thr Glu 1105 1110 1115 1120 Glu Phe Ser Ser Glu Ser Asp Met Glu Glu Ser Lys Glu Lys Leu Asn 1125 1130 1135 Ala Thr Ser Ser Ser Glu Gly Ser Thr Val Asp Ile Gly Ala Pro Ala 1140 1145 1150 Glu Gly Glu Gln Pro Glu Ala Glu Pro Glu Glu Ser Leu Glu Pro Glu 1155 1160 1165 Ala Cys Phe Thr Glu Asp Cys Val Arg Lys Phe Lys Cys Cys Gln Ile 1170 1175 1180 Ser Ile Glu Glu Gly Lys Gly Lys Leu Trp Trp Asn Leu Arg Lys Thr 1185 1190 1195 1200 Cys Tyr Lys Ile Val Glu His Asn Trp Phe Glu Ile Phe Ile Val Phe 1205 1210 1215 Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile 1220 1225 1230 Glu Gln Arg Lys Thr Ile Lys Thr Met Leu Glu Tyr Ala Asp Lys Val 1235 1240 1245 Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr 1250 1255 1260 Gly Phe Gln Met Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1265 1270 1275 1280 Ile Val Asp Val Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr 1285 1290 1295 Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg 1300 1305 1310 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn 1315 1320 1325 Ala Leu Leu Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys 1330 1335 1340 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala 1345 1350 1355 1360 Gly Lys Phe Tyr His Cys Ile Asn Tyr Thr Ile Gly Glu Met Phe Asp 1365 1370 1375 Val Ser Val Val Asn Asn Tyr Ser Glu Cys Gln Ala Leu Ile Glu Ser 1380 1385 1390 Asn Gln Thr Ala Arg Trp Lys Asn Val Lys Val Asn Phe Asp Asn Val 1395 1400 1405 Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1410 1415 1420 Met Asp Ile Met Tyr Ala Ala Val Asp Ser Arg Asn Val Glu Leu Gln 1425 1430 1435 1440 Pro Lys Tyr Glu Asp Asn Leu Tyr Met Tyr Leu Tyr Phe Val Ile Phe 1445 1450 1455 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile 1460 1465 1470 Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile 1475 1480 1485 Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu 1490 1495 1500 Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Ala Asn Lys Phe 1505 1510 1515 1520 Gln Gly Met Val Phe Asp Phe Val Thr Lys Gln Val Phe Asp Ile Ser 1525 1530 1535 Ile Met Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr 1540 1545 1550 Asp Asp Gln Ser Gln Glu Met Thr Asn Ile Leu Tyr Trp Ile Asn Leu 1555 1560 1565 Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser 1570 1575 1580 Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp Phe Val 1585 1590 1595 1600 Val Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Glu Leu Ile Glu 1605 1610 1615 Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg 1620 1625 1630 Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr 1635 1640 1645 Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1650 1655 1660 Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 1665 1670 1675 1680 Asn Phe Ala Tyr Val Lys Arg Glu Val Gly Ile Asp Asp Met Phe Asn 1685 1690 1695 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr 1700 1705 1710 Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Gly Pro 1715 1720 1725 Pro Asp Cys Asp Pro Glu Lys Asp His Pro Gly Ser Ser Val Lys Gly 1730 1735 1740 Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Phe Val Ser Tyr Ile 1745 1750 1755 1760 Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu 1765 1770 1775 Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu 1780 1785 1790 Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 1795 1800 1805 Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala Ala Ala 1810 1815 1820 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile 1825 1830 1835 1840 Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp 1845 1850 1855 Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly Glu Ser Gly Glu Met 1860 1865 1870 Asp Ala Leu Arg Ile Gln Met Glu Glu Arg Phe Met Ala Ser Asn Pro 1875 1880 1885 Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1890 1895 1900 Glu Glu Val Ser Ala Ile Val Ile Gln Arg Ala Tyr Arg Arg Tyr Leu 1905 1910 1915 1920 Leu Lys Gln Lys Val Lys Lys Val Ser Ser Ile Tyr Lys Lys Asp Lys 1925 1930 1935 Gly Lys Glu Asp Glu Gly Thr Pro Ile Lys Glu Asp Ile Ile Thr Asp 1940 1945 1950 Lys Leu Asn Glu Asn Ser Thr Pro Glu Lys Thr Asp Val Thr Pro Ser 1955 1960 1965 Thr Thr Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys Pro Glu Lys Glu 1970 1975 1980 Lys Phe Glu Lys Asp Lys Ser Glu Lys Glu Asp Lys Gly Lys Asp Ile 1985 1990 1995 2000 Arg Glu Ser Lys Lys 2005 813 amino acids amino acid single linear peptide 8 Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 1 5 10 15 Ser Ser Asp Asn Leu Ala Asp Asn Asn Leu Gln Ile Ala Val Arg Gly 20 25 30 Ile Val Lys Arg Glu Phe Ile Lys Phe Lys Lys Lys Asp Asn Asn Lys 35 40 45 Lys Ile Ser Asn Thr Glu Lys Asp Asn Leu Lys Ser Gly Gly Ser Ser 50 55 60 Lys Asp Glu Asp Tyr Ser Phe Ile Asn Pro Ser Leu Thr Val Thr Val 65 70 75 80 Pro Ile Ala Gly Glu Ser Asp Glu Asn Thr Glu Glu Ser Ser Ser Asp 85 90 95 Ser Lys Glu Lys Asn Ser Ser Ser Glu Ser Thr Val Asp Pro Glu Glu 100 105 110 Glu Ala Glu Pro Glu Pro Glu Ala Cys Phe Thr Cys Val Arg Phe Cys 115 120 125 Cys Gln Gly Lys Gly Lys Trp Trp Arg Lys Thr Cys Tyr Ile Val Glu 130 135 140 His Trp Phe Glu Phe Ile Val Met Ile Leu Leu Ser Ser Gly Ala Leu 145 150 155 160 Ala Phe Glu Asp Ile Tyr Ile Glu Lys Thr Ile Lys Leu Glu Tyr Ala 165 170 175 Asp Lys Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val 180 185 190 Ala Tyr Gly Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile 195 200 205 Val Asp Val Ser Leu Val Leu Ala Asn Leu Gly Tyr Ser Leu Gly Ile 210 215 220 Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser 225 230 235 240 Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu Gly Ala Ile Pro 245 250 255 Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe 260 265 270 Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys Phe Tyr Cys Asn Thr 275 280 285 Gly Phe Ser Val Asn Ser Glu Cys Ala Leu Arg Trp Lys Asn Lys Val 290 295 300 Asn Phe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 305 310 315 320 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Asn 325 330 335 Val Gln Pro Lys Tyr Glu Leu Tyr Met Tyr Tyr Phe Val Ile Phe Ile 340 345 350 Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile 355 360 365 Asp Asn Phe Asn Gln Gln Lys Lys Lys Gly Gly Gln Asp Ile Phe Met 370 375 380 Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser 385 390 395 400 Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Asn Lys Phe Gln Gly Phe 405 410 415 Asp Val Thr Gln Phe Asp Ile Ile Met Leu Ile Cys Leu Asn Met Val 420 425 430 Thr Met Met Val Glu Gln Met Leu Trp Ile Asn Val Phe Ile Leu Phe 435 440 445 Thr Gly Glu Cys Val Leu Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe 450 455 460 Thr Gly Trp Asn Ile Phe Val Val Val Ile Leu Ser Ile Val Gly Met 465 470 475 480 Phe Leu Ala Glu Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg 485 490 495 Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly 500 505 510 Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro 515 520 525 Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr 530 535 540 Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Glu Gly Ile Asp 545 550 555 560 Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln 565 570 575 Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn 580 585 590 Ser Pro Pro Asp Cys Asp Pro Lys His Pro Gly Ser Ser Val Gly Asp 595 600 605 Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Val Ser Tyr Ile Ile Ile 610 615 620 Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 625 630 635 640 Phe Ser Val Ala Thr Glu Glu Ser Glu Pro Leu Ser Glu Asp Asp Phe 645 650 655 Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp Ala Thr Gln 660 665 670 Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro 675 680 685 Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile Ala Met Asp 690 695 700 Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp Ile Leu Phe 705 710 715 720 Ala Phe Thr Lys Arg Val Leu Gly Glu Gly Glu Met Asp Leu Arg Gln 725 730 735 Met Glu Glu Arg Phe Met Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile 740 745 750 Thr Thr Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Ile Gln Arg 755 760 765 Ala Tyr Arg Arg Tyr Leu Gln Val Lys Ser Ser Ile Tyr Lys Asp Asp 770 775 780 Pro Lys Glu Asp Asp Asn Glu Asn Ser Pro Glu Lys Thr Asp Val Thr 785 790 795 800 Ser Thr Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys Pro 805 810 6452 base pairs nucleic acid double both DNA (genomic) CDS 326..6277 9 GTCGCCTCAT CCTGAGCAGA CTGGAAACAG ACTCCGTGCA GGCCTCGCCC GCGCTCCAGT 60 TGCGACTGTA GGGTTTTCAT TCCTGCCCAC TGCGCAGACT GGGCTGAGCT AGCCTGGGTA 120 TCCACGATTC GCGACTCGTA GTAACAGGCA CTCTGAGCAA CAGGATTTCA GAGAAAGAAG 180 CAGAGGCAAG AAAGAAGCCT GGGGAGAGAG GAAGACTTTC CTTGGATCAG ACTCCGCAGG 240 TGCACACACC GGGTGGGCAT GATCCGTGGG GCCAGGCCTC TTAGGTAAGG AGTCAAAGGG 300 GAAATAAAAC ATACAGGATG AAAAG ATG GCG ATG CTG CCT CCT CCA GGA CCT 352 Met Ala Met Leu Pro Pro Pro Gly Pro 1015 1020 CAG AGT TTC GTT CAC TTC ACA AAA CAG TCC CTT GCC CTC ATT GAA CAG 400 Gln Ser Phe Val His Phe Thr Lys Gln Ser Leu Ala Leu Ile Glu Gln 1025 1030 1035 CGT ATT TCT GAA GAA AAA GCC AAG GAA CAC AAA GAC GAA AAG AAA GAT 448 Arg Ile Ser Glu Glu Lys Ala Lys Glu His Lys Asp Glu Lys Lys Asp 1040 1045 1050 GAT GAG GAA GAA GGC CCC AAG CCC AGC AGT GAC TTG GAA GCT GGG AAA 496 Asp Glu Glu Glu Gly Pro Lys Pro Ser Ser Asp Leu Glu Ala Gly Lys 1055 1060 1065 CAG CTC CCC TTC ATC TAT GGA GAC ATT CCC CCT GGA ATG GTG TCA GAG 544 Gln Leu Pro Phe Ile Tyr Gly Asp Ile Pro Pro Gly Met Val Ser Glu 1070 1075 1080 CCC CTG GAG GAC CTG GAC CCA TAC TAT GCT GAC AAA AAA ACT TTT ATA 592 Pro Leu Glu Asp Leu Asp Pro Tyr Tyr Ala Asp Lys Lys Thr Phe Ile 1085 1090 1095 1100 GTA TTG AAC AAA GGG AAA GCA ATC TTC CGT TTC AAC GCC ACC CCT GCT 640 Val Leu Asn Lys Gly Lys Ala Ile Phe Arg Phe Asn Ala Thr Pro Ala 1105 1110 1115 TTG TAC ATG CTG TCT CCC TTC AGT CCT CTA AGA AGA ATA TCT ATT AAG 688 Leu Tyr Met Leu Ser Pro Phe Ser Pro Leu Arg Arg Ile Ser Ile Lys 1120 1125 1130 ATC TTA GTG CAC TCC TTA TTC AGC ATG CTA ATC ATG TGC ACA ATT CTG 736 Ile Leu Val His Ser Leu Phe Ser Met Leu Ile Met Cys Thr Ile Leu 1135 1140 1145 ACG AAC TGC ATA TTC ATG ACC TTG AGC AAC CCT CCA GAA TGG ACC AAA 784 Thr Asn Cys Ile Phe Met Thr Leu Ser Asn Pro Pro Glu Trp Thr Lys 1150 1155 1160 AAT GTA GAG TAC ACT TTT ACT GGG ATA TAT ACT TTT GAA TCA CTC ATA 832 Asn Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile 1165 1170 1175 1180 AAA ATC CTT GCA AGA GGC TTT TGC GTG GGA GAA TTC ACC TTC CTC CGT 880 Lys Ile Leu Ala Arg Gly Phe Cys Val Gly Glu Phe Thr Phe Leu Arg 1185 1190 1195 GAC CCT TGG AAC TGG CTG GAC TTT GTT GTC ATT GTT TTT GCG TAT TTA 928 Asp Pro Trp Asn Trp Leu Asp Phe Val Val Ile Val Phe Ala Tyr Leu 1200 1205 1210 ACA GAA TTT GTA AAC CTA GGC AAT GTT TCA GCT CTT CGA ACT TTC AGA 976 Thr Glu Phe Val Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg 1215 1220 1225 GTC TTG AGA GCT TTG AAA ACT ATT TCT GTA ATC CCA GGA CTA AAG ACC 1024 Val Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr 1230 1235 1240 ATC GTG GGG GCC CTG ATC CAG TCA GTG AAG AAG CTC TCT GAC GTC ATG 1072 Ile Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met 1245 1250 1255 1260 ATC CTC ACT GTG TTC TGT CTC AGT GTG TTT GCA CTA ATT GGA CTA CAG 1120 Ile Leu Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln 1265 1270 1275 CTG TTT ATG GGC AAC TTG AAG CAT AAA TGT TTC AGG AAG GAA CTC GAA 1168 Leu Phe Met Gly Asn Leu Lys His Lys Cys Phe Arg Lys Glu Leu Glu 1280 1285 1290 GAG AAT GAA ACA TTA GAA AGT ATC ATG AAT ACT GCT GAG AGT GAA GAA 1216 Glu Asn Glu Thr Leu Glu Ser Ile Met Asn Thr Ala Glu Ser Glu Glu 1295 1300 1305 GAA TTG AAA AAA TAT TTT TAT TAC TTG GAG GGA TCC AAA GAT GCT CTA 1264 Glu Leu Lys Lys Tyr Phe Tyr Tyr Leu Glu Gly Ser Lys Asp Ala Leu 1310 1315 1320 CTC TGC GGC TTC AGC ACA GAT TCA GGG CAG TGT CCA GAA GGC TAC ATC 1312 Leu Cys Gly Phe Ser Thr Asp Ser Gly Gln Cys Pro Glu Gly Tyr Ile 1325 1330 1335 1340 TGT GTG AAG GCT GGC AGA AAC CCG GAT TAT GGC TAC ACG AGC TTT GAC 1360 Cys Val Lys Ala Gly Arg Asn Pro Asp Tyr Gly Tyr Thr Ser Phe Asp 1345 1350 1355 ACA TTC AGC TGG GCC TTC TTG GCC TTG TTT CGG CTA ATG ACT CAG GAC 1408 Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met Thr Gln Asp 1360 1365 1370 TAC TGG GAG AAC CTT TAC CAA CAG ACT CTG CGT GCT GCT GGC AAA ACC 1456 Tyr Trp Glu Asn Leu Tyr Gln Gln Thr Leu Arg Ala Ala Gly Lys Thr 1375 1380 1385 TAC ATG ATT TTC TTT GTC GTG GTT ATT TTT CTG GGC TCC TTT TAC CTG 1504 Tyr Met Ile Phe Phe Val Val Val Ile Phe Leu Gly Ser Phe Tyr Leu 1390 1395 1400 ATA AAC TTG ATC CTG GCT GTG GTA GCC ATG GCG TAT GAG GAA CAG AAC 1552 Ile Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu Gln Asn 1405 1410 1415 1420 CAG GCC AAC ATC GAA GAA GCT AAA CAG AAA GAG TTA GAA TTT CAG CAG 1600 Gln Ala Asn Ile Glu Glu Ala Lys Gln Lys Glu Leu Glu Phe Gln Gln 1425 1430 1435 ATG TTA GAC CGA CTC AAA AAG GAG CAG GAA GAA GCT GAG GCG ATC GCT 1648 Met Leu Asp Arg Leu Lys Lys Glu Gln Glu Glu Ala Glu Ala Ile Ala 1440 1445 1450 GCA GCT GCT GCT GAG TTC ACG AGT ATA GGG CGG AGC AGG ATC ATG GGA 1696 Ala Ala Ala Ala Glu Phe Thr Ser Ile Gly Arg Ser Arg Ile Met Gly 1455 1460 1465 CTC TCT GAG AGC TCT TCA GAA ACC TCC AGG CTG AGC TCA AAG AGT GCC 1744 Leu Ser Glu Ser Ser Ser Glu Thr Ser Arg Leu Ser Ser Lys Ser Ala 1470 1475 1480 AAG GAG AGA AGA AAC CGA AGA AAG AAA AAG AAA CAG AAG ATG TCC AGT 1792 Lys Glu Arg Arg Asn Arg Arg Lys Lys Lys Lys Gln Lys Met Ser Ser 1485 1490 1495 1500 GGC GAG GAA AAG GGT GAC GAT GAG AAG CTG TCC AAG TCA GGA TCA GAG 1840 Gly Glu Glu Lys Gly Asp Asp Glu Lys Leu Ser Lys Ser Gly Ser Glu 1505 1510 1515 GAA AGC ATC CGA AAG AAA AGC TTC CAT CTC GGT GTG GAA GGG CAC CAC 1888 Glu Ser Ile Arg Lys Lys Ser Phe His Leu Gly Val Glu Gly His His 1520 1525 1530 CGG ACC CGG GAA AAG AGG CTG TCC ACC CCC AAC CAG TCG CCA CTC AGC 1936 Arg Thr Arg Glu Lys Arg Leu Ser Thr Pro Asn Gln Ser Pro Leu Ser 1535 1540 1545 ATT CGC GGG TCC CTG TTT TCT GCC AGG CGC AGC AGC AGG ACG AGT CTC 1984 Ile Arg Gly Ser Leu Phe Ser Ala Arg Arg Ser Ser Arg Thr Ser Leu 1550 1555 1560 TTC AGT TTT AAG GGG CGA GGA AGA GAT CTG GGA TCT GAG ACA GAA TTC 2032 Phe Ser Phe Lys Gly Arg Gly Arg Asp Leu Gly Ser Glu Thr Glu Phe 1565 1570 1575 1580 GCT GAT GAT GAG CAT AGC ATT TTT GGA GAC AAC GAG AGC AGA AGG GGT 2080 Ala Asp Asp Glu His Ser Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly 1585 1590 1595 TCA CTA TTC GTA CCC CAT AGA CCC CGG GAG CGG CGC AGC AGT AAC ATC 2128 Ser Leu Phe Val Pro His Arg Pro Arg Glu Arg Arg Ser Ser Asn Ile 1600 1605 1610 AGT CAG GCC AGT AGG TCC CCG CCA GTG CTA CCG GTG AAC GGG AAG ATG 2176 Ser Gln Ala Ser Arg Ser Pro Pro Val Leu Pro Val Asn Gly Lys Met 1615 1620 1625 CAC AGT GCA GTG GAC TGC AAT GGA GTC GTG TCG CTT GTT GAT GGA CCC 2224 His Ser Ala Val Asp Cys Asn Gly Val Val Ser Leu Val Asp Gly Pro 1630 1635 1640 TCA GCC CTC ATG CTC CCC AAT GGA CAG CTT CTT CCA GAG GTG ATA ATA 2272 Ser Ala Leu Met Leu Pro Asn Gly Gln Leu Leu Pro Glu Val Ile Ile 1645 1650 1655 1660 GAT AAG GCA ACT TCC GAC GAC AGC GGC ACG ACT AAT CAG ATG CGC AAA 2320 Asp Lys Ala Thr Ser Asp Asp Ser Gly Thr Thr Asn Gln Met Arg Lys 1665 1670 1675 AAA AGG CTC TCT AGT TCT TAC TTC TTG TCT GAG GAC ATG CTG AAT GAC 2368 Lys Arg Leu Ser Ser Ser Tyr Phe Leu Ser Glu Asp Met Leu Asn Asp 1680 1685 1690 CCG CAT CTC AGG CAA AGG GCC ATG AGC AGG GCG AGC ATA CTG ACC AAC 2416 Pro His Leu Arg Gln Arg Ala Met Ser Arg Ala Ser Ile Leu Thr Asn 1695 1700 1705 ACT GTG GAA GAA CTT GAA GAA TCT AGA CAA AAA TGT CCA CCA TGG TGG 2464 Thr Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro Pro Trp Trp 1710 1715 1720 TAC AGA TTT GCT CAC ACA TTT TTA ATC TGG AAT TGC TCT CCA TAT TGG 2512 Tyr Arg Phe Ala His Thr Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp 1725 1730 1735 1740 ATA AAA TTC AAA AAG CTC ATC TAT TTT ATT GTG ATG GAT CCT TTT GTA 2560 Ile Lys Phe Lys Lys Leu Ile Tyr Phe Ile Val Met Asp Pro Phe Val 1745 1750 1755 GAT CTT GCA ATT ACC ATT TGC ATA GTT TTA AAC ACC TTA TTT ATG GCT 2608 Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala 1760 1765 1770 ATG GAG CAC CAC CCA ATG ACT GAA GAA TTC AAA AAT GTC CTT GCA GTG 2656 Met Glu His His Pro Met Thr Glu Glu Phe Lys Asn Val Leu Ala Val 1775 1780 1785 GGG AAC TTG ATC TTT ACA GGG ATC TTC GCA GCT GAA ATG GTA CTG AAG 2704 Gly Asn Leu Ile Phe Thr Gly Ile Phe Ala Ala Glu Met Val Leu Lys 1790 1795 1800 TTA ATA GCC ATG GAC CCC TAT GAG TAT TTC CAA GTA GGG TGG AAT ATT 2752 Leu Ile Ala Met Asp Pro Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile 1805 1810 1815 1820 TTT GAC AGC CTA ATT GTG ACG CTG AGT TTG ATA GAG CTT TTC CTA GCA 2800 Phe Asp Ser Leu Ile Val Thr Leu Ser Leu Ile Glu Leu Phe Leu Ala 1825 1830 1835 GAT GTG GAA GGA TTA TCA GTT CTG CGG TCA TTC AGA TTG CTC CGA GTC 2848 Asp Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val 1840 1845 1850 TTC AAG TTG GCA AAG TCC TGG CCC ACA CTG AAC ATG CTC ATT AAG ATC 2896 Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile 1855 1860 1865 ATC GGC AAC TCG GTG GGC GCA CTG GGC AAC CTG ACC CTG GTG CTG GCC 2944 Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala 1870 1875 1880 ATC ATC GTC TTC ATT TTT GCC GTG GTC GGC ATG CAG CTG TTT GGA AAG 2992 Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys 1885 1890 1895 1900 AGC TAC AAG GAG TGT GTC TGC AAG ATC AAT GTG GAC TGC AAG CTG CCG 3040 Ser Tyr Lys Glu Cys Val Cys Lys Ile Asn Val Asp Cys Lys Leu Pro 1905 1910 1915 CGC TGG CAC ATG AAC GAC TTC TTC CAC TCC TTC CTC ATC GTG TTC CGA 3088 Arg Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg 1920 1925 1930 GTG CTG TGT GGG GAG TGG ATA GAG ACC ATG TGG GAC TGC ATG GAG GTC 3136 Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val 1935 1940 1945 GCG GGC CAG ACC ATG TGC CTT ATT GTT TAC ATG ATG GTC ATG GTG ATT 3184 Ala Gly Gln Thr Met Cys Leu Ile Val Tyr Met Met Val Met Val Ile 1950 1955 1960 GGG AAC CTT GTG GTC CTG AAC CTG TTT CTG GCT CTT TTG CTG AGT TCC 3232 Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser 1965 1970 1975 1980 TTT AGT TCT GAC AAT CTT ACA GCA ATT GAG GAA GAC ACC GAT GCA AAC 3280 Phe Ser Ser Asp Asn Leu Thr Ala Ile Glu Glu Asp Thr Asp Ala Asn 1985 1990 1995 AAC CTC CAG ATC GCA GTG GCC AGA ATT AAG AGG GGA ATC AAT TAC GTG 3328 Asn Leu Gln Ile Ala Val Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val 2000 2005 2010 AAA CAG ACC CTG CGT GAA TTC ATT CTA AAA TCA TTT TCC AAA AAG CCA 3376 Lys Gln Thr Leu Arg Glu Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro 2015 2020 2025 AAG GGC TCC AAG GAC ACA AAA CGA ACA GCA GAT CCC AAC AAC AAG AAA 3424 Lys Gly Ser Lys Asp Thr Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys 2030 2035 2040 GAA AAC TAT ATT TCA AAC CGT ACC CTT GCG GAG ATG AGC AAG GAT CAC 3472 Glu Asn Tyr Ile Ser Asn Arg Thr Leu Ala Glu Met Ser Lys Asp His 2045 2050 2055 2060 AAT TTC CTC AAA GAA AAG GAT AGG ATC AGT GGT TAT GGC AGC AGT CTA 3520 Asn Phe Leu Lys Glu Lys Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu 2065 2070 2075 GAC AAA AGC TTT ATG GAT GAA AAT GAT TAC CAG TCC TTT ATC CAT AAC 3568 Asp Lys Ser Phe Met Asp Glu Asn Asp Tyr Gln Ser Phe Ile His Asn 2080 2085 2090 CCC AGC CTC ACA GTG ACA GTG CCA ATT GCA CCT GGG GAG TCT GAT TTG 3616 Pro Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu 2095 2100 2105 GAG ATT ATG AAC ACA GAA GAG CTT AGC AGT GAC TCA GAC AGT GAC TAC 3664 Glu Ile Met Asn Thr Glu Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr 2110 2115 2120 AGC AAA GAG AAA CGG AAC CGA TCA AGC TCT TCT GAG TGC AGC ACT GTT 3712 Ser Lys Glu Lys Arg Asn Arg Ser Ser Ser Ser Glu Cys Ser Thr Val 2125 2130 2135 2140 GAC AAC CCT CTG CCA GGA GAA GAG GAG GCT GAA GCA GAG CCC GTA AAC 3760 Asp Asn Pro Leu Pro Gly Glu Glu Glu Ala Glu Ala Glu Pro Val Asn 2145 2150 2155 GCA GAT GAG CCT GAA GCC TGC TTT ACA GAT GGT TGT GTG AGG AGA TTT 3808 Ala Asp Glu Pro Glu Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe 2160 2165 2170 CCA TGC TGC CAA GTT AAT GTA GAC TCT GGG AAA GGG AAA GTT TGG TGG 3856 Pro Cys Cys Gln Val Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp 2175 2180 2185 ACC ATC AGG AAG ACG TGC TAC AGG ATA GTT GAA CAC AGC TGG TTT GAA 3904 Thr Ile Arg Lys Thr Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu 2190 2195 2200 AGC TTC ATC GTT CTC ATG ATC CTG CTC AGC AGT GGA GCT CTG GCT TTT 3952 Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe 2205 2210 2215 2220 GAA GAT ATC TAT ATT GAA AAG AAA AAG ACC ATT AAG ATT ATC CTG GAG 4000 Glu Asp Ile Tyr Ile Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu 2225 2230 2235 TAT GCT GAC AAG ATA TTC ACC TAC ATC TTC ATT CTG GAA ATG CTT CTA 4048 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 2240 2245 2250 AAA TGG GTC GCA TAT GGG TAT AAA ACA TAT TTC ACT AAT GCC TGG TGT 4096 Lys Trp Val Ala Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys 2255 2260 2265 TGG CTG GAC TTC TTA ATT GTT GAT GTG TCT CTA GTT ACT TTA GTA GCC 4144 Trp Leu Asp Phe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala 2270 2275 2280 AAC ACT CTT GGC TAC TCA GAC CTT GGC CCC ATT AAA TCT CTA CGG ACA 4192 Asn Thr Leu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr 2285 2290 2295 2300 CTG AGG GCC CTA AGA CCC CTA AGA GCC TTG TCT AGA TTT GAA GGA ATG 4240 Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met 2305 2310 2315 AGG GTA GTG GTC AAC GCA CTC ATA GGA GCA ATC CCT TCC ATC ATG AAC 4288 Arg Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn 2320 2325 2330 GTG CTT CTC GTG TGC CTT ATA TTC TGG CTA ATA TTT AGC ATC ATG GGA 4336 Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly 2335 2340 2345 GTC AAT CTG TTT GCT GGC AAG TTC TAT GAG TGT GTC AAC ACC ACC GAT 4384 Val Asn Leu Phe Ala Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp 2350 2355 2360 GGG TCA CGA TTT CCT ACA TCT CAA GTT GCA AAC CGT TCT GAG TGT TTT 4432 Gly Ser Arg Phe Pro Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe 2365 2370 2375 2380 GCC CTG ATG AAC GTT AGT GGA AAT GTG CGA TGG AAA AAC CTG AAA GTA 4480 Ala Leu Met Asn Val Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val 2385 2390 2395 AAC TTC GAC AAC GTT GGG CTT GGT TAC CTG TCG CTG CTT CAA GTT GCA 4528 Asn Phe Asp Asn Val Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala 2400 2405 2410 ACA TTC AAG GGC TGG ATG GAT ATT ATG TAT GCA GCA GTT GAC TCT GTT 4576 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val 2415 2420 2425 AAT GTA AAT GAA CAG CCG AAA TAC GAA TAC AGT CTC TAC ATG TAC ATT 4624 Asn Val Asn Glu Gln Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile 2430 2435 2440 TAC TTT GTC ATC TTC ATC ATC TTC GGC TCA TTC TTC ACG TTG AAC CTG 4672 Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 2445 2450 2455 2460 TTC ATT GGT GTC ATC ATA GAT AAT TTC AAC CAA CAG AAA AAA AAG CTT 4720 Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu 2465 2470 2475 GGA GGT CAA GAT ATC TTT ATG ACA GAA GAA CAG AAG AAA TAC TAT AAT 4768 Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 2480 2485 2490 GCA ATG AAG AAG CTT GGG TCC AAA AAA CCA CAA AAA CCA ATT CCA AGG 4816 Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg 2495 2500 2505 CCA GGG AAC AAA TTC CAA GGA TGT ATA TTT GAC TTA GTG ACA AAC CAA 4864 Pro Gly Asn Lys Phe Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln 2510 2515 2520 GCT TTT GAT ATC ACC ATC ATG GTT CTT ATA TGC CTC AAC ATG GTA ACC 4912 Ala Phe Asp Ile Thr Ile Met Val Leu Ile Cys Leu Asn Met Val Thr 2525 2530 2535 2540 ATG ATG GTA GAA AAA GAG GGG CAA ACT GAG TAC ATG GAT TAT GTT TTA 4960 Met Met Val Glu Lys Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu 2545 2550 2555 CAC TGG ATC AAC ATG GTC TTC ATT ATC CTG TTC ACT GGG GAG TGT GTG 5008 His Trp Ile Asn Met Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val 2560 2565 2570 CTG AAG CTA ATC TCC CTC AGA CAT TAC TAC TTC ACT GTG GGT TGG AAC 5056 Leu Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn 2575 2580 2585 ATT TTT GAT TTT GTG GTA GTG ATC CTC TCC ATT GTA GGA ATG TTT CTC 5104 Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 2590 2595 2600 GCT GAG ATG ATA GAG AAG TAT TTC GTG TCC CCT ACC CTG TTC CGA GTC 5152 Ala Glu Met Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val 2605 2610 2615 2620 ATC CGC CTG GCC AGG ATT GGA CGA ATC CTA CGC CTG ATC AAA GGC GCC 5200 Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 2625 2630 2635 AAG GGG ATC CGC ACT CTG CTC TTT GCT TTG ATG ATG TCC CTT CCT GCG 5248 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 2640 2645 2650 CTG TTC AAC ATC GGC CTC CTG CTT TTC CTG GTC ATG TTC ATC TAC GCC 5296 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala 2655 2660 2665 ATC TTT GGG ATG TCC AAC TTT GCC TAC GTT AAA AAG GAG GCT GGA ATT 5344 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile 2670 2675 2680 AAT GAC ATG TTC AAC TTT GAG ACT TTT GGC AAC AGC ATG ATC TGC TTG 5392 Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu 2685 2690 2695 2700 TTC CAA ATC ACC ACC TCT GCC GGC TGG GAC GGA CTG CTG GCC CCC ATC 5440 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile 2705 2710 2715 CTC AAC AGC GCA CCT CCC GAC TGT GAC CCT AAA AAA GTT CAC CCA GGA 5488 Leu Asn Ser Ala Pro Pro Asp Cys Asp Pro Lys Lys Val His Pro Gly 2720 2725 2730 AGT TCA GTG GAA GGG GAC TGT GGG AAC CCA TCC GTG GGG ATT TTT TAC 5536 Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Tyr 2735 2740 2745 TTT GTC AGC TAC ATC ATC ATA TCC TTC CTG GTG GTG GTG AAC ATG TAC 5584 Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr 2750 2755 2760 ATC GCT GTC ATC CTG GAG AAC TTC AGC GTC GCC ACC GAA GAG AGC ACT 5632 Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Thr 2765 2770 2775 2780 GAG CCT CTG AGT GAG GAC GAC TTT GAG ATG TTC TAC GAG GTC TGG GAG 5680 Glu Pro Leu Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu 2785 2790 2795 AAG TTC GAC CCT GAC GCC ACT CAG TTC ATA GAG TTC TGC AAG CTC TCT 5728 Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser 2800 2805 2810 GAC TTT GCA GCT GCC CTG GAT CCT CCC CTC CTC ATC GCA AAG CCA AAC 5776 Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn 2815 2820 2825 AAA GTC CAG CTC ATT GCC ATG GAC CTG CCC ATG GTG AGT GGA GAC CGC 5824 Lys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg 2830 2835 2840 ATC CAC TGC CTG GAC ATC TTG TTT GCT TTT ACA AAG CGG GTC CTG GGT 5872 Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly 2845 2850 2855 2860 GAG GGT GGA GAG ATG GAT TCT CTT CGT TCA CAG ATG GAA GAA AGG TTC 5920 Glu Gly Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe 2865 2870 2875 ATG TCA GCC AAT CCT TCT AAA GTG TCC TAT GAA CCC ATC ACG ACC ACA 5968 Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr 2880 2885 2890 CTG AAG AGA AAA CAA GAG GAG GTG TCC GCG ACT ATC ATT CAG CGT GCT 6016 Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala 2895 2900 2905 TAC AGA CGG TAT CGC CTC AGA CAA CAC GTC AAG AAT ATA TCG AGT ATA 6064 Tyr Arg Arg Tyr Arg Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile 2910 2915 2920 TAC ATA AAA GAT GGA GAC AGG GAT GAT GAT TTG CCC AAT AAA GAA GAT 6112 Tyr Ile Lys Asp Gly Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp 2925 2930 2935 2940 ACA GTT TTT GAT AAC GTG AAC GAG AAC TCA AGT CCG GAA AAG ACA GAT 6160 Thr Val Phe Asp Asn Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp 2945 2950 2955 GTA ACT GCC TCA ACC ATC TCG CCA CCT TCC TAT GAC AGT GTC ACA AAG 6208 Val Thr Ala Ser Thr Ile Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys 2960 2965 2970 CCA GAT CAA GAG AAA TAT GAA ACA GAC AAA ACA GAG AAG GAA GAC AAA 6256 Pro Asp Gln Glu Lys Tyr Glu Thr Asp Lys Thr Glu Lys Glu Asp Lys 2975 2980 2985 GAG AAA GAT GAA AGC AGG AAA TAGAGCTTTG GTTTTGATAC ACTGTTGACA 6307 Glu Lys Asp Glu Ser Arg Lys 2990 2995 GCCTGTGAAG GTTGACTCAC TCGTGTTAGT AAGACTCTTT TACGGAGGTC TATCCAAACT 6367 CTTTTATCAA AAATTCTCAA GGCAGCACAG CCATTAGCTC TGATCCAACG AGGCAGAGGG 6427 CAGCATTTAC ACATGGCTAT GTTTT 6452 1984 amino acids amino acid linear protein 10 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Ser Glu Glu Lys Ala 20 25 30 Lys Glu His Lys Asp Glu Lys Lys Asp Asp Glu Glu Glu Gly Pro Lys 35 40 45 Pro Ser Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp Ile Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Ala 85 90 95 Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130 135 140 Leu Ser Asn Pro Pro Glu Trp Thr Lys Asn Val Glu Tyr Thr Phe Thr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Leu Ala Arg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg Lys Glu Leu Glu Glu Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr Ala Glu Ser Glu Glu Glu Leu Lys Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu Gly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser Gly Gln Cys Pro Glu Gly Tyr Ile Cys Val Lys Ala Gly Arg Asn 325 330 335 Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val 385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp Arg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala Ala Ala Glu Phe Thr 435 440 445 Ser Ile Gly Arg Ser Arg Ile Met Gly Leu Ser Glu Ser Ser Ser Glu 450 455 460 Thr Ser Arg Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Lys Gln Lys Met Ser Ser Gly Glu Glu Lys Gly Asp Asp 485 490 495 Glu Lys Leu Ser Lys Ser Gly Ser Glu Glu Ser Ile Arg Lys Lys Ser 500 505 510 Phe His Leu Gly Val Glu Gly His His Arg Thr Arg Glu Lys Arg Leu 515 520 525 Ser Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe Ser 530 535 540 Ala Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg Gly 545 550 555 560 Arg Asp Leu Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser Ile 565 570 575 Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His Arg 580 585 590 Pro Arg Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser Pro 595 600 605 Pro Val Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys Asn 610 615 620 Gly Val Val Ser Leu Val Asp Gly Pro Ser Ala Leu Met Leu Pro Asn 625 630 635 640 Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys Ala Thr Ser Asp Asp 645 650 655 Ser Gly Thr Thr Asn Gln Met Arg Lys Lys Arg Leu Ser Ser Ser Tyr 660 665 670 Phe Leu Ser Glu Asp Met Leu Asn Asp Pro His Leu Arg Gln Arg Ala 675 680 685 Met Ser Arg Ala Ser Ile Leu Thr Asn Thr Val Glu Glu Leu Glu Glu 690 695 700 Ser Arg Gln Lys Cys Pro Pro Trp Trp Tyr Arg Phe Ala His Thr Phe 705 710 715 720 Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile Lys Phe Lys Lys Leu Ile 725 730 735 Tyr Phe Ile Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr Ile Cys 740 745 750 Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His His Pro Met Thr 755 760 765 Glu Glu Phe Lys Asn Val Leu Ala Val Gly Asn Leu Ile Phe Thr Gly 770 775 780 Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro Tyr 785 790 795 800 Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile Val Thr 805 810 815 Leu Ser Leu Ile Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser Val 820 825 830 Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser Trp 835 840 845 Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly Ala 850 855 860 Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe Ala 865 870 875 880 Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys Val Cys 885 890 895 Lys Ile Asn Val Asp Cys Lys Leu Pro Arg Trp His Met Asn Asp Phe 900 905 910 Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu Trp Ile 915 920 925 Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Thr Met Cys Leu 930 935 940 Ile Val Tyr Met Met Val Met Val Ile Gly Asn Leu Val Val Leu Asn 945 950 955 960 Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu Thr 965 970 975 Ala Ile Glu Glu Asp Thr Asp Ala Asn Asn Leu Gln Ile Ala Val Ala 980 985 990 Arg Ile Lys Arg Gly Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu Phe 995 1000 1005 Ile Leu Lys Ser Phe Ser Lys Lys Pro Lys Gly Ser Lys Asp Thr Lys 1010 1015 1020 Arg Thr Ala Asp Pro Asn Asn Lys Lys Glu Asn Tyr Ile Ser Asn Arg 1025 1030 1035 1040 Thr Leu Ala Glu Met Ser Lys Asp His Asn Phe Leu Lys Glu Lys Asp 1045 1050 1055 Arg Ile Ser Gly Tyr Gly Ser Ser Leu Asp Lys Ser Phe Met Asp Glu 1060 1065 1070 Asn Asp Tyr Gln Ser Phe Ile His Asn Pro Ser Leu Thr Val Thr Val 1075 1080 1085 Pro Ile Ala Pro Gly Glu Ser Asp Leu Glu Ile Met Asn Thr Glu Glu 1090 1095 1100 Leu Ser Ser Asp Ser Asp Ser Asp Tyr Ser Lys Glu Lys Arg Asn Arg 1105 1110 1115 1120 Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly Glu 1125 1130 1135 Glu Glu Ala Glu Ala Glu Pro Val Asn Ala Asp Glu Pro Glu Ala Cys 1140 1145 1150 Phe Thr Asp Gly Cys Val Arg Arg Phe Pro Cys Cys Gln Val Asn Val 1155 1160 1165 Asp Ser Gly Lys Gly Lys Val Trp Trp Thr Ile Arg Lys Thr Cys Tyr 1170 1175 1180 Arg Ile Val Glu His Ser Trp Phe Glu Ser Phe Ile Val Leu Met Ile 1185 1190 1195 1200 Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile Glu Lys 1205 1210 1215 Lys Lys Thr Ile Lys Ile Ile Leu Glu Tyr Ala Asp Lys Ile Phe Thr 1220 1225 1230 Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Tyr 1235 1240 1245 Lys Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val 1250 1255 1260 Asp Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr Ser Asp 1265 1270 1275 1280 Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu 1285 1290 1295 Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu 1300 1305 1310 Ile Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile 1315 1320 1325 Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys 1330 1335 1340 Phe Tyr Glu Cys Val Asn Thr Thr Asp Gly Ser Arg Phe Pro Thr Ser 1345 1350 1355 1360 Gln Val Ala Asn Arg Ser Glu Cys Phe Ala Leu Met Asn Val Ser Gly 1365 1370 1375 Asn Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val Gly Leu 1380 1385 1390 Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp 1395 1400 1405 Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Asn Glu Gln Pro Lys 1410 1415 1420 Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val Ile Phe Ile Ile 1425 1430 1435 1440 Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp 1445 1450 1455 Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met 1460 1465 1470 Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser 1475 1480 1485 Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly 1490 1495 1500 Cys Ile Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Thr Ile Met 1505 1510 1515 1520 Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys Glu Gly 1525 1530 1535 Gln Thr Glu Tyr Met Asp Tyr Val Leu His Trp Ile Asn Met Val Phe 1540 1545 1550 Ile Ile Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser Leu Arg 1555 1560 1565 His Tyr Tyr Phe Thr Val Gly Trp Asn Ile Phe Asp Phe Val Val Val 1570 1575 1580 Ile Leu Ser Ile Val Gly Met Phe Leu Ala Glu Met Ile Glu Lys Tyr 1585 1590 1595 1600 Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly 1605 1610 1615 Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu 1620 1625 1630 Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu 1635 1640 1645 Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser Asn Phe 1650 1655 1660 Ala Tyr Val Lys Lys Glu Ala Gly Ile Asn Asp Met Phe Asn Phe Glu 1665 1670 1675 1680 Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala 1685 1690 1695 Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Ala Pro Pro Asp 1700 1705 1710 Cys Asp Pro Lys Lys Val His Pro Gly Ser Ser Val Glu Gly Asp Cys 1715 1720 1725 Gly Asn Pro Ser Val Gly Ile Phe Tyr Phe Val Ser Tyr Ile Ile Ile 1730 1735 1740 Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 1745 1750 1755 1760 Phe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp 1765 1770 1775 Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp Ala Thr 1780 1785 1790 Gln Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp 1795 1800 1805 Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile Ala Met 1810 1815 1820 Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp Ile Leu 1825 1830 1835 1840 Phe Ala Phe Thr Lys Arg Val Leu Gly Glu Gly Gly Glu Met Asp Ser 1845 1850 1855 Leu Arg Ser Gln Met Glu Glu Arg Phe Met Ser Ala Asn Pro Ser Lys 1860 1865 1870 Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln Glu Glu 1875 1880 1885 Val Ser Ala Thr Ile Ile Gln Arg Ala Tyr Arg Arg Tyr Arg Leu Arg 1890 1895 1900 Gln His Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly Asp Arg 1905 1910 1915 1920 Asp Asp Asp Leu Pro Asn Lys Glu Asp Thr Val Phe Asp Asn Val Asn 1925 1930 1935 Glu Asn Ser Ser Pro Glu Lys Thr Asp Val Thr Ala Ser Thr Ile Ser 1940 1945 1950 Pro Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Gln Glu Lys Tyr Glu 1955 1960 1965 Thr Asp Lys Thr Glu Lys Glu Asp Lys Glu Lys Asp Glu Ser Arg Lys 1970 1975 1980 1989 amino acids amino acid Not Relevant Not Relevant protein 11 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Xaa Glu Xaa Lys Xaa 20 25 30 Lys Glu Xaa Lys Xaa Glu Lys Lys Asp Asp Xaa Glu Glu Xaa Pro Lys 35 40 45 Pro Ser Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp Ile Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Xaa 85 90 95 Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130 135 140 Xaa Xaa Asn Pro Pro Xaa Trp Thr Lys Asn Val Xaa Tyr Thr Phe Thr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Xaa Lys Ile Leu Ala Arg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg Xaa Xaa Leu Glu Xaa Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr Xaa Glu Ser Glu Glu Xaa Xaa Xaa Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu Gly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser Gly Gln Cys Pro Glu Gly Tyr Xaa Cys Val Lys Xaa Gly Arg Asn 325 330 335 Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val 385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp Arg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala Ala Ala Glu Xaa Thr 435 440 445 Ser Ile Xaa Arg Ser Arg Ile Met Gly Leu Ser Glu Ser Ser Ser Glu 450 455 460 Thr Ser Xaa Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Xaa Gln Lys Lys Xaa Ser Ser Gly Glu Glu Lys Gly Asp 485 490 495 Xaa Glu Lys Leu Ser Lys Ser Xaa Ser Glu Xaa Ser Ile Arg Xaa Lys 500 505 510 Ser Phe His Leu Gly Val Glu Gly His Xaa Arg Xaa Xaa Glu Lys Arg 515 520 525 Leu Ser Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 Gly Arg Asp Xaa Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575 Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585 590 Arg Pro Xaa Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595 600 605 Pro Pro Xaa Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys 610 615 620 Asn Gly Val Val Ser Leu Val Asp Gly Xaa Ser Ala Leu Met Leu Pro 625 630 635 640 Asn Gly Gln Leu Leu Pro Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 645 650 655 Xaa Xaa Gly Thr Thr Asn Gln Xaa Xaa Lys Lys Arg Xaa Xaa Ser Ser 660 665 670 Tyr Xaa Leu Ser Glu Asp Met Leu Asn Asp Pro Xaa Leu Arg Gln Arg 675 680 685 Ala Met Ser Arg Ala Ser Ile Leu Thr Asn Thr Val Glu Glu Leu Glu 690 695 700 Glu Ser Arg Gln Lys Cys Xaa Xaa Xaa Xaa Tyr Arg Phe Ala His Xaa 705 710 715 720 Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile Lys Phe Lys Lys Xaa 725 730 735 Ile Tyr Phe Ile Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr Ile 740 745 750 Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His His Pro Met 755 760 765 Thr Glu Glu Phe Lys Asn Val Leu Ala Xaa Gly Asn Leu Xaa Phe Thr 770 775 780 Gly Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro 785 790 795 800 Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile Val 805 810 815 Thr Leu Ser Leu Xaa Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser 820 825 830 Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser 835 840 845 Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly 850 855 860 Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe 865 870 875 880 Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys Val 885 890 895 Cys Lys Ile Asn Xaa Asp Cys Xaa Leu Pro Arg Trp His Met Asn Asp 900 905 910 Phe Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu Trp 915 920 925 Ile Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Xaa Met Cys 930 935 940 Leu Ile Val Tyr Met Met Val Met Val Ile Gly Asn Leu Val Val Leu 945 950 955 960 Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu 965 970 975 Thr Ala Ile Glu Glu Asp Xaa Asp Ala Asn Asn Leu Gln Ile Ala Val 980 985 990 Xaa Arg Ile Lys Xaa Gly Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu 995 1000 1005 Phe Ile Leu Lys Xaa Phe Ser Lys Lys Pro Lys Xaa Ser Xaa Xaa Xaa 1010 1015 1020 Xaa Xaa Xaa Xaa Asp Xaa Asn Xaa Lys Lys Glu Asn Tyr Ile Ser Asn 1025 1030 1035 1040 Xaa Thr Leu Ala Glu Met Ser Lys Xaa His Asn Phe Leu Lys Glu Lys 1045 1050 1055 Asp Xaa Ile Ser Gly Xaa Gly Ser Ser Xaa Asp Lys Xaa Xaa Met Xaa 1060 1065 1070 Xaa Xaa Asp Xaa Gln Ser Phe Ile His Asn Pro Ser Leu Thr Val Thr 1075 1080 1085 Val Pro Ile Ala Pro Gly Glu Ser Asp Leu Glu Xaa Met Asn Xaa Glu 1090 1095 1100 Glu Leu Ser Ser Asp Ser Asp Ser Xaa Tyr Ser Lys Xaa Xaa Xaa Asn 1105 1110 1115 1120 Arg Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly 1125 1130 1135 Glu Gly Glu Glu Ala Glu Ala Glu Pro Xaa Asn Xaa Asp Glu Pro Glu 1140 1145 1150 Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe Xaa Cys Cys Gln Val 1155 1160 1165 Asn Xaa Xaa Ser Gly Lys Gly Lys Xaa Trp Trp Xaa Ile Arg Lys Thr 1170 1175 1180 Cys Tyr Xaa Ile Val Glu His Ser Trp Phe Glu Ser Phe Ile Val Leu 1185 1190 1195 1200 Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile 1205 1210 1215 Glu Xaa Lys Lys Thr Ile Lys Ile Ile Leu Glu Tyr Ala Asp Lys Ile 1220 1225 1230 Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Xaa Ala Tyr 1235 1240 1245 Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1250 1255 1260 Ile Val Asp Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr 1265 1270 1275 1280 Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg 1285 1290 1295 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn 1300 1305 1310 Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys 1315 1320 1325 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala 1330 1335 1340 Gly Lys Phe Tyr Glu Cys Xaa Asn Thr Thr Asp Gly Ser Arg Phe Pro 1345 1350 1355 1360 Xaa Ser Gln Val Xaa Asn Arg Ser Glu Cys Phe Ala Leu Met Asn Val 1365 1370 1375 Ser Xaa Asn Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val 1380 1385 1390 Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1395 1400 1405 Xaa Xaa Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Xaa Xaa Gln 1410 1415 1420 Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val Xaa Phe 1425 1430 1435 1440 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile 1445 1450 1455 Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile 1460 1465 1470 Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu 1475 1480 1485 Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Xaa 1490 1495 1500 Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Xaa 1505 1510 1515 1520 Ile Met Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys 1525 1530 1535 Glu Gly Gln Xaa Xaa Xaa Met Xaa Xaa Val Leu Xaa Trp Ile Asn Xaa 1540 1545 1550 Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser 1555 1560 1565 Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn Ile Xaa Xaa Phe Val 1570 1575 1580 Val Val Ile Xaa Ser Ile Val Gly Met Phe Leu Ala Xaa Xaa Ile Glu 1585 1590 1595 1600 Xaa Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg 1605 1610 1615 Ile Gly Arg Ile Leu Arg Leu Xaa Lys Gly Ala Lys Gly Ile Arg Thr 1620 1625 1630 Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1635 1640 1645 Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 1650 1655 1660 Asn Phe Ala Tyr Val Lys Lys Glu Xaa Gly Ile Asn Asp Met Phe Asn 1665 1670 1675 1680 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr 1685 1690 1695 Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Xaa Pro 1700 1705 1710 Pro Asp Cys Asp Pro Lys Lys Val His Pro Gly Ser Ser Val Glu Gly 1715 1720 1725 Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Tyr Phe Val Ser Tyr Ile 1730 1735 1740 Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu 1745 1750 1755 1760 Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu 1765 1770 1775 Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 1780 1785 1790 Ala Thr Gln Phe Ile Glu Phe Xaa Lys Leu Ser Asp Phe Ala Ala Ala 1795 1800 1805 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile 1810 1815 1820 Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp 1825 1830 1835 1840 Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly Glu Xaa Gly Glu Met 1845 1850 1855 Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe Met Ser Ala Asn Pro 1860 1865 1870 Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1875 1880 1885 Glu Xaa Val Ser Ala Thr Xaa Ile Gln Arg Ala Tyr Arg Arg Tyr Arg 1890 1895 1900 Leu Arg Gln Xaa Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly 1905 1910 1915 1920 Asp Arg Asp Asp Asp Leu Xaa Asn Lys Xaa Asp Xaa Xaa Phe Asp Asn 1925 1930 1935 Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp Xaa Thr Xaa Ser Thr 1940 1945 1950 Xaa Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Xaa Glu Lys 1955 1960 1965 Tyr Glu Xaa Asp Xaa Thr Glu Lys Glu Asp Lys Xaa Lys Asp Ser Lys 1970 1975 1980 Glu Ser Xaa Lys Xaa 1985 1989 amino acids amino acid Not Relevant Not Relevant protein 12 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Ser Glu Glu Lys Ala 20 25 30 Lys Glu His Lys Asp Glu Lys Lys Asp Asp Glu Glu Glu Gly Pro Lys 35 40 45 Pro Ser Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp Ile Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Ala 85 90 95 Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130 135 140 Leu Ser Asn Pro Pro Glu Trp Thr Lys Asn Val Gly Tyr Thr Phe Thr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Leu Ala Arg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg Lys Glu Leu Glu Glu Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr Ala Glu Ser Glu Glu Glu Leu Lys Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu Gly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser Gly Gln Cys Pro Glu Gly Tyr Ile Cys Val Lys Ala Gly Arg Asn 325 330 335 Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val 385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp Arg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala Ala Ala Glu Phe Thr 435 440 445 Ser Ile Arg Arg Ser Arg Ile Met Gly Leu Ser Glu Ser Ser Ser Glu 450 455 460 Thr Ser Arg Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Lys Gln Lys Xaa Met Ser Ser Gly Glu Glu Lys Gly Asp 485 490 495 Asp Glu Lys Leu Ser Lys Ser Gly Ser Glu Glu Ser Ile Arg Lys Lys 500 505 510 Ser Phe His Leu Gly Val Glu Gly His His Arg Thr Arg Glu Lys Arg 515 520 525 Leu Ser Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 Gly Arg Asp Leu Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575 Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585 590 Arg Pro Arg Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595 600 605 Pro Pro Val Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys 610 615 620 Asn Gly Val Val Ser Leu Val Asp Gly Pro Ser Ala Leu Met Leu Pro 625 630 635 640 Asn Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys Ala Thr Ser Asp 645 650 655 Asp Ser Gly Thr Thr Asn Gln Met Arg Lys Lys Arg Leu Ser Ser Ser 660 665 670 Tyr Phe Leu Ser Glu Asp Met Leu Asn Asp Pro His Leu Arg Gln Arg 675 680 685 Ala Met Ser Arg Ala Ser Ile Leu Thr Asn Thr Val Glu Glu Leu Glu 690 695 700 Glu Ser Arg Gln Lys Cys His Gln Leu Leu Tyr Arg Phe Ala His Thr 705 710 715 720 Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile Lys Phe Lys Lys Leu 725 730 735 Ile Tyr Phe Ile Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr Ile 740 745 750 Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His His Pro Met 755 760 765 Thr Glu Glu Phe Lys Asn Val Leu Ala Val Gly Asn Leu Ile Phe Thr 770 775 780 Gly Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp Pro 785 790 795 800 Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile Val 805 810 815 Thr Leu Ser Leu Ile Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser 820 825 830 Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser 835 840 845 Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly 850 855 860 Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe 865 870 875 880 Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys Val 885 890 895 Cys Lys Ile Asn Val Asp Cys Lys Leu Pro Arg Trp His Met Asn Asp 900 905 910 Phe Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu Trp 915 920 925 Ile Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Thr Met Cys 930 935 940 Leu Ile Val Tyr Met Met Val Met Val Ile Gly Asn Leu Val Val Leu 945 950 955 960 Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu 965 970 975 Thr Ala Ile Glu Glu Asp Thr Asp Ala Asn Asn Leu Gln Ile Ala Val 980 985 990 Ala Arg Ile Lys Arg Gly Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu 995 1000 1005 Phe Ile Leu Lys Ser Phe Ser Lys Lys Pro Lys Gly Ser Lys Asp Thr 1010 1015 1020 Lys Arg Thr Ala Asp Pro Asn Asn Lys Lys Glu Asn Tyr Ile Ser Asn 1025 1030 1035 1040 Arg Thr Leu Ala Glu Met Ser Lys Asp His Asn Phe Leu Lys Glu Lys 1045 1050 1055 Asp Arg Ile Ser Gly Tyr Gly Ser Ser Leu Asp Lys Ser Phe Met Asp 1060 1065 1070 Glu Asn Asp Tyr Gln Ser Phe Ile His Asn Pro Ser Leu Thr Val Thr 1075 1080 1085 Val Pro Ile Ala Pro Gly Glu Ser Asp Leu Glu Ile Met Asn Thr Glu 1090 1095 1100 Glu Leu Ser Ser Asp Ser Asp Ser Asp Tyr Ser Lys Glu Lys Arg Asn 1105 1110 1115 1120 Arg Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly 1125 1130 1135 Glu Xaa Glu Glu Ala Glu Ala Glu Pro Val Asn Ala Asp Glu Pro Glu 1140 1145 1150 Ala Cys Phe Thr Asp Gly Cys Val Arg Arg Phe Pro Cys Cys Gln Val 1155 1160 1165 Asn Val Asp Ser Gly Lys Gly Lys Val Trp Trp Thr Ile Arg Lys Thr 1170 1175 1180 Cys Tyr Arg Ile Val Glu His Ser Trp Phe Glu Ser Phe Ile Val Leu 1185 1190 1195 1200 Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile 1205 1210 1215 Glu Lys Lys Lys Thr Ile Lys Ile Ile Leu Glu Tyr Ala Asp Lys Ile 1220 1225 1230 Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr 1235 1240 1245 Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1250 1255 1260 Ile Val Asp Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr 1265 1270 1275 1280 Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg 1285 1290 1295 Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn 1300 1305 1310 Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys 1315 1320 1325 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala 1330 1335 1340 Gly Lys Phe Tyr Glu Cys Val Asn Thr Thr Asp Gly Ser Arg Phe Pro 1345 1350 1355 1360 Thr Ser Gln Val Ala Asn Arg Ser Glu Cys Phe Ala Leu Met Asn Val 1365 1370 1375 Ser Gly Asn Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val 1380 1385 1390 Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1395 1400 1405 Met Asp Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Asn Glu Gln 1410 1415 1420 Pro Lys Tyr Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val Ile Phe 1425 1430 1435 1440 Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile 1445 1450 1455 Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile 1460 1465 1470 Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu 1475 1480 1485 Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe 1490 1495 1500 Gln Gly Cys Ile Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Thr 1505 1510 1515 1520 Ile Met Val Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys 1525 1530 1535 Glu Gly Gln Thr Glu Tyr Met Asp Tyr Val Leu His Trp Ile Asn Met 1540 1545 1550 Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser 1555 1560 1565 Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn Ile Leu Tyr Phe Val 1570 1575 1580 Val Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Glu Met Ile Glu 1585 1590 1595 1600 Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg 1605 1610 1615 Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr 1620 1625 1630 Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly 1635 1640 1645 Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser 1650 1655 1660 Asn Phe Ala Tyr Val Lys Lys Glu Ala Gly Ile Asn Asp Met Phe Asn 1665 1670 1675 1680 Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr 1685 1690 1695 Ser Ala Gly Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Ala Pro 1700 1705 1710 Pro Asp Cys Asp Pro Lys Lys Val His Pro Gly Ser Ser Val Glu Gly 1715 1720 1725 Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Tyr Phe Val Ser Tyr Ile 1730 1735 1740 Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu 1745 1750 1755 1760 Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu 1765 1770 1775 Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 1780 1785 1790 Ala Thr Gln Phe Ile Glu Phe Cys Lys Leu Ser Asp Phe Ala Ala Ala 1795 1800 1805 Leu Asp Pro Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile 1810 1815 1820 Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp 1825 1830 1835 1840 Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly Glu Gly Gly Glu Met 1845 1850 1855 Asp Ser Leu Arg Ser Gln Met Glu Glu Arg Phe Met Ser Ala Asn Pro 1860 1865 1870 Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln 1875 1880 1885 Glu Glu Val Ser Ala Thr Ile Ile Gln Arg Ala Tyr Arg Arg Tyr Arg 1890 1895 1900 Leu Arg Gln His Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly 1905 1910 1915 1920 Asp Arg Asp Asp Asp Leu Pro Asn Lys Glu Asp Thr Val Phe Asp Asn 1925 1930 1935 Val Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp Val Thr Ala Ser Thr 1940 1945 1950 Ile Ser Pro Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Gln Glu Lys 1955 1960 1965 Tyr Glu Thr Asp Lys Thr Glu Lys Glu Asp Lys Glu Lys Asp Xaa Xaa 1970 1975 1980 Glu Ser Arg Lys Xaa 1985 6371 base pairs nucleic acid both both DNA (genomic) 13 CTCTTATGTG AGGAGCTGAA GAGGAATTAA AATATACAGG ATGAAAAGAT GGCAATGTTG 60 CCTCCCCCAG GACCTCAGAG CTTTGTCCAT TTCACAAAAC AGTCTCTTGC CCTCATTGAA 120 CAACGCATTG CTGAAAGAAA ATCAAAGGAA CCCAAAGAAG AAAAGAAAGA TGATGATGAA 180 GAAGCCCCAA AGCCAAGCAG TGACTTGGAA GCTGGCAAAC AACTGCCCTT CATCTATGGG 240 GACATTCCTC CCGGCATGGT GTCAGAGCCC CTGGAGGACT TGGACCCCTA CTATGCAGAC 300 AAAAAGACTT TCATAGTATT GAACAAAGGG AAAACAATCT TCCGTTTCAA TGCCACACCT 360 GCTTTATATA TGCTTTCTCC TTTCAGTCCT CTAAGAAGAA TATCTATTAA GATTTTAGTA 420 CACTCCTTAT TCAGCATGCT CATCATGTGC ACTATTCTGA CAAACTGCAT ATTTATGACC 480 ATGAATAACC CGCCGGACTG GACCAAAAAT GTCGAGTACA CTTTTACTGG AATATATACT 540 TTTGAATCAC TTGTAAAAAT CCTTGCAAGA GGCTTCTGTG TAGGAGAATT CACTTTTCTT 600 CGTGACCCGT GGAACTGGCT GGATTTTGTC GTCATTGTTT TTGCGTATTT AACAGAATTT 660 GTAAACCTAG GCAATGTTTC AGCTCTTCGA ACTTTCAGAG TATTGAGAGC TTTGAAAACT 720 ATTTCTGTAA TCCCAGGCCT GAAGACAATT GTAGGGGCTT TGATCCAGTC AGTGAAGAAG 780 CTTTCTGATG TCATGATCCT GACTGTGTTC TGTCTGAGTG TGTTTGCACT AATTGGACTA 840 CAGCTGTTCA TGGGAAACCT GAAGCATAAA TGTTTTCGAA ATTCACTTGA AAATAATGAA 900 ACATTAGAAA GCATAATGAA TACCCTAGAG AGTGAAGAAG ACTTTAGAAA ATATTTTTAT 960 TACTTGGAAG GATCCAAAGA TGCTCTCCTT TGTGGTTTCA GCACAGATTC AGGTCAGTGT 1020 CCAGAGGGGT ACACCTGTGT GAAAATTGGC AGAAACCCTG ATTATGGCTA CACGAGCTTT 1080 GACACTTTCA GCTGGGCCTT CTTAGCCTTG TTTAGGCTAA TGACCCAAGA TTACTGGGAA 1140 AACCTTTACC AACAGACGCT GCGTGCTGCT GGCAAAACCT ACATGATCTT CTTTGTCGTA 1200 GTGATTTTCC TGGGCTCCTT TTATCTAATA AACTTGATCC TGGCTGTGGT TGCCATGGCA 1260 TATGAAGAAC AGAACCAGGC AAACATTGAA GAAGCTAAAC AGAAAGAATT AGAATTTCAA 1320 CAGATGTTAG ACCGTCTTAA AAAAGAGCAA GAAGAAGCTG AGGCAATTGC AGCGGCAGCG 1380 GCTGAATATA CAAGTATTAG GAGAAGCAGA ATTATGGGCC TCTCAGAGAG TTCTTCTGAA 1440 ACATCCAAAC TGAGCTCTAA AAGTGCTAAA GAAAGAAGAA ACAGAAGAAA GAAAAAGAAT 1500 CAAAAGAAGC TCTCCAGTGG AGAGGAAAAG GGAGATGCTG AGAAATTGTC GAAATCAGAA 1560 TCAGAGGACA GCATCAGAAG AAAAAGTTTC CACCTTGGTG TCGAAGGGCA TAGGCGAGCA 1620 CATGAAAAGA GGTTGTCTAC CCCCAATCAG TCACCACTCA GCATTCGTGG CTCCTTGTTT 1680 TCTGCAAGGC GAAGCAGCAG AACAAGTCTT TTTAGTTTCA AAGGCAGAGG AAGAGATATA 1740 GGATCTGAGA CTGAATTTGC CGATGATGAG CACAGCATTT TTGGAGACAA TGAGAGCAGA 1800 AGGGGCTCAC TGTTTGTGCC CCACAGACCC CAGGAGCGAC GCAGCAGTAA CATCAGCCAA 1860 GCCAGTAGGT CCCCACCAAT GCTGCCGGTG AACGGGAAAA TGCACAGTGC TGTGGACTGC 1920 AACGGTGTGG TCTCCCTGGT TGATGGACGC TCAGCCCTCA TGCTCCCCAA TGGACAGCTT 1980 CTGCCAGAGG GCACGACCAA TCAAATACAC AAGAAAAGGC GTTGTAGTTC CTATCTCCTT 2040 TCAGAGGATA TGCTGAATGA TCCCAACCTC AGACAGAGAG CAATGAGTAG AGCAAGCATA 2100 TTAACAAACA CTGTGGAAGA ACTTGAAGAG TCCAGACAAA AATGTCCACC TTGGTGGTAC 2160 AGATTTGCAC ACAAATTCTT GATCTGGAAT TGCTCTCCAT ATTGGATAAA ATTCAAAAAG 2220 TGTATCTATT TTATTGTAAT GGATCCTTTT GTAGATCTTG CAATTACCAT TTGCATAGTT 2280 TTAAACACAT TATTTATGGC TATGGAACAC CACCCAATGA CTGAGGAATT CAAAAATGTA 2340 CTTGCTATAG GAAATTTGGT CTTTACTGGA ATCTTTGCAG CTGAAATGGT ATTAAAACTG 2400 ATTGCCATGG ATCCATATGA GTATTTCCAA GTAGGCTGGA ATATTTTTGA CAGCCTTATT 2460 GTGACTTTAA GTTTAGTGGA GCTCTTTCTA GCAGATGTGG AAGGATTGTC AGTTCTGCGA 2520 TCATTCAGAC TGCTCCGAGT CTTCAAGTTG GCAAAATCCT GGCCAACATT GAACATGCTG 2580 ATTAAGATCA TTGGTAACTC AGTAGGGGCT CTAGGTAACC TCACCTTAGT GTTGGCCATC 2640 ATCGTCTTCA TTTTTGCTGT GGTCGGCATG CAGCTCTTTG GTAAGAGCTA CAAAGAATGT 2700 GTCTGCAAGA TCAATGATGA CTGTACGCTC CCACGGTGGC ACATGAACGA CTTCTTCCAC 2760 TCCTTCCTGA TTGTGTTCCG CGTGCTGTGT GGAGAGTGGA TAGAGACCAT GTGGGACTGT 2820 ATGGAGGTCG CTGGTCAAGC TATGTGCCTT ATTGTTTACA TGATGGTCAT GGTCATTGGA 2880 AACCTGGTGG TCCTAAACCT ATTTCTGGCC TTATTATTGA GCTCATTTAG TTCAGACAAT 2940 CTTACAGCAA TTGAAGAAGA CCCTGATGCA AACAACCTCC AGATTGCAGT GACTAGAATT 3000 AAAAAGGGAA TAAATTATGT GAAACAAACC TTACGTGAAT TTATTCTAAA AGCATTTTCC 3060 AAAAAGCCAA AGATTTCCAG GGAGATAAGA CAAGCAGAAG ATCTGAATAC TAAGAAGGAA 3120 AACTATATTT CTAACCATAC ACTTGCTGAA ATGAGCAAAG GTCACAATTT CCTCAAGGAA 3180 AAAGATAAAA TCAGTGGTTT TGGAAGCAGC GTGGACAAAC ACTTGATGGA AGACAGTGAT 3240 GGTCAATCAT TTATTCACAA TCCCAGCCTC ACAGTGACAG TGCCAATTGC ACCTGGGGAA 3300 TCCGATTTGG AAAATATGAA TGCTGAGGAA CTTAGCAGTG ATTCGGATAG TGAATACAGC 3360 AAAGTGAGAT TAAACCGGTC AAGCTCCTCA GAGTGCAGCA CAGTTGATAA CCCTTTGCCT 3420 GGAGAAGGAG AAGAAGCAGA GGCTGAACCT ATGAATTCCG ATGAGCCAGA GGCCTGTTTC 3480 ACAGATGGTT GTGTACGGAG GTTCTCATGC TGCCAAGTTA ACATAGAGTC AGGGAAAGGA 3540 AAAATCTGGT GGAACATCAG GAAAACCTGC TACAAGATTG TTGAACACAG TTGGTTTGAA 3600 AGCTTCATTG TCCTCATGAT CCTGCTCAGC AGTGGTGCCC TGGCTTTTGA AGATATTTAT 3660 ATTGAAAGGA AAAAGACCAT TAAGATTATC CTGGAGTATG CAGACAAGAT CTTCACTTAC 3720 ATCTTCATTC TGGAAATGCT TCTAAAATGG ATAGCATATG GTTATAAAAC ATATTTCACC 3780 AATGCCTGGT GTTGGCTGGA TTTCCTAATT GTTGATGTTT CTTTGGTTAC TTTAGTGGCA 3840 AACACTCTTG GCTACTCAGA TCTTGGCCCC ATTAAATCCC TTCGGACACT GAGAGCTTTA 3900 AGACCTCTAA GAGCCTTATC TAGATTTGAA GGAATGAGGG TCGTTGTGAA TGCACTCATA 3960 GGAGCAATTC CTTCCATCAT GAATGTGCTA CTTGTGTGTC TTATATTCTG GCTGATATTC 4020 AGCATCATGG GAGTAAATTT GTTTGCTGGC AAGTTCTATG AGTGTATTAA CACCACAGAT 4080 GGGTCACGGT TTCCTGCAAG TCAAGTTCCA AATCGTTCCG AATGTTTTGC CCTTATGAAT 4140 GTTAGTCAAA ATGTGCGATG GAAAAACCTG AAAGTGAACT TTGATAATGT CGGACTTGGT 4200 TACCTATCTC TGCTTCAAGT TGCAACTTTT AAGGGATGGA CGATTATTAT GTATGCAGCA 4260 GTGGATTCTG TTAATGTAGA CAAGCAGCCC AAATATGAAT ATAGCCTCTA CATGTATATT 4320 TATTTTGTCG TCTTTATCAT CTTTGGGTCA TTCTTCACTT TGAACTTGTT CATTGGTGTC 4380 ATCATAGATA ATTTCAACCA ACAGAAAAAG AAGCTTGGAG GTCAAGACAT CTTTATGACA 4440 GAAGAACAGA AGAAATACTA TAATGCAATG AAAAAGCTGG GGTCCAAGAA GCCACAAAAG 4500 CCAATTCCTC GACCAGGGAA CAAAATCCAA GGATGTATAT TTGACCTAGT GACAAATCAA 4560 GCCTTTGATA TTAGTATCAT GGTTCTTATC TGTCTCAACA TGGTAACCAT GATGGTAGAA 4620 AAGGAGGGTC AAAGTCAACA TATGACTGAA GTTTTATATT GGATAAATGT GGTTTTTATA 4680 ATCCTTTTCA CTGGAGAATG TGTGCTAAAA CTGATCTCCC TCAGACACTA CTACTTCACT 4740 GTAGGATGGA ATATTTTTGA TTTTGTGGTT GTGATTATCT CCATTGTAGG TATGTTTCTA 4800 GCTGATTTGA TTGAAACGTA TTTTGTGTCC CCTACCCTGT TCCGAGTGAT CCGTCTTGCC 4860 AGGATTGGCC GAATCCTACG TCTAGTCAAA GGAGCAAAGG GGATCCGCAC GCTGCTCTTT 4920 GCTTTGATGA TGTCCCTTCC TGCGTTGTTT AACATCGGCC TCCTGCTCTT CCTGGTCATG 4980 TTCATCTACG CCATCTTTGG AATGTCCAAC TTTGCCTATG TTAAAAAGGA AGATGGAATT 5040 AATGACATGT TCAATTTTGA GACCTTTGGC AACAGTATGA TTTGCCTGTT CCAAATTACA 5100 ACCTCTGCTG GCTGGGATGG ATTGCTAGCA CCTATTCTTA ACAGTAAGCC ACCCGACTGT 5160 GACCCAAAAA AAGTTCATCC TGGAAGTTCA GTTGAAGGAG ACTGTGGTAA CCCATCTGTT 5220 GGAATATTCT ACTTTGTTAG TTATATCATC ATATCCTTCC TGGTTGTGGT GAACATGTAC 5280 ATTGCAGTCA TACTGGAGAA TTTTAGTGTT GCCACTGAAG AAAGTACTGA ACCTCTGAGT 5340 GAGGATGACT TTGAGATGTT CTATGAGGTT TGGGAGAAGT TTGATCCCGA TGCGACCCAG 5400 TTTATAGAGT TCTCTAAACT CTCTGATTTT GCAGCTGCCC TGGATCCTCC TCTTCTCATA 5460 GCAAAACCCA ACAAAGTCCA GCTCATTGCC ATGGATCTGC CCATGGTTAG TGGTGACCGG 5520 ATCCATTGTC TTGACATCTT ATTTGCTTTT ACAAAGCGTG TTTTGGGTGA GAGTGGGGAG 5580 ATGGATTCTC TTCGTTCACA GATGGAAGAA AGGTTCATGT CTGCAAATCC TTCCAAAGTG 5640 TCCTATGAAC CCATCACAAC CACACTAAAA CGGAAACAAG AGGATGTGTC TGCTACTGTC 5700 ATTCAGCGTG CTTATAGACG TTACCGCTTA AGGCAAAATG TCAAAAATAT ATCAAGTATA 5760 TACATAAAAG ATGGAGACAG AGATGATGAT TTACTCAATA AAAAAGATAT GGCTTTTGAT 5820 AATGTTAATG AGAACTCAAG TCCAGAAAAA ACAGATGCCA CTTCATCCAC CACCTCTCCA 5880 CCTTCATATG ATAGTGTAAC AAAGCCAGAC AAAGAGAAAT ATGAACAAGA CAGAACAGAA 5940 AAGGAAGACA AAGGGAAAGA CAGCAAGGAA AGCAAAAAAT AGAGCTTCAT TTTTGATATA 6000 TTGTTTACAG CCTGTGAAAG TGATTTATTT GTGTTAATAA AACTCTTTTG AGGAAGTCTA 6060 TGCCAAAATC CTTTTTATCA AAATATTCTC GAAGGCAGTG CAGTCACTAA CTCTGATTTC 6120 CTAAGAAAGG TGGGCAGCAT TAGCAGATGG TTATTTTTGC ACTGATGATT CTTTAAGAAT 6180 CGTAAGAGAA CTCTGTAGGA ATTATTGATT ATAGCATACA AAAGTGATTG ATTCAGTTTT 6240 TTGGTTTTTA ATAAATCAGA AGACCATGTA GAAAACTTTT ACATCTGCCT TGTCATCTTT 6300 TCACAGGATT GTAATTAGTC TTGTTTCCCA TGTAAATAAA CAACACACGC ATACAGAAAA 6360 AAAAAAAAAA A 6371 6404 base pairs nucleic acid both both DNA (genomic) 14 CTCTTATGTG AGGAGCTGAA GAGGAATTAA AATATACAGG ATGAAAAGAT GGCAATGTTG 60 CCTCCCCCAG GACCTCAGAG CTTTGTCCAT TTCACAAAAC AGTCTCTTGC CCTCATTGAA 120 CAACGCATTG CTGAAAGAAA ATCAAAGGAA CCCAAAGAAG AAAAGAAAGA TGATGATGAA 180 GAAGCCCCAA AGCCAAGCAG TGACTTGGAA GCTGGCAAAC AACTGCCCTT CATCTATGGG 240 GACATTCCTC CCGGCATGGT GTCAGAGCCC CTGGAGGACT TGGACCCCTA CTATGCAGAC 300 AAAAAGACTT TCATAGTATT GAACAAAGGG AAAACAATCT TCCGTTTCAA TGCCACACCT 360 GCTTTATATA TGCTTTCTCC TTTCAGTCCT CTAAGAAGAA TATCTATTAA GATTTTAGTA 420 CACTCCTTAT TCAGCATGCT CATCATGTGC ACTATTCTGA CAAACTGCAT ATTTATGACC 480 ATGAATAACC CGCCGGACTG GACCAAAAAT GTCGAGTACA CTTTTACTGG AATATATACT 540 TTTGAATCAC TTGTAAAAAT CCTTGCAAGA GGCTTCTGTG TAGGAGAATT CACTTTTCTT 600 CGTGACCCGT GGAACTGGCT GGATTTTGTC GTCATTGTTT TTGCGTATTT AACAGAATTT 660 GTAAACCTAG GCAATGTTTC AGCTCTTCGA ACTTTCAGAG TATTGAGAGC TTTGAAAACT 720 ATTTCTGTAA TCCCAGGCCT GAAGACAATT GTAGGGGCTT TGATCCAGTC AGTGAAGAAG 780 CTTTCTGATG TCATGATCCT GACTGTGTTC TGTCTGAGTG TGTTTGCACT AATTGGACTA 840 CAGCTGTTCA TGGGAAACCT GAAGCATAAA TGTTTTCGAA ATTCACTTGA AAATAATGAA 900 ACATTAGAAA GCATAATGAA TACCCTAGAG AGTGAAGAAG ACTTTAGAAA ATATTTTTAT 960 TACTTGGAAG GATCCAAAGA TGCTCTCCTT TGTGGTTTCA GCACAGATTC AGGTCAGTGT 1020 CCAGAGGGGT ACACCTGTGT GAAAATTGGC AGAAACCCTG ATTATGGCTA CACGAGCTTT 1080 GACACTTTCA GCTGGGCCTT CTTAGCCTTG TTTAGGCTAA TGACCCAAGA TTACTGGGAA 1140 AACCTTTACC AACAGACGCT GCGTGCTGCT GGCAAAACCT ACATGATCTT CTTTGTCGTA 1200 GTGATTTTCC TGGGCTCCTT TTATCTAATA AACTTGATCC TGGCTGTGGT TGCCATGGCA 1260 TATGAAGAAC AGAACCAGGC AAACATTGAA GAAGCTAAAC AGAAAGAATT AGAATTTCAA 1320 CAGATGTTAG ACCGTCTTAA AAAAGAGCAA GAAGAAGCTG AGGCAATTGC AGCGGCAGCG 1380 GCTGAATATA CAAGTATTAG GAGAAGCAGA ATTATGGGCC TCTCAGAGAG TTCTTCTGAA 1440 ACATCCAAAC TGAGCTCTAA AAGTGCTAAA GAAAGAAGAA ACAGAAGAAA GAAAAAGAAT 1500 CAAAAGAAGC TCTCCAGTGG AGAGGAAAAG GGAGATGCTG AGAAATTGTC GAAATCAGAA 1560 TCAGAGGACA GCATCAGAAG AAAAAGTTTC CACCTTGGTG TCGAAGGGCA TAGGCGAGCA 1620 CATGAAAAGA GGTTGTCTAC CCCCAATCAG TCACCACTCA GCATTCGTGG CTCCTTGTTT 1680 TCTGCAAGGC GAAGCAGCAG AACAAGTCTT TTTAGTTTCA AAGGCAGAGG AAGAGATATA 1740 GGATCTGAGA CTGAATTTGC CGATGATGAG CACAGCATTT TTGGAGACAA TGAGAGCAGA 1800 AGGGGCTCAC TGTTTGTGCC CCACAGACCC CAGGAGCGAC GCAGCAGTAA CATCAGCCAA 1860 GCCAGTAGGT CCCCACCAAT GCTGCCGGTG AACGGGAAAA TGCACAGTGC TGTGGACTGC 1920 AACGGTGTGG TCTCCCTGGT TGATGGACGC TCAGCCCTCA TGCTCCCCAA TGGACAGCTT 1980 CTGCCAGAGG TGATAATAGA TAAGACAACT TCTGATGACA GCGGCACGAC CAATCAAATA 2040 CACAAGAAAA GGCGTTGTAG TTCCTATCTC CTTTCAGAGG ATATGCTGAA TGATCCCAAC 2100 CTCAGACAGA GAGCAATGAG TAGAGCAAGC ATATTAACAA ACACTGTGGA AGAACTTGAA 2160 GAGTCCAGAC AAAAATGTCC ACCTTGGTGG TACAGATTTG CACACAAATT CTTGATCTGG 2220 AATTGCTCTC CATATTGGAT AAAATTCAAA AAGTGTATCT ATTTTATTGT AATGGATCCT 2280 TTTGTAGATC TTGCAATTAC CATTTGCATA GTTTTAAACA CATTATTTAT GGCTATGGAA 2340 CACCACCCAA TGACTGAGGA ATTCAAAAAT GTACTTGCTA TAGGAAATTT GGTCTTTACT 2400 GGAATCTTTG CAGCTGAAAT GGTATTAAAA CTGATTGCCA TGGATCCATA TGAGTATTTC 2460 CAAGTAGGCT GGAATATTTT TGACAGCCTT ATTGTGACTT TAAGTTTAGT GGAGCTCTTT 2520 CTAGCAGATG TGGAAGGATT GTCAGTTCTG CGATCATTCA GACTGCTCCG AGTCTTCAAG 2580 TTGGCAAAAT CCTGGCCAAC ATTGAACATG CTGATTAAGA TCATTGGTAA CTCAGTAGGG 2640 GCTCTAGGTA ACCTCACCTT AGTGTTGGCC ATCATCGTCT TCATTTTTGC TGTGGTCGGC 2700 ATGCAGCTCT TTGGTAAGAG CTACAAAGAA TGTGTCTGCA AGATCAATGA TGACTGTACG 2760 CTCCCACGGT GGCACATGAA CGACTTCTTC CACTCCTTCC TGATTGTGTT CCGCGTGCTG 2820 TGTGGAGAGT GGATAGAGAC CATGTGGGAC TGTATGGAGG TCGCTGGTCA AGCTATGTGC 2880 CTTATTGTTT ACATGATGGT CATGGTCATT GGAAACCTGG TGGTCCTAAA CCTATTTCTG 2940 GCCTTATTAT TGAGCTCATT TAGTTCAGAC AATCTTACAG CAATTGAAGA AGACCCTGAT 3000 GCAAACAACC TCCAGATTGC AGTGACTAGA ATTAAAAAGG GAATAAATTA TGTGAAACAA 3060 ACCTTACGTG AATTTATTCT AAAAGCATTT TCCAAAAAGC CAAAGATTTC CAGGGAGATA 3120 AGACAAGCAG AAGATCTGAA TACTAAGAAG GAAAACTATA TTTCTAACCA TACACTTGCT 3180 GAAATGAGCA AAGGTCACAA TTTCCTCAAG GAAAAAGATA AAATCAGTGG TTTTGGAAGC 3240 AGCGTGGACA AACACTTGAT GGAAGACAGT GATGGTCAAT CATTTATTCA CAATCCCAGC 3300 CTCACAGTGA CAGTGCCAAT TGCACCTGGG GAATCCGATT TGGAAAATAT GAATGCTGAG 3360 GAACTTAGCA GTGATTCGGA TAGTGAATAC AGCAAAGTGA GATTAAACCG GTCAAGCTCC 3420 TCAGAGTGCA GCACAGTTGA TAACCCTTTG CCTGGAGAAG GAGAAGAAGC AGAGGCTGAA 3480 CCTATGAATT CCGATGAGCC AGAGGCCTGT TTCACAGATG GTTGTGTACG GAGGTTCTCA 3540 TGCTGCCAAG TTAACATAGA GTCAGGGAAA GGAAAAATCT GGTGGAACAT CAGGAAAACC 3600 TGCTACAAGA TTGTTGAACA CAGTTGGTTT GAAAGCTTCA TTGTCCTCAT GATCCTGCTC 3660 AGCAGTGGTG CCCTGGCTTT TGAAGATATT TATATTGAAA GGAAAAAGAC CATTAAGATT 3720 ATCCTGGAGT ATGCAGACAA GATCTTCACT TACATCTTCA TTCTGGAAAT GCTTCTAAAA 3780 TGGATAGCAT ATGGTTATAA AACATATTTC ACCAATGCCT GGTGTTGGCT GGATTTCCTA 3840 ATTGTTGATG TTTCTTTGGT TACTTTAGTG GCAAACACTC TTGGCTACTC AGATCTTGGC 3900 CCCATTAAAT CCCTTCGGAC ACTGAGAGCT TTAAGACCTC TAAGAGCCTT ATCTAGATTT 3960 GAAGGAATGA GGGTCGTTGT GAATGCACTC ATAGGAGCAA TTCCTTCCAT CATGAATGTG 4020 CTACTTGTGT GTCTTATATT CTGGCTGATA TTCAGCATCA TGGGAGTAAA TTTGTTTGCT 4080 GGCAAGTTCT ATGAGTGTAT TAACACCACA GATGGGTCAC GGTTTCCTGC AAGTCAAGTT 4140 CCAAATCGTT CCGAATGTTT TGCCCTTATG AATGTTAGTC AAAATGTGCG ATGGAAAAAC 4200 CTGAAAGTGA ACTTTGATAA TGTCGGACTT GGTTACCTAT CTCTGCTTCA AGTTGCAACT 4260 TTTAAGGGAT GGACGATTAT TATGTATGCA GCAGTGGATT CTGTTAATGT AGACAAGCAG 4320 CCCAAATATG AATATAGCCT CTACATGTAT ATTTATTTTG TCGTCTTTAT CATCTTTGGG 4380 TCATTCTTCA CTTTGAACTT GTTCATTGGT GTCATCATAG ATAATTTCAA CCAACAGAAA 4440 AAGAAGCTTG GAGGTCAAGA CATCTTTATG ACAGAAGAAC AGAAGAAATA CTATAATGCA 4500 ATGAAAAAGC TGGGGTCCAA GAAGCCACAA AAGCCAATTC CTCGACCAGG GAACAAAATC 4560 CAAGGATGTA TATTTGACCT AGTGACAAAT CAAGCCTTTG ATATTAGTAT CATGGTTCTT 4620 ATCTGTCTCA ACATGGTAAC CATGATGGTA GAAAAGGAGG GTCAAAGTCA ACATATGACT 4680 GAAGTTTTAT ATTGGATAAA TGTGGTTTTT ATAATCCTTT TCACTGGAGA ATGTGTGCTA 4740 AAACTGATCT CCCTCAGACA CTACTACTTC ACTGTAGGAT GGAATATTTT TGATTTTGTG 4800 GTTGTGATTA TCTCCATTGT AGGTATGTTT CTAGCTGATT TGATTGAAAC GTATTTTGTG 4860 TCCCCTACCC TGTTCCGAGT GATCCGTCTT GCCAGGATTG GCCGAATCCT ACGTCTAGTC 4920 AAAGGAGCAA AGGGGATCCG CACGCTGCTC TTTGCTTTGA TGATGTCCCT TCCTGCGTTG 4980 TTTAACATCG GCCTCCTGCT CTTCCTGGTC ATGTTCATCT ACGCCATCTT TGGAATGTCC 5040 AACTTTGCCT ATGTTAAAAA GGAAGATGGA ATTAATGACA TGTTCAATTT TGAGACCTTT 5100 GGCAACAGTA TGATTTGCCT GTTCCAAATT ACAACCTCTG CTGGCTGGGA TGGATTGCTA 5160 GCACCTATTC TTAACAGTAA GCCACCCGAC TGTGACCCAA AAAAAGTTCA TCCTGGAAGT 5220 TCAGTTGAAG GAGACTGTGG TAACCCATCT GTTGGAATAT TCTACTTTGT TAGTTATATC 5280 ATCATATCCT TCCTGGTTGT GGTGAACATG TACATTGCAG TCATACTGGA GAATTTTAGT 5340 GTTGCCACTG AAGAAAGTAC TGAACCTCTG AGTGAGGATG ACTTTGAGAT GTTCTATGAG 5400 GTTTGGGAGA AGTTTGATCC CGATGCGACC CAGTTTATAG AGTTCTCTAA ACTCTCTGAT 5460 TTTGCAGCTG CCCTGGATCC TCCTCTTCTC ATAGCAAAAC CCAACAAAGT CCAGCTCATT 5520 GCCATGGATC TGCCCATGGT TAGTGGTGAC CGGATCCATT GTCTTGACAT CTTATTTGCT 5580 TTTACAAAGC GTGTTTTGGG TGAGAGTGGG GAGATGGATT CTCTTCGTTC ACAGATGGAA 5640 GAAAGGTTCA TGTCTGCAAA TCCTTCCAAA GTGTCCTATG AACCCATCAC AACCACACTA 5700 AAACGGAAAC AAGAGGATGT GTCTGCTACT GTCATTCAGC GTGCTTATAG ACGTTACCGC 5760 TTAAGGCAAA ATGTCAAAAA TATATCAAGT ATATACATAA AAGATGGAGA CAGAGATGAT 5820 GATTTACTCA ATAAAAAAGA TATGGCTTTT GATAATGTTA ATGAGAACTC AAGTCCAGAA 5880 AAAACAGATG CCACTTCATC CACCACCTCT CCACCTTCAT ATGATAGTGT AACAAAGCCA 5940 GACAAAGAGA AATATGAACA AGACAGAACA GAAAAGGAAG ACAAAGGGAA AGACAGCAAG 6000 GAAAGCAAAA AATAGAGCTT CATTTTTGAT ATATTGTTTA CAGCCTGTGA AAGTGATTTA 6060 TTTGTGTTAA TAAAACTCTT TTGAGGAAGT CTATGCCAAA ATCCTTTTTA TCAAAATATT 6120 CTCGAAGGCA GTGCAGTCAC TAACTCTGAT TTCCTAAGAA AGGTGGGCAG CATTAGCAGA 6180 TGGTTATTTT TGCACTGATG ATTCTTTAAG AATCGTAAGA GAACTCTGTA GGAATTATTG 6240 ATTATAGCAT ACAAAAGTGA TTGATTCAGT TTTTTGGTTT TTAATAAATC AGAAGACCAT 6300 GTAGAAAACT TTTACATCTG CCTTGTCATC TTTTCACAGG ATTGTAATTA GTCTTGTTTC 6360 CCATGTAAAT AAACAACACA CGCATACAGA AAAAAAAAAA AAAA 6404 1835 amino acids amino acid Not Relevant Not Relevant protein 15 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Glu Lys Lys Glu Lys 20 25 30 Glu Lys Lys Asp Asp Glu Glu Pro Lys Pro Ser Ser Asp Leu Glu Ala 35 40 45 Gly Lys Gln Leu Pro Phe Ile Tyr Gly Asp Ile Pro Pro Gly Met Val 50 55 60 Ser Glu Pro Leu Glu Asp Leu Asp Pro Tyr Tyr Ala Asp Lys Lys Thr 65 70 75 80 Phe Ile Val Leu Asn Lys Gly Lys Ile Phe Arg Phe Asn Ala Thr Pro 85 90 95 Ala Leu Tyr Met Leu Ser Pro Phe Ser Pro Leu Arg Arg Ile Ser Ile 100 105 110 Lys Ile Leu Val His Ser Leu Phe Ser Met Leu Ile Met Cys Thr Ile 115 120 125 Leu Thr Asn Cys Ile Phe Met Thr Asn Pro Pro Trp Thr Lys Asn Val 130 135 140 Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Lys Ile Leu Ala 145 150 155 160 Arg Gly Phe Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn 165 170 175 Trp Leu Asp Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val 180 185 190 Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala 195 200 205 Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala 210 215 220 Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val 225 230 235 240 Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly 245 250 255 Asn Leu Lys His Lys Cys Phe Arg Leu Glu Asn Glu Thr Leu Glu Ser 260 265 270 Ile Met Asn Thr Glu Ser Glu Glu Lys Tyr Phe Tyr Tyr Leu Glu Gly 275 280 285 Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp Ser Gly Gln Cys 290 295 300 Pro Glu Gly Tyr Cys Val Lys Gly Arg Asn Pro Asp Tyr Gly Tyr Thr 305 310 315 320 Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met 325 330 335 Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Gln Thr Leu Arg Ala Ala 340 345 350 Gly Lys Thr Tyr Met Ile Phe Phe Val Val Val Ile Phe Leu Gly Ser 355 360 365 Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu 370 375 380 Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala Lys Gln Lys Glu Leu Glu 385 390 395 400 Phe Gln Gln Met Leu Asp Arg Leu Lys Lys Glu Gln Glu Glu Ala Glu 405 410 415 Ala Ile Ala Ala Ala Ala Ala Glu Thr Ser Ile Arg Ser Arg Ile Met 420 425 430 Gly Leu Ser Glu Ser Ser Ser Glu Thr Ser Leu Ser Ser Lys Ser Ala 435 440 445 Lys Glu Arg Arg Asn Arg Arg Lys Lys Lys Gln Lys Lys Ser Ser Gly 450 455 460 Glu Glu Lys Gly Asp Glu Lys Leu Ser Lys Ser Ser Glu Ser Ile Arg 465 470 475 480 Lys Ser Phe His Leu Gly Val Glu Gly His Arg Glu Lys Arg Leu Ser 485 490 495 Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe Ser Ala 500 505 510 Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg Gly Arg 515 520 525 Asp Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser Ile Phe Gly 530 535 540 Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His Arg Pro Glu 545 550 555 560 Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser Pro Pro Leu Pro 565 570 575 Val Asn Gly Lys Met His Ser Ala Val Asp Cys Asn Gly Val Val Ser 580 585 590 Leu Val Asp Gly Ser Ala Leu Met Leu Pro Asn Gly Gln Leu Leu Pro 595 600 605 Glu Gly Thr Thr Asn Gln Lys Lys Arg Ser Ser Tyr Leu Ser Glu Asp 610 615 620 Met Leu Asn Asp Pro Leu Arg Gln Arg Ala Met Ser Arg Ala Ser Ile 625 630 635 640 Leu Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Tyr 645 650 655 Arg Phe Ala His Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile Lys 660 665 670 Phe Lys Lys Ile Tyr Phe Ile Val Met Asp Pro Phe Val Asp Leu Ala 675 680 685 Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His 690 695 700 His Pro Met Thr Glu Glu Phe Lys Asn Val Leu Ala Gly Asn Leu Phe 705 710 715 720 Thr Gly Ile Phe Ala Ala Glu Met Val Leu Lys Leu Ile Ala Met Asp 725 730 735 Pro Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe Asp Ser Leu Ile 740 745 750 Val Thr Leu Ser Leu Glu Leu Phe Leu Ala Asp Val Glu Gly Leu Ser 755 760 765 Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser 770 775 780 Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val Gly 785 790 795 800 Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile Phe 805 810 815 Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys Val 820 825 830 Cys Lys Ile Asn Asp Cys Leu Pro Arg Trp His Met Asn Asp Phe Phe 835 840 845 His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu Trp Ile Glu 850 855 860 Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Met Cys Leu Ile Val 865 870 875 880 Tyr Met Met Val Met Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe 885 890 895 Leu Ala Leu Leu Leu Ser Ser Phe Ser Ser Asp Asn Leu Thr Ala Ile 900 905 910 Glu Glu Asp Asp Ala Asn Asn Leu Gln Ile Ala Val Arg Ile Lys Gly 915 920 925 Ile Asn Tyr Val Lys Gln Thr Leu Arg Glu Phe Ile Leu Lys Phe Ser 930 935 940 Lys Lys Pro Lys Ser Asp Asn Lys Lys Glu Asn Tyr Ile Ser Asn Thr 945 950 955 960 Leu Ala Glu Met Ser Lys His Asn Phe Leu Lys Glu Lys Asp Ile Ser 965 970 975 Gly Gly Ser Ser Asp Lys Met Asp Gln Ser Phe Ile His Asn Pro Ser 980 985 990 Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu Glu Met 995 1000 1005 Asn Glu Glu Leu Ser Ser Asp Ser Asp Ser Tyr Ser Lys Asn Arg Ser 1010 1015 1020 Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly Glu Gly 1025 1030 1035 1040 Glu Glu Ala Glu Ala Glu Pro Asn Asp Glu Pro Glu Ala Cys Phe Thr 1045 1050 1055 Asp Gly Cys Val Arg Arg Phe Cys Cys Gln Val Asn Ser Gly Lys Gly 1060 1065 1070 Lys Trp Trp Ile Arg Lys Thr Cys Tyr Ile Val Glu His Ser Trp Phe 1075 1080 1085 Glu Ser Phe Ile Val Leu Met Ile Leu Leu Ser Ser Gly Ala Leu Ala 1090 1095 1100 Phe Glu Asp Ile Tyr Ile Glu Lys Lys Thr Ile Lys Ile Ile Leu Glu 1105 1110 1115 1120 Tyr Ala Asp Lys Ile Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu 1125 1130 1135 Lys Trp Ala Tyr Gly Tyr Lys Thr Tyr Phe Thr Asn Ala Trp Cys Trp 1140 1145 1150 Leu Asp Phe Leu Ile Val Asp Val Ser Leu Val Thr Leu Val Ala Asn 1155 1160 1165 Thr Leu Gly Tyr Ser Asp Leu Gly Pro Ile Lys Ser Leu Arg Thr Leu 1170 1175 1180 Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg 1185 1190 1195 1200 Val Val Val Asn Ala Leu Ile Gly Ala Ile Pro Ser Ile Met Asn Val 1205 1210 1215 Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val 1220 1225 1230 Asn Leu Phe Ala Gly Lys Phe Tyr Glu Cys Asn Thr Thr Asp Gly Ser 1235 1240 1245 Arg Phe Pro Ser Gln Val Asn Arg Ser Glu Cys Phe Ala Leu Met Asn 1250 1255 1260 Val Ser Asn Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val 1265 1270 1275 1280 Gly Leu Gly Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp 1285 1290 1295 Ile Met Tyr Ala Ala Val Asp Ser Val Asn Val Gln Pro Lys Tyr Glu 1300 1305 1310 Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val Phe Ile Ile Phe Gly Ser 1315 1320 1325 Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn 1330 1335 1340 Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met Thr Glu Glu 1345 1350 1355 1360 Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro 1365 1370 1375 Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Gln Gly Cys Ile Phe Asp 1380 1385 1390 Leu Thr Asn Gln Ala Phe Asp Ile Ile Met Val Leu Ile Cys Leu Asn 1395 1400 1405 Met Val Thr Met Met Val Glu Lys Glu Gly Gln Met Val Leu Trp Ile 1410 1415 1420 Asn Val Phe Ile Ile Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile 1425 1430 1435 1440 Ser Leu Arg His Tyr Tyr Phe Thr Val Gly Trp Asn Ile Phe Val Val 1445 1450 1455 Val Ile Ser Ile Val Gly Met Phe Leu Ala Ile Glu Tyr Phe Val Ser 1460 1465 1470 Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu 1475 1480 1485 Arg Leu Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met 1490 1495 1500 Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val 1505 1510 1515 1520 Met Phe Ile Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys 1525 1530 1535 Lys Glu Gly Ile Asn Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser 1540 1545 1550 Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu 1555 1560 1565 Leu Ala Pro Ile Leu Asn Ser Pro Pro Asp Cys Asp Pro Lys Lys Val 1570 1575 1580 His Pro Gly Ser Ser Val Glu Gly Asp Cys Gly Asn Pro Ser Val Gly 1585 1590 1595 1600 Ile Phe Tyr Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val Val 1605 1610 1615 Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe Ser Val Ala Thr Glu 1620 1625 1630 Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu 1635 1640 1645 Val Trp Glu Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Phe Lys 1650 1655 1660 Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Leu Ile Ala Lys 1665 1670 1675 1680 Pro Asn Lys Val Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly 1685 1690 1695 Asp Arg Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val 1700 1705 1710 Leu Gly Glu Gly Glu Met Asp Ser Leu Arg Ser Gln Met Glu Glu Arg 1715 1720 1725 Phe Met Ser Ala Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr 1730 1735 1740 Thr Leu Lys Arg Lys Gln Glu Val Ser Ala Thr Ile Gln Arg Ala Tyr 1745 1750 1755 1760 Arg Arg Tyr Arg Leu Arg Gln Val Lys Asn Ile Ser Ser Ile Tyr Ile 1765 1770 1775 Lys Asp Gly Asp Arg Asp Asp Asp Leu Asn Lys Asp Phe Asp Asn Val 1780 1785 1790 Asn Glu Asn Ser Ser Pro Glu Lys Thr Asp Thr Ser Thr Ser Pro Pro 1795 1800 1805 Ser Tyr Asp Ser Val Thr Lys Pro Asp Glu Lys Tyr Glu Asp Thr Glu 1810 1815 1820 Lys Glu Asp Lys Lys Asp Ser Lys Glu Ser Lys 1825 1830 1835 1969 amino acids amino acid Not Relevant linear protein 16 Met Ala Met Leu Pro Pro Pro Gly Pro Gln Ser Phe Val His Phe Thr 1 5 10 15 Lys Gln Ser Leu Ala Leu Ile Glu Gln Arg Ile Ala Glu Arg Lys Ser 20 25 30 Lys Glu Pro Lys Glu Glu Lys Lys Asp Asp Asp Glu Glu Ala Pro Lys 35 40 45 Pro Ser Ser Asp Leu Glu Ala Gly Lys Gln Leu Pro Phe Ile Tyr Gly 50 55 60 Asp Ile Pro Pro Gly Met Val Ser Glu Pro Leu Glu Asp Leu Asp Pro 65 70 75 80 Tyr Tyr Ala Asp Lys Lys Thr Phe Ile Val Leu Asn Lys Gly Lys Ala 85 90 95 Ile Phe Arg Phe Asn Ala Thr Pro Ala Leu Tyr Met Leu Ser Pro Phe 100 105 110 Ser Pro Leu Arg Arg Ile Ser Ile Lys Ile Leu Val His Ser Leu Phe 115 120 125 Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Ile Phe Met Thr 130 135 140 Met Asn Asn Pro Pro Asp Trp Thr Lys Asn Val Gly Tyr Thr Phe Thr 145 150 155 160 Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys Ile Leu Ala Arg Gly Phe 165 170 175 Cys Val Gly Glu Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp 180 185 190 Phe Val Val Ile Val Phe Ala Tyr Leu Thr Glu Phe Val Asn Leu Gly 195 200 205 Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys Thr 210 215 220 Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu Ile Gln 225 230 235 240 Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe Cys Leu 245 250 255 Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn Leu Lys 260 265 270 His Lys Cys Phe Arg Asn Ser Leu Glu Asn Asn Glu Thr Leu Glu Ser 275 280 285 Ile Met Asn Thr Leu Glu Ser Glu Glu Asp Phe Arg Lys Tyr Phe Tyr 290 295 300 Tyr Leu Glu Gly Ser Lys Asp Ala Leu Leu Cys Gly Phe Ser Thr Asp 305 310 315 320 Ser Gly Gln Cys Pro Glu Gly Tyr Thr Cys Val Lys Ile Gly Arg Asn 325 330 335 Pro Asp Tyr Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu 340 345 350 Ala Leu Phe Arg Leu Met Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln 355 360 365 Gln Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met Ile Phe Phe Val Val 370 375 380 Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala Val 385 390 395 400 Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Asn Ile Glu Glu Ala 405 410 415 Lys Gln Lys Glu Leu Glu Phe Gln Gln Met Leu Asp Arg Leu Lys Lys 420 425 430 Glu Gln Glu Glu Ala Glu Ala Ile Ala Ala Ala Ala Ala Glu Tyr Thr 435 440 445 Ser Ile Arg Arg Ser Arg Ile Met Gly Leu Ser Glu Ser Ser Ser Glu 450 455 460 Thr Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 465 470 475 480 Lys Lys Lys Asn Gln Lys Lys Leu Ser Ser Gly Glu Glu Lys Gly Asp 485 490 495 Ala Glu Lys Leu Ser Lys Ser Glu Ser Glu Asp Ser Ile Arg Arg Lys 500 505 510 Ser Phe His Leu Gly Val Glu Gly His Arg Arg Ala His Glu Lys Arg 515 520 525 Leu Ser Thr Pro Asn Gln Ser Pro Leu Ser Ile Arg Gly Ser Leu Phe 530 535 540 Ser Ala Arg Arg Ser Ser Arg Thr Ser Leu Phe Ser Phe Lys Gly Arg 545 550 555 560 Gly Arg Asp Xaa Gly Ser Glu Thr Glu Phe Ala Asp Asp Glu His Ser 565 570 575 Ile Phe Gly Asp Asn Glu Ser Arg Arg Gly Ser Leu Phe Val Pro His 580 585 590 Arg Pro Xaa Glu Arg Arg Ser Ser Asn Ile Ser Gln Ala Ser Arg Ser 595 600 605 Pro Pro Met Leu Pro Val Asn Gly Lys Met His Ser Ala Val Asp Cys 610 615 620 Asn Gly Val Val Ser Leu Val Asp Gly Xaa Ser Ala Leu Met Leu Pro 625 630 635 640 Asn Gly Gln Leu Leu Pro Glu Gly Thr Thr Asn Gln Ile His Lys Lys 645 650 655 Arg Arg Cys Ser Ser Tyr Leu Leu Ser Glu Asp Met Leu Asn Asp Pro 660 665 670 Asn Leu Arg Gln Arg Ala Met Ser Arg Ala Ser Ile Leu Thr Asn Thr 675 680 685 Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro Pro Trp Trp Tyr 690 695 700 Arg Phe Ala His Lys Phe Leu Ile Trp Asn Cys Ser Pro Tyr Trp Ile 705 710 715 720 Lys Phe Lys Lys Cys Ile Tyr Phe Ile Val Met Asp Pro Phe Val Asp 725 730 735 Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met 740 745 750 Glu His His Pro Met Thr Glu Glu Phe Lys Asn Val Leu Ala Ile Gly 755 760 765 Asn Leu Val Phe Thr Gly Ile Phe Ala Ala Glu Met Val Leu Lys Leu 770 775 780 Ile Ala Met Asp Pro Tyr Glu Tyr Phe Gln Val Gly Trp Asn Ile Phe 785 790 795 800 Asp Ser Leu Ile Val Thr Leu Ser Leu Val Glu Leu Phe Leu Ala Asp 805 810 815 Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe 820 825 830 Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile 835 840 845 Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile 850 855 860 Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser 865 870 875 880 Tyr Lys Glu Cys Val Cys Lys Ile Asn Asp Asp Cys Thr Leu Pro Arg 885 890 895 Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg Val 900 905 910 Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val Ala 915 920 925 Gly Gln Ala Met Cys Leu Ile Val Tyr Met Met Val Met Val Ile Gly 930 935 940 Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 945 950 955 960 Ser Ser Asp Asn Leu Thr Ala Ile Glu Glu Asp Pro Asp Ala Asn Asn 965 970 975 Leu Gln Ile Ala Val Thr Arg Ile Lys Lys Gly Ile Asn Tyr Val Lys 980 985 990 Gln Thr Leu Arg Glu Phe Ile Leu Lys Ala Phe Ser Lys Lys Pro Lys 995 1000 1005 Ile Ser Arg Glu Ile Arg Gln Ala Glu Asp Leu Asn Thr Lys Lys Glu 1010 1015 1020 Asn Tyr Ile Ser Asn Met Thr Leu Ala Glu Met Ser Lys Gly His Asn 1025 1030 1035 1040 Phe Leu Lys Glu Lys Asp Lys Ile Ser Gly Phe Gly Ser Ser Xaa Asp 1045 1050 1055 Lys His Leu Met Glu Asp Ser Asp Gly Gln Ser Phe Ile His Asn Pro 1060 1065 1070 Ser Leu Thr Val Thr Val Pro Ile Ala Pro Gly Glu Ser Asp Leu Glu 1075 1080 1085 Met Asn Glu Glu Leu Ser Ser Asp Ser Asp Ser Tyr Ser Lys Asn Arg 1090 1095 1100 Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Asn Pro Leu Pro Gly Glu 1105 1110 1115 1120 Gly Glu Glu Ala Glu Ala Glu Pro Asn Asp Glu Pro Glu Ala Cys Phe 1125 1130 1135 Thr Asp Gly Cys Val Arg Arg Phe Ser Cys Cys Gln Val Asn Ile Glu 1140 1145 1150 Ser Gly Lys Gly Lys Ile Trp Trp Asn Ile Arg Lys Thr Cys Tyr Lys 1155 1160 1165 Ile Val Glu His Ser Trp Phe Glu Ser Phe Ile Val Leu Met Ile Leu 1170 1175 1180 Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile Glu Arg Lys 1185 1190 1195 1200 Lys Thr Ile Lys Ile Ile Leu Glu Tyr Ala Asp Lys Ile Phe Thr Tyr 1205 1210 1215 Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Ile Ala Tyr Gly Tyr Lys 1220 1225 1230 Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asp 1235 1240 1245 Val Ser Leu Val Thr Leu Val Ala Asn Thr Leu Gly Tyr Ser Asp Leu 1250 1255 1260 Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg 1265 1270 1275 1280 Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu Ile 1285 1290 1295 Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile Phe 1300 1305 1310 Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys Phe 1315 1320 1325 Tyr Glu Cys Ile Asn Thr Thr Asp Gly Ser Arg Phe Pro Ala Ser Gln 1330 1335 1340 Val Pro Asn Arg Ser Glu Cys Phe Ala Leu Met Asn Val Ser Gln Asn 1345 1350 1355 1360 Val Arg Trp Lys Asn Leu Lys Val Asn Phe Asp Asn Val Gly Leu Gly 1365 1370 1375 Tyr Leu Ser Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Thr Ile Ile 1380 1385 1390 Met Tyr Ala Ala Val Asp Ser Val Asn Val Asp Lys Gln Pro Lys Tyr 1395 1400 1405 Glu Tyr Ser Leu Tyr Met Tyr Ile Tyr Phe Val Val Phe Ile Ile Phe 1410 1415 1420 Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn 1425 1430 1435 1440 Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met Thr 1445 1450 1455 Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys 1460 1465 1470 Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Ile Gln Gly Cys 1475 1480 1485 Ile Phe Asp Leu Val Thr Asn Gln Ala Phe Asp Ile Ser Ile Met Val 1490 1495 1500 Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Lys Glu Gly Gln 1505 1510 1515 1520 Ser Gln His Met Thr Glu Val Leu Tyr Trp Ile Asn Val Val Phe Ile 1525 1530 1535 Ile Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile Ser Leu Arg His 1540 1545 1550 Tyr Tyr Phe Thr Val Gly Trp Asn Ile Phe Asp Phe Val Val Val Ile 1555 1560 1565 Ile Ser Ile Val Gly Met Phe Leu Ala Asp Leu Ile Glu Thr Tyr Phe 1570 1575 1580 Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg 1585 1590 1595 1600 Ile Leu Arg Leu Val Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe 1605 1610 1615 Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu 1620 1625 1630 Phe Leu Val Met Phe Ile Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala 1635 1640 1645 Tyr Val Lys Lys Glu Asp Gly Ile Asn Asp Met Phe Asn Phe Glu Thr 1650 1655 1660 Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly 1665 1670 1675 1680 Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Lys Pro Pro Asp Cys 1685 1690 1695 Asp Pro Lys Lys Val His Pro Gly Ser Ser Val Glu Gly Asp Cys Gly 1700 1705 1710 Asn Pro Ser Val Gly Ile Phe Tyr Phe Val Ser Tyr Ile Ile Ile Ser 1715 1720 1725 Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe 1730 1735 1740 Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe 1745 1750 1755 1760 Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp Ala Thr Gln 1765 1770 1775 Phe Ile Glu Phe Ser Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro 1780 1785 1790 Pro Leu Leu Ile Ala Lys Pro Asn Lys Val Gln Leu Ile Ala Met Asp 1795 1800 1805 Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp Ile Leu Phe 1810 1815 1820 Ala Phe Thr Lys Arg Val Leu Gly Glu Ser Gly Glu Met Asp Ser Leu 1825 1830 1835 1840 Arg Ser Gln Met Glu Glu Arg Phe Met Ser Ala Asn Pro Ser Lys Val 1845 1850 1855 Ser Tyr Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln Glu Xaa Val 1860 1865 1870 Ser Ala Thr Val Ile Gln Arg Ala Tyr Arg Arg Tyr Arg Leu Arg Gln 1875 1880 1885 Asn Val Lys Asn Ile Ser Ser Ile Tyr Ile Lys Asp Gly Asp Arg Asp 1890 1895 1900 Asp Asp Leu Leu Asn Lys Glu Asp Met Ala Phe Asp Asn Val Asn Glu 1905 1910 1915 1920 Asn Ser Ser Pro Glu Lys Thr Asp Ala Thr Ser Ser Thr Thr Ser Pro 1925 1930 1935 Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Lys Glu Lys Tyr Glu Xaa 1940 1945 1950 Asp Gln Thr Glu Lys Glu Asp Lys Gly Lys Asp Ser Lys Glu Ser Lys 1955 1960 1965 Lys 21 base pairs nucleic acid single linear cDNA 17 TTTGTGCCCC ACAGACCCCA G 21 26 base pairs nucleic acid single linear cDNA 18 ACACAAATTC TTGATCTGGA ATTGCT 26 23 base pairs nucleic acid single linear cDNA 19 CAACCTCAGA CAGAGAGCAA TGA 23

Claims (7)

1-16. (Cancelled)
17. A bioassay for assessing a candidate modulating agent of a PNS SCP, comprising:
(A) contacting a candidate agent with a cell line expressing in the cell membrane of said cell a PNS SCP; and
(B) evaluating the modulation of the SC biological activity of said cell mediated by said contacting of said candidate agent.
18. A method according to claim 17, wherein said cell line is selected from PC12 cells or a recombinant form thereof comprising an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule encodes a polypeptide monomer of a peripheral nervous system specific (PNS) sodium channel the monomer: (i) having an amino acid sequence that has at least 87% identity to the full amino acid sequence disclosed in SEQ ID NO:10 and (ii) forming a PNS type I voltage gated sodium channel sodium ions when the monomer is expressed in Xenopus oocytes.
19. A PNS SCP modulating agent, identified by a method according to claim 17.
20. A PNS SCP modulating agent according to claim 19, wherein said agent is a methyl-phenylhalophenyl-substituted piperizine compound.
21. A PNS SCP modulating agent according to claim 20, wherein said piperizine compound is lidoflazine (Merck Index Monograph 5311) or a derivative thereof.
22-39. (Cancelled)
US10/768,798 1994-11-02 2004-01-29 Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof Abandoned US20040229792A1 (en)

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US11/810,919 US8012748B2 (en) 1994-11-02 2007-06-06 Peripheral nervous system specific sodium channel nucleic acids
US11/810,882 US20090029415A1 (en) 1994-11-02 2007-06-06 Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof

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US09/457,571 US6703486B2 (en) 1994-11-02 1999-12-09 Peripheral nervous system specific sodium channels
US10/768,798 US20040229792A1 (en) 1994-11-02 2004-01-29 Peripheral nervous system specific sodium channels, DNA encoding therefor, crystallization, X-ray diffraction, computer molecular modeling, rational drug design, drug screening, and methods of making and using thereof

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Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE407207T1 (en) * 1994-11-02 2008-09-15 Trophix Pharm Inc SODIUM ION CHANNELS SPECIFIC TO THE PERIPHERAL NERVOUS SYSTEM, DNA WHICH ENCODES THEM, SEARCH FOR ACTIVE INGREDIENTS AND METHODS FOR THEIR PRODUCTION AND USE
US6184349B1 (en) 1995-10-11 2001-02-06 Syntex (Usa) Inc. Cloned peripheral nerve, tetrodotoxin-resistant sodium channel α-subunit
US6030810A (en) 1997-02-26 2000-02-29 Delgado; Stephen Gregory Cloned tetrodotoxin-sensitive sodium channel α-subunit and a splice variant thereof
EP0875570A3 (en) * 1997-05-01 1999-12-22 Smithkline Beecham Plc Neurodegenerative polypeptides HHPDZ65
CH693031A5 (en) * 1997-11-20 2003-01-31 Hoffmann La Roche Nucleic acid encoding a sodium channel nerve tissue.
AU3908700A (en) * 1999-03-26 2000-10-16 Human Genome Sciences, Inc. 48 human secreted proteins
US7083777B1 (en) * 1999-04-02 2006-08-01 The Brigham And Women's Hospital, Inc. Immunomodulating polymers
CA2394229C (en) 1999-11-26 2010-04-13 Mcgill University Loci for idiopathic generalized epilepsy, mutations thereof and method using same to assess, diagnose, prognose or treat epilepsy
WO2002002608A2 (en) * 2000-07-05 2002-01-10 Neurosearch A/S NEW SODIUM CHANNEL (rNaV1.5a) AND USE THEREOF
GB0021617D0 (en) * 2000-09-02 2000-10-18 Imp College Innovations Ltd Diagnosis and treatment of cancer
AU2001294141A1 (en) * 2000-09-19 2002-04-02 Gbf Gesellschaft Fur Biotechnologische Forschung Mbh Crystalline fgf9 dimer and methods of use
DE10054878A1 (en) * 2000-11-06 2002-05-08 Boehringer Ingelheim Int Peripheral nerve type sodium channels
SV2003000753A (en) * 2000-12-05 2003-06-16 Brigham & Womens Hospital USE OF ZWITTERIONIC POLYSACARIDS FOR THE SPECIFIC MODULATION OF IMMUNE PROGRESS
EP1468293A1 (en) * 2002-01-17 2004-10-20 EVOTEC Neurosciences GmbH Diagnostic and therapeutic use of the voltage-gated ion channel scn2a for neurodegenerative diseases
US9278088B2 (en) 2002-02-19 2016-03-08 Xenon Pharmaceuticals Inc. Methods for identifying analgesic agents
US7659082B2 (en) 2002-02-19 2010-02-09 Xenon Pharmaceuticals Inc. Methods for identifying analgesic agents
CA2474751A1 (en) 2002-02-25 2003-09-04 Vanderbilt University Expression system for human brain-specific voltage-gated sodium channel, type 1
WO2004050857A2 (en) * 2002-12-04 2004-06-17 Euro-Celtique S.A. Splice variant of human sodium iii channel (hnaiii18)
EP1638581A2 (en) * 2003-03-31 2006-03-29 The Brigham And Women's Hospital, Inc. Zwitterionic immunomodulators for the treatment of asthma and allergy
WO2005069969A2 (en) * 2004-01-21 2005-08-04 University Of Utah Research Foundation Mutant sodium channel nav1.7 and methods related thereto
US20050201995A1 (en) * 2004-02-05 2005-09-15 Board Of Regents, The University Of Texas System Methods and compositions related to neuronal differentiation
US7485468B2 (en) 2004-10-15 2009-02-03 Galapagos Bv Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
US8206726B2 (en) 2006-02-06 2012-06-26 The Brigham And Women's Hospital, Inc. Zwitterionic polysaccharides for promotion of immune system maturation and health
US20100247435A1 (en) * 2007-06-14 2010-09-30 Erik Arstad Measurement of neural activity
US20120283116A1 (en) 2009-09-11 2012-11-08 Leppert Mark F Mutant Sodium Channel Nav1.7 and Methods Related Thereto
WO2013009945A1 (en) 2011-07-12 2013-01-17 The Brigham And Women's Hospital, Inc. Lipid-containing psa compositions, methods of isolation and methods of use thereof
WO2017031431A1 (en) 2015-08-19 2017-02-23 President And Fellows Of Harvard College Lipidated psa compositions and methods
EP3484441A4 (en) 2016-07-15 2020-03-18 President and Fellows of Harvard College Glycolipid compositions and methods of use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3267104A (en) * 1964-06-09 1966-08-16 Janssen Pharmaceutica Nv 1, 4-disubstituted piperazines and diazepines
US5356777A (en) * 1992-10-01 1994-10-18 University Of Pittsburgh Methods of detecting periodic paralysis in horses

Family Cites Families (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4500530A (en) * 1980-11-26 1985-02-19 Boucher John H Method of treating horses to inhibit or reduce increases in crenated red blood cells during exercise
US4405712A (en) 1981-07-01 1983-09-20 The United States Of America As Represented By The Department Of Health And Human Services LTR-Vectors
NZ201918A (en) 1981-09-18 1987-04-30 Genentech Inc N-terminal methionyl analogues of bovine growth hormone
US4656134A (en) 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
JPS6147500A (en) 1984-08-15 1986-03-07 Res Dev Corp Of Japan Chimera monoclonal antibody and its preparation
EP0173494A3 (en) 1984-08-27 1987-11-25 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by dna splicing and expression
GB8422238D0 (en) 1984-09-03 1984-10-10 Neuberger M S Chimeric proteins
US4699880A (en) 1984-09-25 1987-10-13 Immunomedics, Inc. Method of producing monoclonal anti-idiotype antibody
JPS61134325A (en) 1984-12-04 1986-06-21 Teijin Ltd Expression of hybrid antibody gene
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4766067A (en) 1985-05-31 1988-08-23 President And Fellows Of Harvard College Gene amplification
WO1987002671A1 (en) 1985-11-01 1987-05-07 International Genetic Engineering, Inc. Modular assembly of antibody genes, antibodies prepared thereby and use
GB8601597D0 (en) 1986-01-23 1986-02-26 Wilson R H Nucleotide sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
US5322770A (en) 1989-12-22 1994-06-21 Hoffman-Laroche Inc. Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription
US4921794A (en) 1987-01-14 1990-05-01 President And Fellows Of Harvard College T7 DNA polymerase
US4795699A (en) 1987-01-14 1989-01-03 President And Fellows Of Harvard College T7 DNA polymerase
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5142033A (en) 1988-09-23 1992-08-25 Hoffmann-La Roche Inc. Structure-independent DNA amplification by the polymerase chain reaction
US5091310A (en) 1988-09-23 1992-02-25 Cetus Corporation Structure-independent dna amplification by the polymerase chain reaction
US5066584A (en) 1988-09-23 1991-11-19 Cetus Corporation Methods for generating single stranded dna by the polymerase chain reaction
US5082670A (en) 1988-12-15 1992-01-21 The Regents Of The University Of California Method of grafting genetically modified cells to treat defects, disease or damage or the central nervous system
US4994370A (en) 1989-01-03 1991-02-19 The United States Of America As Represented By The Department Of Health And Human Services DNA amplification technique
WO1990009441A1 (en) 1989-02-01 1990-08-23 The General Hospital Corporation Herpes simplex virus type i expression vector
WO1990009391A1 (en) * 1989-02-13 1990-08-23 Arch Development Corporation Nucleic acid encoding sodium channel protein
US5380836A (en) * 1989-02-13 1995-01-10 Arch Development Corporation Nucleic acid encoding sodium channel protein
DE69120699T2 (en) * 1990-03-08 1997-01-09 Fujisawa Pharmaceutical Co N-Monosubstituted cyclopentenylamines, a process for their preparation, and their use as a medicament
IE911869A1 (en) 1990-06-01 1991-12-04 Regeneron Pharma A family of map2 protein kinases
DE69131321T2 (en) 1990-09-28 1999-12-16 F. Hoffmann-La Roche Ag, Basel MUTATIONS IN 5'-3'-EXONUCLEASE ACTIVITY OF THERMOSTABLE DNA POLYMERASES
WO1992006693A1 (en) 1990-10-22 1992-04-30 Fox Chase Cancer Center Dna construct for providing rna therapy
AU2468992A (en) 1991-08-16 1993-03-16 General Hospital Corporation, The Method of gene delivery to post-mitotic cells
US5338671A (en) 1992-10-07 1994-08-16 Eastman Kodak Company DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody
US5340728A (en) 1992-12-09 1994-08-23 E. I. Du Pont De Nemours And Company Method for amplification of targeted segments of nucleic acid using nested polymerase chain reaction
WO1994014978A1 (en) 1992-12-18 1994-07-07 Strack Hans B Improved yielding of pcr products in the presence of single-strand dna binding protein and preparation thereof from thermus flavus
ATE407207T1 (en) * 1994-11-02 2008-09-15 Trophix Pharm Inc SODIUM ION CHANNELS SPECIFIC TO THE PERIPHERAL NERVOUS SYSTEM, DNA WHICH ENCODES THEM, SEARCH FOR ACTIVE INGREDIENTS AND METHODS FOR THEIR PRODUCTION AND USE

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3267104A (en) * 1964-06-09 1966-08-16 Janssen Pharmaceutica Nv 1, 4-disubstituted piperazines and diazepines
US5356777A (en) * 1992-10-01 1994-10-18 University Of Pittsburgh Methods of detecting periodic paralysis in horses

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US6703486B2 (en) 2004-03-09
EP0789575B1 (en) 2008-09-03
CA2204355C (en) 2001-01-16
CA2204355A1 (en) 1996-05-17
US20090029415A1 (en) 2009-01-29
AU697465B2 (en) 1998-10-08
US20090203122A1 (en) 2009-08-13
DK0789575T3 (en) 2009-01-12
DE69535830D1 (en) 2008-10-16
JP2007169286A (en) 2007-07-05

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