Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6478—Aspartic endopeptidases (3.4.23)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• TECHNICAL FIELD This invention relates to nucleic acid and amino acid sequences of aspartic proteases and to the use of these sequences in the diagnosis, treatment, and prevention of respiratory, endocrinological, and immunological disorders, and cancer.
• Proteolytic processing is an essential component of normal cell growth, differentiation, remodeling, and homeostasis.
• the cleavage of peptide bonds within cells is necessary for the maturation of precursor proteins to their active form, the removal of signal sequences from targeted proteins, the degradation of incorrectly folded proteins, and the controlled turnover of peptides within the cell.
• Proteases participate in apoptosis, inflammation, and in tissue remodeling during embryonic development, wound healing, and normal growth. They are necessary components of bacterial, parasitic, and viral invasion and replication within a host.
• Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (Beynon, R.J. and J.S. Bond ( 1994) Proteolytic Enzymes: A Practical Approach. Oxford University Press, New York, NY, pp. 1-5.)
• cysteine proteases involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation of proteins. Cysteine proteases are produced by monocytes, macrophages and other cells ofthe immune system which migrate to sites of inflammation and in their protective role secrete various molecules to repair damaged tissue. These cells may overproduce the same molecules and cause tissue destruction in certain disorders.
• the cathepsin family of lysosomal proteases includes the cysteine proteases, including cathepsins B, H, K, L, 02, and S, and the aspartic proteases, including pepsin A, gastricsin, chymosin, renin, and cathepsins D and E.
• cathepsin D have a ubiquitous tissue distribution while others, such as cathepsin L, are found only in monocytes, macrophages, and other cells of the immune system.
• the characteristic active site residues of aspartic proteases are a pair of aspartic acid residues, e.g., asp33 and asp213 in penicillopepsin.
• Aspartic proteases are also called acid proteases because the optimum pH for activity is between 2 and 3. In this pH range, one ofthe aspartate residues is ionized, the other un-ionized.
• a potent inhibitor of aspartic proteases is the hexapeptide, pepstatin, which in the transition state resembles the normal substrate. Abnormal regulation and expression of cathepsins is evident in various inflammatory disease states.
• autoimmune diseases such as rheumatoid arthritis
• the secretion ofthe cysteine protease, cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones.
• the mRNA for stromelysin, cytokines, TIMP-1, cathepsin, gelatinase, and other molecules is preferentially expressed.
• Expression of cathepsins L and D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis.
• Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction ofthe rheumatoid synovium.
• the increased expression and differential regulation ofthe cathepsins is linked to the metastatic potential of a variety of cancers and as such is of therapeutic and prognostic interest.
• the invention features substantially purified polypeptides, aspartic proteases, referred to collectively as "NHAP” and individually as “NHAP-1 " and “NHAP-2.”
• NHAP substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1 , and a fragment of SEQ ID NO:3.
• the invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO:3, or to a fragment of either of these sequences.
• the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: 1 , and a fragment of SEQ ID NO:3.
• the invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, a fragment of SEQ ID NO: 1 , and a fragment of SEQ ID NO:3.
• the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: l, and a fragment of SEQ ID NO:3, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, a fragment of SEQ ID NO:l, and a fragment of SEQ ID NO:3.
• the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.
• the invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.
• the invention further provides an expression vector containing at least a fragment ofthe polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3.
• the expression vector is contained within a host cell.
• the invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression ofthe polypeptide; and (b) recovering the polypeptide from the host cell culture.
• the invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1 , and a fragment of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
• the invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, a fragment of SEQ ID NO: l, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to the polypeptide.
• the invention also provides a method for treating or preventing an endocrinological disorder associated with decreased expression or activity of NHAP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO:l, and a fragment of SEQ ID NO:3.
• the invention also provides a method for treating or preventing an endocrinological disorder associated with increased expression or activity of NHAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist ofthe polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3
• the invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist ofthe polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3.
• the invention also provides a method for treating or preventing an immunological disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist ofthe polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3.
• the invention also provides a method for treating or preventing a respiratory disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist ofthe polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3.
• the invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1 , and a fragment of SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement ofthe polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3 to at least one ofthe nucleic acids ofthe biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample.
• the method further comprises amplifying the polynucle
• Figures 1A, IB, IC, and I D show the amino acid sequence (SEQ ID NO:l) and nucleic acid sequence (SEQ ID NO:2) of NHAP- 1.
• the alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd., San Bruno, CA).
• Figures 2A, 2B, 2C, and 2D show the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of NHAP-2.
• the alignment was produced using MacDNASIS PROTM software
• Figures 3 A, 3B, and 3C show the amino acid sequence alignments among NHAP-1 (372637; SEQ ID NO: l), NHAP-2 (2435410; SEQ ID NO:3), and a mouse kidney-derived, aspartic protease-like protein (GI 1906810; SEQ ID NO: 10), produced using the multisequence alignment program of LASERGENETM software (DNASTAR Inc, Madison WI).
• Figure 4 shows the northern analysis of NHAP-1 and NHAP-2 probed with NHAP-2 cDNA. Tissue blots were obtained from Clontech, Palo Alto, CA.
• Figure 5 shows the northern analysis of NHAP-1 probed with NHAP-1 -specific oligonucleotide using the same tissue blots as in Figure 4.
• Figure 6 shows western analysis of recombinant NHAP-1 protein expression in Escherichia coli. Competent E. Coli strain BL21 (DE3) was transformed with either vector (pETl 5b) or with NHAP- 1 expression construct (pETl 5b/NHAP- 1 ). Cell lysates from cultures before IPTG induction (P) or after IPTG induction (I) were separated using polyacrylamide gel electrophoresis under reduced denatured conditions, and probed with preimmune and immune serums (IC620).
• P IPTG induction
• I IPTG induction
• Table 1 shows the Incyte clone and the associated library in which nucleic acid sequences encoding NHAP were identified, a brief description ofthe library, and the vector into which each cDNA was cloned.
• Table 2 summarizes the databases and tools used to assemble and analyze the sequences ofthe invention.
• the first column of Table 2 shows the tool, program, or algorithm; the second column, the database; the third column, a brief description; and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).
• NHAP refers to the amino acid sequences, or variant thereof, of substantially purified NHAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
• agonist refers to a molecule which, when bound to NHAP, increases or prolongs the duration ofthe effect of NHAP.
• Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of NHAP.
• allelic variant is an alternative form ofthe gene encoding NHAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• altered nucleic acid sequences encoding NHAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as NHAP or a polypeptide with at least one functional characteristic of NHAP. Included within this definition are polymo ⁇ hisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NHAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding NHAP.
• the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent NHAP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NHAP is retained.
• negatively charged amino acids may include aspartic acid and glutamic acid
• positively charged amino acids may include lysine and arginine
• amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
• amino acid or amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
• fragments refer to fragments of NHAP which are preferably at least 5 to about 1 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of NHAP.
• amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
• Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C.W. and G.S. Dveksler (1995) PCR Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview, NY, pp.1-5.)
• PCR polymerase chain reaction
• Antagonist refers to a molecule which, when bound to NHAP, decreases the amount or the duration ofthe effect ofthe biological or immunological activity of NHAP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of NHAP.
• antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
• Antibodies that bind NHAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
• antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
• an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
• biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
• immunologically active refers to the capability ofthe natural, recombinant, or synthetic NHAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• complementarity refers to the natural binding of polynucleotides by base pairing.
• sequence 5' A-G-T 3'
• complementary sequence 3' T-C-A 5'.
• Complementarity between two single-stranded molecules may be "partial,” such that only some ofthe nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules.
• the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength ofthe hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
• PNA peptide nucleic acid
• composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
• the composition may comprise a dry formulation or an aqueous solution.
• Compositions comprising polynucleotide sequences encoding NHAP or fragments of NHAP may be employed as hybridization probes.
• the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
• the probe may be deployed in an aqueous solution containing salts, e.g., NaCl, detergents, e.g.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
• salts e.g., NaCl
• detergents e.g.,sodium dodecyl sulfate (SDS)
• SDS sodium dodecyl sulfate
• Denhardt's solution e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
• Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (The Perkin-Elmer Corp., Norwalk, CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GEL VIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
• the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding NHAP, by Northern analysis is indicative ofthe presence of nucleic acids encoding NHAP in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding NHAP.
• a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
• a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
• similarity refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.”
• a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
• the inhibition of hybridization ofthe completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency.
• a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency.
• Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlignTM program (DNASTAR, Inc., Madison WI). The MegAlignTM program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
• the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum ofthe residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
• HACs Human artificial chromosomes
• HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J.J. et al. (1997) Nat Genet. 15:345-355.)
• humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
• Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
• a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
• insertion or “addition,” as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
• Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
• microarray refers to an arrangement of distinct polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
• element or “array element” as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
• modulate refers to a change in the activity of NHAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NHAP.
• nucleic acid refers to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
• fragment refers to those nucleic acid sequences which, comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:2 and SEQ ID NO:4, for example, as distinct from any other sequence in the same genome.
• a fragment of SEQ ID NO:2 or a fragment of SEQ ID NO:4 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:2 or SEQ ID NO:4 from related polynucleotide sequences.
• a fragment of SEQ ID NO:2 or a fragment of SEQ ID NO:4 is at least about 15-20 nucleotides in length.
• the precise length ofthe fragments of SEQ ID NO:2 or SEQ ID NO:4 and the regions of SEQ ID NO:2 and SEQ ID NO:4 to which the fragments correspond are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
• a fragment when translated would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, ofthe full-length polypeptide.
• operably associated refers to functionally related nucleic acid sequences.
• a promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
• oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
• oligonucleotide is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
• PNA protein nucleic acid
• PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
• PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)
• sample as used herein, is used in its broadest sense.
• a biological sample suspected of containing nucleic acids encoding NHAP, or fragments thereof, or NHAP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solid support; a tissue; a tissue print; etc.
• the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure ofthe protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
• stringent conditions refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides.
• Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
• substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
• substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
• Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection. and particle bombardment.
• transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
• a “variant" of NHAP polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
• the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
• Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENETM software.
• variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to NHAP. This definition may also include, for example, "allelic” (as defined above), “splice,” “species,” or “polymorphic” variants.
• a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
• the corresponding polypeptide may possess additional functional domains or an absence of domains.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
• a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
• Polymorphic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• the invention is based on the discovery of two new human aspartic proteases (NHAP), the polynucleotides encoding NHAP, and the use of these compositions for the diagnosis, treatment, or prevention of respiratory, endocrinological, and immunological disorders, and cancer.
• NHAP human aspartic proteases
• Nucleic acids encoding the NHAP-1 and NHAP-2 ofthe present invention were identified in the following Incyte Clones: (SEQ ID NO:5 through 9) 372637H1 (LUNGNOT02), 1242901H1 (LUNGNOT03), 2222291H1 (LUNGNOT18), 2435410H1 (EOSINOT03), and 2756549H1 (THP1AZS08) using a computer search, e.g., BLAST, for amino acid sequence alignments.
• the full length cDNA sequence of NHAP-1 was obtained from a human lung cDNA library using the GeneTrapperTM method (Life Technologies, Gaithersburg MD) and oligonucleotides derived from Incyte clone 2756549 (THP1AZS08).
• the full length cDNA sequence of NHAP-2 was obtained from a human leukocyte cDNA library using the GeneTrapperTM method (Life Technologies) and the same oligonucleotides as were used for NHAP- 1.
• the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:l, as shown in Figures 1A, IB, IC, and ID.
• NHAP-1 is 420 amino acids in length and has a potential signal peptide sequence extending from residues Ml to P21.
• Potential N- glycosylation sites are found at residues N90, N133, and N336.
• Potential phosphorylation sites are found for casein kinase II at S60 and T338, and for protein kinase C at S106, T143, T346, and S393.
• Two potential leucine zipper patterns are found beginning at L309 and L316, and a potential cell attachment site is found in the sequence R387GD.
• NHAP-1 has chemical and structural similarity with a mouse aspartic protease-like protein (GI 1906810; SEQ ID NO: 10).
• GI 1906810 mouse aspartic protease-like protein
• NHAP-1 and the mouse aspartic protease-like protein share 69% identity.
• the two proteins share the signal sequence, the three potential glycosylation sites, and the potential phosphorylations sites found in NHAP-1 at S106, T143, and T338.
• the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:3, as shown in Figures 2A, 2B, 2C, and 2D.
• NHAP-2 is 433 amino acids in length and has a potential signal sequence extending from residues Ml to P21 , three potential N- glycosylation sites at N90, N125, and N336, potential phosphorylation sites for cAMP- cGMP-dependent protein kinase at T413, for casein kinase at S60, S181, T338, and T383, for protein kinase C at SI 06, SI 29, and T143, and for tyrosine kinase at Y78, and a potential cell attachment site is found in the sequence R387GD.
• Two potential active site aspartate residues, characteristic of aspartic proteases, are found at residues D96 and D283.
• BLOCKS and PRINTS analyses also identify sequences encompassing the two aspartate residues as characteristic of aspartic proteases.
• NHAP-2 has chemical and structural similarity with a mouse aspartic protease- like protein (GI 1906810; SEQ ID NO: 10).
• GI 1906810 mouse aspartic protease-like protein
• NHAP-2 and the mouse aspartic protease-like protein share 69% identity, the two potential glycosylation sites at N90 and N336, and the potential phosphorylations sites found in NHAP-2 at S106, S129, T143 and T338.
• the two potential active site aspartate residues found in NHAP-2, and their surrounding sequences, are also conserved in the mouse protein.
• sequence of SEQ ID NO:4 from about nucleotide 190 to about nucleotide 258, which encodes a fragment of SEQ ID NO:3 from about amino acid residue P54 to about amino acid residue A76, is useful, for example, as a hybridization probe.
• Electronic northern analysis shows clones clustered with NHAP expressed in a variety of cDNA libraries at least 59% of which involve cancer and immortalized cell lines, and at least 22% of which involve inflammation and the immune response.
• NHAP in lung tissue (37%).
• Membrane based northern analysis using NHAP-2 cDNA showed the expression of an ⁇ 1.3 kb RNA species in kidney, lung, and tissues associated with the immune response, including spleen, bone marrow, and peripheral blood leukocytes ( Figure 4). Since the NHAP-2 probe has -90% homology to NHAP-1, the analysis represents the expression of both NHAP-1 and NHAP-2.
• Membrane based northern analysis using an oligonucleotide probe specific for NHAP-1 ( Figure 5) showed the expression ofthe ⁇ 1.3 kb RNA species only in lung.
• Immunocytochemical staining of normal and diseased human tissue samples using NHAP-1 specific rabbit immune serum demonstrated the expression ofthe protein in pituitary gland, thyroid follicular cells, normal lung alveoli, bronchioloalveolar carcinoma and lung adenocarcinoma.
• Figure 6 shows the western analysis of recombinant NHAP-1 protein expressed in E. Coli.
• NHAP- 1 was detected as a band of around 45 kDa using immune, but not preimmune, serum and was found predominantly in IPTG-induced cells containing the NHAP-1 expression construct.
• the invention also encompasses NHAP variants.
• a preferred NHAP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the NHAP amino acid sequence, and which contains at least one functional or structural characteristic of NHAP.
• the invention also encompasses polynucleotides which encode NHAP.
• the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:2, which encodes an NHAP.
• the invention encompasses the polynucleotide sequence comprising the sequence of SEQ ID NO:4, which encodes an NHAP.
• the invention also encompasses a variant of a polynucleotide sequence encoding NHAP.
• such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding NHAP.
• a particular aspect ofthe invention encompasses a variant of SEQ ID NO:2 which has at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:2.
• the invention further encompasses a polynucleotide variant of SEQ ID NO:4 having at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:4. Any one ofthe polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of NHAP.
• nucleotide sequences which encode NHAP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring NHAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding NHAP possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression ofthe peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
• the invention also encompasses production of DNA sequences which encode NHAP and NHAP derivatives, or fragments thereof, entirely by synthetic chemistry.
• the synthetic sequence may be inserted into any ofthe many available expression vectors and cell systems using reagents well known in the art.
• synthetic chemistry may be used to introduce mutations into a sequence encoding NHAP or any fragment thereof.
• polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, or a fragment of SEQ ID NO:4 under various conditions of stringency.
• stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
• Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
• Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C.
• Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
• concentration of detergent e.g., sodium dodecyl sulfate (SDS)
• SDS sodium dodecyl sulfate
• Various levels of stringency are accomplished by combining these various conditions as needed.
• hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
• hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 g/ml denatured salmon sperm DNA (ssDNA).
• hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
• wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
• stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
• Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C.
• wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
• the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE ® (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), Taq polymerase (The Perkin-Elmer Co ⁇ ., Norwalk, CT), thermostable T7 polymerase (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), or combinations of polymerases and proofreading exonucleases, such as those found in the ELONGASETM amplification system (Life Technologies).
• Klenow fragment of DNA polymerase I SEQUENASE ®
• Taq polymerase The Perkin-Elmer Co ⁇ ., Norwalk, CT
• thermostable T7 polymerase Amersham Pharmacia Biotech Ltd., Uppsala, Sweden
• combinations of polymerases and proofreading exonucleases such as those found in the ELONGASETM amplification system (Life Technologies).
• sequence preparation is automated with machines, e.g., the ABI CATALYSTTM 800 (The Perkin-Elmer Co ⁇ ., Norwalk, CT) or MICROLAB® 2200 (Hamilton Co., Reno, NV) systems, in combination with thermal cyclers. Sequencing can also be automated, such as by ABI PRISMTM 373 or 377 systems (The Perkin-Elmer Co ⁇ ., Norwalk, CT) or the MEGABACETM 1000 capillary electrophoresis system (Molecular Dynamics, Inc., Sunnyvale, CA). Sequences can be analyzed using computer programs and algorithms well known in the art. (See, e.g., Ausubel. supra, unit 7.7; and Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, Inc, New York, NY.)
• the nucleic acid sequences encoding NHAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• one method which may be employed restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
• Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
• the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
• a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
• Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
• primers may be designed using commercially available software, such as OLIGOTM 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
• Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
• Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
• capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection ofthe emitted wavelengths.
• Output/light intensity may be converted to electrical signal using appropriate software (e.g., GenotyperTM and Sequence NavigatorTM, (The Perkin-Elmer Co ⁇ ., Norwalk, CT)), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
• Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
• polynucleotide sequences or fragments thereof which encode NHAP may be cloned in recombinant DNA molecules that direct expression of NHAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express NHAP.
• nucleotide sequences ofthe present invention can be engineered using methods generally known in the art in order to alter NHAP-encoding sequences for a variety of pu ⁇ oses including, but not limited to, modification ofthe cloning, processing, and/or expression ofthe gene product.
• DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
• oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
• sequences encoding NHAP may be synthesized, in whole or in part, using chemical methods well known in the art.
• chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.
• NHAP itself or a fragment thereof may be synthesized using chemical methods.
• peptide synthesis can be performed using various solid-phase techniques.
• Automated synthesis may be achieved using the ABI 431 A Peptide Synthesizer (The Perkin-Elmer Co ⁇ ., Norwalk, CT). Additionally, the amino acid sequence of NHAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
• the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392- 421.)
• the composition ofthe synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties. WH Freeman and Co., New York, NY.)
• the nucleotide sequences encoding NHAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control ofthe inserted coding sequence in a suitable host.
• these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding NHAP.
• Such elements may vary in their strength and specificity.
• Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NHAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding NHAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
• the invention is not limited by the host cell employed.
• cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NHAP.
• routine cloning, subcloning, and propagation of polynucleotide sequences encoding NHAP can be achieved using a multifunctional E. coli vector such as Bluescript® (Stratagene) or pSportlTM plasmid (GlBCO BRL). Ligation of sequences encoding NHAP into the vector's multiple cloning site disrupts the lac ⁇ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
• these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
• vectors which direct high level expression of NHAP may be used.
• vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of NHAP.
• a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
• yeast Saccharomyces cerevisiae or Pichia pastoris may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
• Plant systems may also be used for expression of NHAP. Transcription of sequences encoding NHAP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 3:17-31 1.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
• viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV.
• plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used
• constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
• pathogen-mediated transfection See, e.g., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York, NY; pp. 191-196.
• a number of viral-based expression systems may be utilized.
• sequences encoding NHAP may be ligated into an adenovirus transcription/translation complex consisting ofthe late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region ofthe viral genome may be used to obtain infective virus which expresses NHAP in host cells.
• transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
• RSV Rous sarcoma virus
• SV40 or EBV-based vectors may also be used for high-level protein expression.
• Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses.
• sequences encoding NHAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction ofthe vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
• the pu ⁇ ose ofthe selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
• Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
• Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk or apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11 :223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
• dhfr confers resistance to methotrexate
• neo confers resistance to the aminoglycosides neomycin and G-418
• als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
• Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
• Visible markers e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto, CA), ⁇ glucuronidase and its substrate ⁇ -D-glucuronoside, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. et al. (1995) Methods Mol. Biol. 55: 121-131.)
• marker gene expression suggests that the gene of interest is also present, the presence and expression ofthe gene may need to be confirmed.
• sequence encoding NHAP is inserted within a marker gene sequence, transformed cells containing sequences encoding NHAP can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding NHAP under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicates expression ofthe tandem gene as well.
• host cells that contain the nucleic acid sequence encoding NHAP and that express NHAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
• Immunological methods for detecting and measuring the expression of NHAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
• ELISAs enzyme-linked immunosorbent assays
• RIAs radioimmunoassays
• FACS fluorescence activated cell sorting
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NHAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
• the sequences encoding NHAP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
• T7, T3, or SP6 an appropriate RNA polymerase
• RNA polymerase such as T7, T3, or SP6
• Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
• Host cells transformed with nucleotide sequences encoding NHAP may be cultured under conditions suitable for the expression and recovery ofthe protein from cell culture.
• the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
• expression vectors containing polynucleotides which encode NHAP may be designed to contain signal sequences which direct secretion of NHAP through a prokaryotic or eukaryotic cell membrane.
• a host cell strain may be chosen for its ability to modulate expression ofthe inserted sequences or to process the expressed protein in the desired fashion.
• Such modifications ofthe polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
• Post-translational processing which cleaves a "prepro" form ofthe protein may also be used to specify protein targeting, folding, and/or activity.
• Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, MD) and may be chosen to ensure the correct modification and processing ofthe foreign protein.
• natural, modified, or recombinant nucleic acid sequences encoding NHAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any ofthe aforementioned host systems.
• a chimeric NHAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NHAP activity.
• Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
• Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6- His, FLAG, c-myc, and hemagglutinin (HA).
• GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
• FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
• a fusion protein may also be engineered to contain a proteolytic cleavage site located between the NHAP encoding sequence and the heterologous protein sequence, so that NHAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
• synthesis of radiolabeled NHAP may be achieved in vitro using the TNTTM rabbit reticulocyte lysate or wheat germ extract systems (Promega, Madison, WI). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35 S-methionine.
• Fragments of NHAP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra pp. 55-60.)
• Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431 A Peptide Synthesizer (The Perkin-Elmer Co ⁇ ., Norwalk, CT). Various fragments of NHAP may be synthesized separately and then combined to produce the full length molecule.
• NHAP is expressed in endocrine tissues, cancer, inflammation and the immune response, and respiratory disorders. Therefore, NHAP appears to play a role in respiratory, endocrinological, and immunological disorders, and cancer.
• NHAP or a fragment or derivative thereof may be administered to a subject to treat or prevent an endocrinological disorder associated with decreased expression or activity of NHAP.
• disorders can include, but are not limited to, disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; and disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism.
• a vector capable of expressing NHAP or a fragment or derivative thereof may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those described above.
• a pharmaceutical composition comprising a substantially purified NHAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those provided above.
• an agonist which modulates the activity of NHAP may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those listed above.
• an antagonist of NHAP may be administered to a subject to treat or prevent an endocrinological disorder associated with increased expression or activity of NHAP.
• disorders associated with hype ⁇ ituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH); disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; and disorders associated with hype ⁇ arathyroidism including Conn disease (chronic hypercalemia).
• an antibody which specifically binds NHAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express NHAP.
• a vector expressing the complement ofthe polynucleotide encoding NHAP may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those described above.
• an antagonist of NHAP may be administered to a subject to treat or prevent a respiratory disorder.
• a respiratory disorder can include, but are not limited to, allergy, asthma, acute and chronic inflammatory lung diseases, Adult Respiratory Distress Syndrome
• ARDS emphysema
• pulmonary congestion edema
• edema Chronic Obstructive Pulmonary Disease
• COPD COPD
• interstitial lung diseases COPD
• lung cancers COPD
• a vector expressing the complement ofthe polynucleotide encoding NHAP may be administered to a subject to treat or prevent a respiratory disorder including, but not limited to, those described above.
• an antagonist of NHAP may be administered to a subject to treat or prevent a cancer.
• a cancer may include, but is not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers ofthe adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
• a vector expressing the complement ofthe polynucleotide encoding NHAP may be administered to a subject to treat or prevent a cancer including, but not limited to, those described above.
• an antagonist of NHAP may be administered to a subject to treat or prevent an immunological disorder.
• Such disorders may include, but are not limited to, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarth
• any ofthe proteins, antagonists, antibodies, agonists, complementary sequences, or vectors ofthe invention may be administered in combination with other appropriate therapeutic agents. Selection ofthe appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
• the combination of therapeutic agents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
• An antagonist of NHAP may be produced using methods which are generally known in the art.
• purified NHAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind NHAP.
• Antibodies to NHAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
• various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with NHAP or with any fragment or oligopeptide thereof which has immunogenic properties. Rats and mice are preferred hosts for downstream applications involving monoclonal antibody production.
• various adjuvants may be used to increase immunological response.
• adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
• BCG Bacilli Calmette-Guerin
• Corvnebacterium parvum are especially preferable.
• the oligopeptides, peptides, or fragments used to induce antibodies to NHAP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 14 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion ofthe amino acid sequence ofthe natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of NHAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
• Monoclonal antibodies to NHAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique.
• the hybridoma technique the human B-cell hybridoma technique
• EBV- hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.
• chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
• techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce NHAP-specific single chain antibodies.
• Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88: 10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et al.
• Antibody fragments which contain specific binding sites for NHAP may also be generated.
• fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion ofthe antibody molecule and Fab fragments generated by reducing the disulfide bridges ofthe F(ab')2 fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
• immunoassays may be used for screening to identify antibodies having the desired specificity and minimal cross-reactivity.
• Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
• Such immunoassays typically involve the measurement of complex formation between NHAP and its specific antibody.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NHAP epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.)
• K a is defined as the molar concentration of NHAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
• K a association constant
• the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular NHAP epitope, represents a true measure of affinity.
• High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the NHAP-antibody complex must withstand rigorous manipulations.
• Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of NHAP, preferably in active form, from the antibody.
• polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
• a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is preferred for use in procedures requiring precipitation of NHAP-antibody complexes.
• Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
• the polynucleotides encoding NHAP, or any fragment or complement thereof may be used for therapeutic pu ⁇ oses.
• the complement ofthe polynucleotide encoding NHAP may be used in situations in which it would be desirable to block the transcription ofthe mRNA.
• cells may be transformed with sequences complementary to polynucleotides encoding NHAP.
• complementary molecules or fragments may be used to modulate NHAP activity, or to achieve regulation of gene function.
• sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NHAP.
• Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding NHAP. (See, e.g., Sambrook, supra: and Ausubel, supra.)
• Genes encoding NHAP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding NHAP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part ofthe vector system.
• modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions ofthe gene encoding NHAP.
• Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, are preferred.
• inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition ofthe ability ofthe double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
• a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
• Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
• the mechanism of ribozyme action involves sequence-specific hybridization ofthe ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding NHAP.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region ofthe target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
• RNA molecules and ribozymes ofthe invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NHAP. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
• RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends ofthe molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule.
• Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.) Any ofthe therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
• An additional embodiment ofthe invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any ofthe therapeutic effects discussed above.
• Such pharmaceutical compositions may consist of NHAP, antibodies to NHAP, and mimetics, agonists, antagonists, or inhibitors of NHAP.
• the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
• the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
• compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
• these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
• Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
• compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
• auxiliaries can be added, if desired.
• Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
• disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
• Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
• Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
• Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
• compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
• Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
• Non-lipid polycationic amino polymers may also be used for delivery.
• the suspension may also contain suitable stabilizers or agents to increase the solubility ofthe compounds and allow for the preparation of highly concentrated solutions.
• penetrants appropriate to the particular barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• compositions ofthe present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
• the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
• the preferred preparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a therapeutically effective dose refers to that amount of active ingredient, for example
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% ofthe population) or LD 50 (the dose lethal to 50% ofthe population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED 5O /LD 50 ratio.
• Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
• the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
• the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity ofthe patient, and the route of administration.
• Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity ofthe disease state, the general health ofthe subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate ofthe particular formulation. Normal dosage amounts may vary from about 0.1 g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
• antibodies which specifically bind NHAP may be used for the diagnosis of disorders characterized by expression of NHAP, or in assays to monitor patients being treated with NHAP or agonists, antagonists, or inhibitors of NHAP.
• Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NHAP include methods which utilize the antibody and a label to detect NHAP in human body fluids or in extracts of cells or tissues.
• the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
• a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
• NHAP NHAP
• ELISAs RIAs
• FACS fluorescence-activated cell sorting
• NHAP expression normal or standard values for NHAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to NHAP under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of NHAP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
• the polynucleotides encoding NHAP may be used for diagnostic pu ⁇ oses.
• the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
• the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of NHAP may be correlated with disease.
• the diagnostic assay may be used to determine absence, presence, and excess expression of NHAP, and to monitor regulation of NHAP levels during therapeutic intervention.
• hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding NHAP or closely related molecules may be used to identify nucleic acid sequences which encode NHAP.
• the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding NHAP, allelic variants, or related sequences.
• Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any ofthe NHAP encoding sequences.
• the hybridization probes ofthe subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:2, SEQ ID NO:4 or from genomic sequences including promoters, enhancers, and introns ofthe NHAP gene.
• Means for producing specific hybridization probes for DNAs encoding NHAP include the cloning of polynucleotide sequences encoding NHAP or NHAP derivatives into vectors for the production of mRNA probes.
• vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means ofthe addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
• Polynucleotide sequences encoding NHAP may be used for the diagnosis of a disorder associated with expression of NHAP.
• a disorder associated with expression of NHAP include, but are not limited to, endocrinological disorders such as disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller- Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; hype ⁇ ituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH); and disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism; disorders associated with hyperthyroidism including thyrotoxicosis and its various forms,
• polynucleotide sequences encoding NHAP may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered NHAP expression. Such qualitative or quantitative methods are well known in the art.
• the nucleotide sequences encoding NHAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
• the nucleotide sequences encoding NHAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding NHAP in the sample indicates the presence ofthe associated disorder.
• Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
• a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NHAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
• hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
• the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development ofthe disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
• oligonucleotides designed from the sequences encoding NHAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding NHAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding NHAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
• Methods which may also be used to quantitate the expression of NHAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
• radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
• the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• oligonucleotides or longer fragments derived from any ofthe polynucleotide sequences described herein may be used as targets in a microarray.
• the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
• Microarrays may be prepared, used, and analyzed using methods known in the art.
• methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, MJ. et al. (1997) U.S. Patent No. 5,605,662.)
• nucleic acid sequences encoding NHAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
• the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constructions or single chromosome cDNA libraries.
• Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
• FISH Fluorescent in situ hybridization
• Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location ofthe gene encoding NHAP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
• the nucleotide sequences ofthe invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
• In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
• any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• the nucleotide sequence ofthe subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
• NHAP its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
• the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between NHAP and the agent being tested may be measured.
• Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
• a solid substrate such as plastic pins or some other surface.
• the test compounds are reacted with NHAP, or fragments thereof, and washed.
• Bound NHAP is then detected by methods well known in the art.
• Purified NHAP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
• non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
• nucleotide sequences which encode NHAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
• RNA was purchased from Clontech (Palo Alto, CA) or isolated at Incyte from tissues described in Table 1. The tissue was homogenized and lysed in guanidinium isothiocyanate, and the lysate was centrifuged over a CsCl cushion. Alternatively, the tissue was homogenized and lysed in phenol or a suitable mixture of denaturants such as TRIzol reagent (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate, and the lysate was extracted with chloroform (1 :5 v/v). RNA was precipitated from lysates with either isopropanol or sodium acetate and ethanol.
• TRIzol reagent Life Technologies
• UNIZAP vector Stratagene, La Jolla, CA
• Superscript plasmid system Life Technologies
• cDNA libraries were constructed by Stratagene using RNA provided by Incyte. Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and cDNA was digested with an appropriate restriction enzyme(s).
• cDNA was size- selected (300-1000 bp) using Sephacryl SI 000 or Sepharose CL2B or CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
• cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., pBluescript (Stratagene), pSPORT 1 (Life Technologies), pINCY (Incyte Pharmaceuticals Inc, Palo Alto, CA).
• pINCY was amplified in JM109 cells and purified using the QiaQuick column (QIAGEN Inc).
• Recombinant plasmids were transformed into competent E. coli cells, e.g., XLl-Blue, XLl-BlueMRF, or SOLR (Stratagene) or DH5 ⁇ , DH10B, or ElectroMAX DH10B (Life Technologies).
• Plasmids were purified using the MAGIC MINIPREPS DNA purification system (Promega, Madison, WI); Miniprep kit (Advanced Genetic Technologies Co ⁇ oration, Gaithersburg, MD); QIAwell-8 Plasmid, QIAwell PLUS DNA, or QIAwell ULTRA DNA purification systems; or REAL Prep 96 plasmid kit (QIAGEN Inc) using the recommended protocol. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
• plasmid DNA was amplified from host cell lysates using direct link PCR (Rao, V.B. (1994) Anal. Biochem. 216: 1-14) in a high-throughput format. Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates (Genetix Ltd, Wales UK) and concentration of amplified plasmid DNA was quantified fluorometrically using Pico Green Dye (Molecular Probes, Eugene OR) and a Fluoroscan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
• the cDNAs were prepared for sequencing using either an ABI CATALYST 800 (Perkin Elmer) or a Hamilton MICRO LAB 2200 (Hamilton, Reno, NV) in combination with Peltier Thermal Cyclers (PTC200; MJ Research, Watertown MA).
• the cDNAs were sequenced on the ABI 373 or 377 DNA Sequencing systems (Perkin Elmer) by the method of Sanger F and A.R. Coulson (1975; J. Mol. Biol. 94:441-448) using standard ABI protocols, base calling software, and kits.
• cDNAs were prepared and sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frame was determined using standard methods (Ausubel, supra).
• nucleotide sequences and/or amino acid sequences ofthe Sequence Listing were queried against databases such as GenBank primate (pri), rodent (rod), mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS, and other databases which contain previously identified and annotated motifs and sequences.
• Algorithms such as Smith Waterman which deal with primary sequence patterns and secondary structure gap penalties (Smith, T. et al. (1992) Protein Engineering 5:35-51) and programs and algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J.
• the first column of Table 2 shows the tool, program, or algorithm; the second column, the database; the third column, a brief description; and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).
• the GeneTrapperTM cDNA Positive Selection System kit (Life Technologies) was employed to isolate full length cDNA clones of NHAP-1 and NHAP-2. Following the manufacturer's instructions, oligonucleotides were designed based on partial nucleic acid sequences from Incyte clone 2756549, biotinylated at the 3' end, and hybridized to single stranded DNA from plasmid cDNA libraries of human lung (Cat. No. 10424-018, Life Technologies) and human leukocytes (Cat. No. 10421-014, Life Technologies).
• cDNA clones Five cDNA clones; gt83, gt86, gt97, gt88, and gt91 were isolated from lung cDNA library, and five cDNA clones; gt4, gt22, gt49, gt53, and gt90 were isolated from the leukocyte library. Sequencing revealed that the clones isolated from the lung library were identical in nucleic acid sequence to Incyte clones 372637 and 1242901 and to the gene subsequently named NHAP-1 (HUPM-4 in the prior application). However, the clones isolated from the lung library differed in nucleic acid sequences from those isolated from the leukocyte library and from Incyte clones 2435410 and 2756549.
• NHAP-1 encompasses cDNA clones gt83, gt86, gt97, gt88, gt91, Incyte clone 372637 and 1242901.
• NHAP-2 encompasses cDNA clones gt4, gt22, gt49, gt53, gt90 and Incyte clones 2435410 and 2756549. Sequence homology analysis showed 89% nucleic acid identity between NHAP- 1 and NHAP-2. V. Northern Analysis
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook. supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)
• RNA samples from a variety of human tissues were labeled with 33 P using the random primer labeling method with the HIGH PRIMER DNA labeling kit (Boerheinger Mannheim, Indianapolis, IN). Hybridization was conducted under high stringency conditions in a solution containing 50% formamide, 5X SSC, 50 mM NaP04, pH 7.4, IX Denhardts, 2% SDS and 100 ug/ml Salmon Sperm DNA at 42° C overnight.
• RNA species of -1.3 kb from kidney, peripheral blood leukocytes, spleen and lymph nodes ( Figure 4). This RNA species was also expressed at a lower level in lung, bone marrow, thymus, and fetal liver. Since NHAP-2 has 89% homology to NHAP-1, the northern analysis reflected the expression profile of both NHAP-1 and NHAP-2. When the above blots were stripped and reprobed with NHAP-1 -specific oligonucleotide, the expression ofthe 1.3 kb RNA species was found only in the lung ( Figure 5).
• the product score takes into account both the degree of similarity between two sequences and the length ofthe sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
• Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
• Oligonucleotides are designed using state-of-the-art software such as OLIGOTM 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
• the labeled oligonucleotides are substantially purified using a SephadexTM G-25 superfine size exclusion dextran bead column (Pharmacia & Upjohn, Kalamazoo, MI).
• the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester, NY) is exposed to the blots to film for several hours, hybridization patterns are compared visually.
• a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
• An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
• a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
• nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis ofthe scanned images.
• Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements ofthe microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENETM.
• Full-length cDNAs, ESTs, or fragments thereof corresponding to one ofthe nucleotide sequences ofthe present invention, or selected at random from a cDNA library relevant to the present invention are arranged on an appropriate substrate, e.g., a glass slide.
• the cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
• Sequences complementary to the NHAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring NHAP.
• oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
• Appropriate oligonucleotides are designed using OLIGOTM 4.06 software and the coding sequence of NHAP.
• a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
• To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the NHAP-encoding transcript. TX. Expression of NHAP-1
• NHAP-1 The cDNA encoding NHAP-1 was used to express full-length NHAP-1 by subcloning the cDNAs into appropriate vectors and introducing the constructs into host cells.
• NHAP-1 cDNA was subcloned into a bacterial expression vector pET15b
• NHAP- 1 cDNA was also subcloned into the baculovirus pFast-bac-HTc (Life Technologies) for expression in Sf9 insect cells, and into pCMV-SPORT (Life Technologies) for expression in mammalian HEK 293 cells.
• Protease activity of NHAP is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantitated by spectrophotometric (or fluorometric) abso ⁇ tion ofthe released chromophore.
• Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), animopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidase A and B, procollagen C-proteinase).
• Chromogens commonly used are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid.
• Assays are performed at ambient temperature and contain an aliquot ofthe enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette and followed by the increase/decrease in absorbance ofthe chromogen released during hydrolysis ofthe peptide substrate. The change in absorbance is proportional to the enzyme activity in the assay.
• NHAP function is assessed by expressing the sequences encoding NHAP at physiologically elevated levels in mammalian cell culture systems.
• cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
• Vectors of choice include pCMV SPORTTM (Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus promoter.
• 5-10 g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation.
• 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
• Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
• Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, CA), CD64, or a CD64-GFP fusion protein.
• Flow cytometry an automated, laser optics- based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state.
• FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York, NY.
• NHAP The influence of NHAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding NHAP and either CD64 or CD64-GFP.
• CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
• Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success, NY).
• mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding NHAP and other genes of interest can be analyzed by Northern analysis or microarray techniques.
• oligopeptide containing 19 amino acid residues from the C-terminus of NHAP-1 was synthesized.
• Two rabbits were immunized with the oligopeptide-KLH complex in complete Freund's adjuvant (Zeneca LifeScience Molecules, Wilmington, DE).
• the resulting antisera, IC619 and IC620 were tested for antipeptide activity by ELISA. Both antisera recognized recombinant protein expressed in E.coli and in Sf9 insect cells by western blot analysis.
• E.coli and Sf9 cells containing the corresponding expression constructs were lysed, and proteins were separated on a denatured PAGE gel (NuPage gels, Novex) and transferred onto a nitrocellulose membrane according to the method previously described. The blot was then probed with antisera IC619 or IC620. Binding ofthe antisera was detected by HRP-conjugated donkey anti-rabbit Ig and visualized using ECL (enhanced chemiluminescence) system (Amersham Pharmacia Biotech).
• ECL enhanced chemiluminescence
• Naturally occurring or recombinant NHAP is substantially purified by immunoaffinity chromatography using antibodies specific for NHAP.
• An immunoaffinity column is constructed by covalently coupling anti-NHAP antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. Media containing NHAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of NHAP (e.g., high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody/NHAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NHAP is collected.
• a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion
• Immunocytochemical analysis was performed to determine protein localization in human tissue samples using NHAP- 1 -specific rabbit immune serum IC619 as the primary antibody.
• the analysis was performed by LifeSpan BioSciences, Inc., Seattle WA.
• the detection system consisted of a DAKO LSAB+ Kit (DAKO co ⁇ ., Ca ⁇ interia CA) containing labelled
• Streptavidin-Biotin Kit with a biotinylated secondary antibody followed by application of a streptavidin-horseradish peroxidase conjugate and DAB substrate. Tissues were also blocked for endogenous biotin and endogenous peroxide. Negative controls performed on each tissue sample included staining with pre-immune sera. In addition, experiments were performed to block staining by incubating Antibody IC619 with a 10 fold excess of immunizing peptide derivedfrom NAP1. The analysis demonstrated that antibody IC619 produced strong positive staining in the anterior lobe ofthe pituitary, in thyroid follicular cells and within the Type II pneumocytes ofthe lung.
• Type II pneumocytes stained positive for antibody IC619.
• the bronchioloalveolar carcinoma and lung adenocarcinoma produced strong positive staining.
• Other lung neoplasms including a small cell, epidermoid cell, adenocarcinoma and metastatic colon adenocarcinoma were negative when stained.
• FISH Fluorescence In situ Hybridization
• NHAP-2 (Genome Systems, Inc., St. Louis, MO). DNA from two genomic clones, corresponding to NHAP-1 and NHAP-2 , were labeled with digoxigenin dUTP by nick translation. Labeled probes were combined with sheared human DNA and independently hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes from a male donor in a solution containing 50% formamide, 10% dextran sulfate and 2X SSC. Specific signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI. These experiments resulted in the specific labeling ofthe long arms of chromosome 19.
• NHAP or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
• Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529.
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled NHAP, washed, and any wells with labeled NHAP complex are assayed. Data obtained using different concentrations of NHAP are used to calculate values for the number, affinity, and association of NHAP with the candidate molecules.
• ABI FA TURA A program thai removes vector sequences and masks Perkin Elmer Applied Biosystems, ambiguous bases in nucleic acid sequences Foster Cay. CA
• Phrcd A base-calling algorithm that examines automated Lwing B el al ( 1998) Genome sequencer traces with high sensitivity and probability Res 8 175 185, Ewmg B and P Green ( 1998) Genome Res 8 186 194

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