EP0821731A1 - Modulator of neuronal cell response to inhibition by cns myelin - Google Patents

Modulator of neuronal cell response to inhibition by cns myelin

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Publication number
EP0821731A1
EP0821731A1 EP96908957A EP96908957A EP0821731A1 EP 0821731 A1 EP0821731 A1 EP 0821731A1 EP 96908957 A EP96908957 A EP 96908957A EP 96908957 A EP96908957 A EP 96908957A EP 0821731 A1 EP0821731 A1 EP 0821731A1
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EP
European Patent Office
Prior art keywords
seq
leu
ser
nucleic acid
cells
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EP96908957A
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German (de)
English (en)
French (fr)
Inventor
Arthur Roach
Andres Lozano
Monika Labes
John Roder
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Mt Sinai Hospital
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Mt Sinai Hospital
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Publication of EP0821731A1 publication Critical patent/EP0821731A1/en
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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C12N9/1211—Thymidine kinase (2.7.1.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates generally to a novel protein, nucleic acid molecule encoding the protein, a novel hybridoma cell line, and particularly to a hybridoma cell line producing monoclonal antibodies against neuronal cell membranes. Also provided are methods for using the protein, nucleic acid molecule, and monoclonal antibodies; methods for identifying substances which modulate the response of neuronal cells to inhibition by mammalian central nervous system myelin; and methods for assaying for neurite growth inhibitory activity. BACKGROUND OF THE INVENTION
  • a remarkable feature of axons in the peripheral nerves of adult mammals is that after interruption, they are able to regenerate through the distal nerve stump to reconnect with their targets and re-establish function.
  • CNS central nervous system
  • Axons injured in the brain, optic nerve or spinal cord of adult mammals do not successfully regrow. This leads to an irreversible disruption of neuronal circuits and permanent neurologic disability.
  • PNS peripheral nervous system
  • the nervous system also contains molecules which function to inhibit or restrict axonal growth.
  • the inhibitory molecules share the property of causing growth cone collapse
  • the growth cone is a specialized structure at the distal tip of the advancing neurite and it is primarily responsible for the transduction of environmental signals which modulate growth cone advancement and axonal extension. Some of these molecules are membrane associated glycoproteins expressed early in development.
  • Posterior sclerotomes for example, contain proteins that cause the collapse of chick dorsal root ganglion (DRG) neuron growth cones (Davies et al , 1990, Neuron 4, 11-20)
  • the posterior tectum of chicks has a 33 kDa phosphoglycerol inositide linked protein that causes the collapse of temporal but not nasal retinal ganglion cell (RGC) growth cones (Stahl et al , 1990, Neuron 5, 735-743)
  • RRC retinal ganglion cell
  • a 88 kDa molecule from chick brain, collapsin causes the collapse of dorsal root ganglion and retinal ganglion cell growth cones (Luo et al., 1993, Cell 75:217-227).
  • tenascin (Lochter et al., 1991, J. Cell. Biol. 113, 1159-1171) and janusin (Pesheva et al., 1989, J.Cell Biol 109, 1765-1778) found in the extracellular matrix, may be anti-adhesive and play a role in neurite guidance.
  • the function of the inhibitory molecules during development of the nervous system may be to focus and restrict axonal outgrowth along specific neural projections and towards appropriate synaptic targets.
  • the adult mammalian CNS also contains inhibitory molecules which may be responsible for the lack of successful axonal regeneration after injury.
  • Two fractions of non-neuronal origin have been identified in adult mammalian central nervous system myelin which inhibit neurite outgrowth. These fractions designated NI-35 and NI-250 (neurite inhibitor; NI) are found in the myelin of the CNS but not that of peripheral nerves (Carom and Schwabb, J. Cell Biol. 106:1281-1, 1988).
  • the fractions are absent in periods of embryonic CNS axonal outgrowth but are produced by oligodendrocytes immediately before myelinahon of established axonal projections (Caroni and Schwabb, J. Cell Biol.
  • NI-35 produces rapid and dramatic growth cone collapse (Bandtlow et al., 1993, Science 259:80-83 and Igarashi et al., 1993, Science 259:77-79 ). The inhibitory effects are seen at low concentrations suggesting that signal amplification may be required.
  • CNS myelin-associated growth inhibiting molecules appear to act by triggering an active biochemical response in neurons which may be transduced by binding to a receptor on the neuronal surface.
  • CNS myelin-associated growth inhibiting molecules appear to act by triggering an active biochemical response in neurons which may be transduced by binding to a receptor on the neuronal surface.
  • the same activity inhibits spreading of a fibroblast cell line (Caroni and Schnell, supra 1988), suggesting that its effects may not be restricted to neural cells.
  • the present inventors developed an in vitro neurite growth inhibition assay which resembles the inhibitory effect of CNS myelin on neurite growth in vivo.
  • the inventors used their neurite growth inhibition assay to scree n a panel of monoclonal antibodies raised against rat neuronal membrane proteins, for clones capable of blocking the inhibitory response.
  • One monoclonal antibody having the laboratory designation 10D, was found to neutralize the inhibition of neurite growth by several neuronal types on CNS myelin substrates.
  • 10D monoclonal antibody when applied to Western Blots recognizes most prominently bands of M r 35,000 and 33,000 expressed in neuronal and fibroblast cell lines, and in rat brain and liver. Proteins recognized by 10D monoclonal antibody play a role in the interaction between cells and their growth substrates, and are novel candidates for cellular receptors for myelin inhibitors.
  • the present inventors also screened a cDNA expression library derived from adult rat brain mRNA using the 10D monoclonal antibody. Resulting clones were tested for their ability to modulate neurite growth on an inhibitory CNS myelm substrate when expressed as antisense transcripts in a neuronal cell line Transfectants containing antisense constructs derived from the clone having the laboratory designation "Dl", showed significant enhancement of neurite growth on myelm. Sequence analysis of the partial Dl cDNA clone indicated that it is a previously unreported gene.
  • Probes derived from sequences in the partial cDNA clone were used to screen a cDNA library, and a gene designated "petrin” encoding a protem involved in modulating neurite growth inhibition was identified.
  • the petrin gene encodes a 60 to 64 kDa protem which is a new member of the protem phosphatase 2C family ("PP2C").
  • the novel protem has been designated "Petrin”
  • the human petrin locus was localized to chromosome 12
  • the present inventors have also shown by in situ hybridization that the petrin gene is expressed in neurons in brain tissue, and in particular, in the Purkinje cells of the cerebellum; in the 3rd and 4th layers of the cerebral cortex; and, dispersed neurons in the hippocampus.
  • RNA blot analysis showed that in the rat brain expression was first detectable at embryonic day 13, and increased to a maximum level in the adult brain Northern and DNA analysis also showed that the protein is present in different mammalian species such as mouse, rat, hamster, and human
  • the biological function of Petrin was investigated using phosphatase assays on immunoprecipitated material, and it was found that Petrin has serine/threonine phosphatase activity and tyrosine phosphatase activity both of which are magnesium dependent. Phosphatase activity was also shown to be highest while NG108 cells are proliferating and growing neurites and was not detected in late growth stages. Serine/threonine-phosphatase and tyrosine phosphatase activities were inhibited by okadaic acid or ortho-vanadate, respectively.
  • the present inventors also prepared antisense oligonucleotides to petrin and found that they enhanced neurite growth in a functional in vitro assay. Therefore, the present invention contemplates a method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelm comprising growing neuronal cells which have a propensity for neurite outgrowth on mammalian central nervous system (CNS) myelin m the presence of a test substance which is suspected of affecting neurite outgrowth, and assaying for neurite outgrowth.
  • CNS mammalian central nervous system
  • hybridoma cell Imes which produce monoclonal antibodies which (a) immunoreact with neuronal membrane proteins; (b) neutralize the inhibition of neurite growth by mammalian central nervous system myelm; and, (c) recognize bands of M r 35,000 and M r 33,000 expressed in neuronal and fibroblast cell Imes and m rat cerebrum and rat liver.
  • Preferred hybridoma cell Imes are those havmg the laboratory designation D10
  • the monoclonal antibodies produced, and the antigens recognized by this cell line are also a part of the present invention Accordingly, the present mvention also contemplates a monoclonal antibody which (a) immunoreacts with neuronal membrane proteins; (b) neutralizes the inhibition of neurite growth by mammalian central nervous system myelm; and, (c) recognizes bands of M r 35,000 and M r 33,000 expressed neuronal and fibroblast cell lines and in rat cerebrum and rat liver.
  • the invention also provides a method for assaying for the presence of an activator or inhibitor of a monoclonal antibody produced by the hybridoma cell line of the mvention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelm in the presence of a known concentration of the monoclonal antibody, and in the presence of a suspected activator or inhibitor of the monoclonal antibody, under conditions which permit neurite outgrowth, and assaying for neurite outgrowth.
  • CNS central nervous system
  • Another aspect of the invention relates to an isolated nucleic acid molecule which is present in neuronal cells, its expression is required for neurite growth inhibition by mammalian central nervous system myelin, and it comprises the nucleic acid sequences shown in the Sequence Listing as SFQ ID No 1, SEQ ID No 3, SEQ ID NO 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7 and SEQ. ID. NO. 8 or as shown in Figures 9 and 11 to 14, and 21.
  • the isolated and purified nucleic acid molecule comprises (a) a nucleic acid sequence as shown in SEQ. ID NO:l, SEQ. ID. NO:3,
  • SEQ. ID. NO:4 SEQ. ID. NO. 5, SEQ. ID. NO.6, SEQ. ID. NO.7 and /or SEQ. ID. NO. 8, or in Figures 9, 11 to 14, and 21 wherein T can also be U;
  • nucleic acid sequences having at least 80-90% identity, preferably 90% identity with SEQ. ID NO:l, SEQ. ID. NO:3, SEQ. ID. NO:4, SEQ. ID. NO. 5, SEQ. ID. NO. 6,
  • nucleic acid molecule differing from any of the nucleic acids of (a) to (d) in codon sequences due to the degeneracy of the genetic code.
  • the mvention also relates to a nucleic acid molecule comprismg
  • nucleic acid sequences complementary to (a); nucleic acid sequences which are at least 80%, preferably 90% identical to (a); or,
  • the isolated and purified nucleic acid molecule comprises (a) a nucleic acid sequence as shown in Figure 23 (or SEQ. ID. NO. 11) , preferably from about nucleotides 486 to 1977 as shown in Figure 23 (or SEQ ID NO:ll), wherein T can also be U;
  • nucleic acid sequences which are at least 80-90% identical, preferably 90% identical to (a); or,
  • a nucleic acid molecule of the invention, or fragments thereof may be mserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence
  • recombinant molecules adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operativelv linked to the nucleic acid molecule
  • the recombinant molecule can be used to prepare transformed host cells expressing the protein or part thereof encoded by a nucleic acid molecule of the invention or a fragment thereof. Therefore, the invention provides host cells containing a recombinant molecule of the invention.
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule of the invention.
  • the invention further provides a method for preparing a protein encoded by the nucleic acid molecule of the invention or parts thereof utilizing the isolated and purified nucleic acid molecules of the invention.
  • a method for preparmg a Petrin protein comprising (a) transferring a recombinant expression vector of the mvention mto a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of Petrin; and (d) isolating Petrin.
  • the present invention also includes a protem encoded by a nucleic acid molecule of the present mvention.
  • Proteins comprise the amino acid sequence as shown in the Sequence Listmg as SEQ. ID. Nos. 2 and 10, and as shown in Figure 10 and the ammo acid sequence as shown m the Sequence Listmg as SEQ. ID. NO. 9; and sequences having at least 80-90% identity, preferably 90% identity thereto.
  • the protem of the invention may be found in brain, NG108, and PC12 cells.
  • a purified Petrin protem which has the ammo acid sequence as shown in Figure 24 or SEQ ID NO: 12.
  • Protems of the mvention include truncations of the purified Petrm protem and analogs, homologs, and isoforms of the protein and truncations thereof.
  • protems of the mvention may be conjugated with other molecules, such as protems to prepare fusion protems. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion protems
  • the mvention also permits the construction of nucleotide probes which are unique to nucleic acid molecules of the mvention and accordingly to a protem of the mvention, or part of a protem of the mvention.
  • the mvention also relates to a probe comprising a nucleic acid molecule of the mvention or a fragment thereof
  • the probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protem which displays the properties of the protem of the mvention, or a part thereof.
  • the mvention further contemplates antibodies having specificity against an epitope of a protem of the mvention, or part of the protem which is unique to the protem Antibodies may be labelled with a detectable substance and they may be used to detect the protem of the mvention m tissues and cells
  • the invention provides a method for assaymg for the presence ot an activator or inhibitor of a protem of the mvention comprising growing neuronal cells w hich have a propensity for neurite growth in the presence of a protein of the mvention, and a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
  • the invention also provides a method for assaying for the presence of an activator or inhibitor of a protein of the mvention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelm and which express a protein of the invention in the presence of a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
  • CNS central nervous system
  • Substances which affect cell neurite growth may also be identified by comparmg the pattern and level of expression of the novel nucleic acid molecule and /or novel protem of the mvention, in tissues and cells in the presence and in the absence of a test substance.
  • the mvention also contemplates a method for assaymg for a c ubstance that affects neuronal growth comp ⁇ smg administering to a non-human animal or to a tissue of an animal, a substance suspected of affectmg neuronal growth, and detectmg, and optionally quantitatmg, the nucleic acid molecule and /or novel protem of the mvention m the non-human animal or tissue.
  • the invention also contemplates a method for identifymg a substance which is capable of b dmg to a protem of the mvention, or a part of the protem, comprising reactmg the protem, or part of the protem, with at least one substance which potentially can bmd with the protem, or part of the protem, under conditions which permit the formation of substance-protem complexes, and assaymg for substance-protein complexes, and /or for free substance, for non-complexed protem.
  • the mvention provides a method for assaymg a medium for the presence of an activator or inhibitor of the mteraction of the protem of the mvention or part thereof, and a substance which bmds to the protem
  • the method comprises providmg a known concentration of a protem of the mvention, or part of the protem, incubating the protem, or part of the protem with a substance which bmds to the protem, or part of the protem, and a suspected activator or inhibitor substance, under conditions which permit the formation of substance-protem complexes, and assaymg for substance-protem complexes
  • the mvention contemplates a method for assaying for a substance that affects the phosphatase activity of a protem of the mvention comprising reactmg a protem of the mvention with a substrate which is capable of bemg dephosphorylated by the protem to produce a dephosphorylated product, in the presence of a substance which is suspected of affecting the phosphatase activity of the protein, under conditions which permit dephosphorvlation of the substrate, assaymg for dephosphorylated product, and comparmg to product obtained in the absence of the substance to determine the affect of the substance on th phosphatase ⁇ t ⁇ of the protein
  • the invention also contemplates pharmaceutical compositions and methods of using (a) the monoclonal antibody produced by the hybridoma cell line of the invention; (b) inhibitors and activators of the monoclonal antibody produced by the hybridoma cell line of the invention; (c) inhibitors and activators of the expression of a nucleic acid molecule of the invention; (
  • Figure 1A shows photomicrographs of representative fields of cultures of dibutyryl cyclic AMP induced NG108 cells plated onto tissue culture plastic coated with poly-L-lysine alone (a), poly-L-lysine followed by 20 ⁇ g/cm 2 bovine serum albumin (BSA) (b), poly-L-lysine followed by 20 ⁇ g/cm.2 CNS myelin (c), and (d) shows a single dbcAMPNG108 cell growing on a myelin-free patch;
  • BSA bovine serum albumin
  • CNS myelin c
  • FIG. 1A shows photomicrographs of representative fields of cultures of dibutyryl cyclic AMP induced NG108 cells plated onto tissue culture plastic coated with poly-L-lysine alone (a), poly-L-lysine followed by 20 ⁇ g/cm 2 bovine serum albumin (BSA) (b), poly-L-lysine followed by 20 ⁇ g/cm.2 CNS myelin (c),
  • Figure IB is a graph showing the proportion of dbcAMP NG108 cells with a process greater than 1 cell diameter after plating on wells coated with bovine serum albumin or extracts from muscle, sciatic nerve and brain;
  • Figure 2 is a graph showing process bear g dbcAMPNG108 cells determined at 24 hrs after plating onto different densities of CNS myelin on poly-L-lysine coated wells;
  • Figure 3A shows photomicrographs of dbcAMPi ⁇ Gl08 ce -* s g ro n on poly-L-lysine alone or 10 ⁇ g/cm 2 of CNS myelin showing that 10D antibody reverses the growth inhibitory effect of CNS myelin;
  • Figure 3B is a graph showing quantitation of process-bearmg cells grown on CNS myelin for 24 or 72 hours with 5 ⁇ l per well of control ascites (filled bars) or 10D ascites (open bars);
  • Figure 4A is a photomicrograph of cells grown on poly-L-lysine coated glass slides for 48 hours, fixed and processed for immunocytochemistry with control ascites diluted 1:1000;
  • Figure 4B is a photomicrograph of cells grown on polv-L-lysinc coated glass slides for 48 hours, fixed and processed for immunocytochemistry with 10D ascites diluted 1:1000;
  • Figure 5 is a photomicrograph showing two identical denaturing 13% polyacrylamide-SDS gels loaded with marker proteins and 10 ⁇ g of protein from liver, cerebrum (both from 2 day old rats), d bcAMPNG108 cells and adult CNS myelin and stained for total proteins with Coomassie Brilliant Blue (left), and one transferred to nitrocellulose and reacted with the 10D monoclonal antibody (right);
  • Figure 6 is a schematic representing the strategy used to characterize clones selected with the 10D monoclonal antibody
  • Figure 7 is a graph showing the number of A3 antisense transformant cells and NG108 parental cells which grew processes on PLL, and myelin with and without the 10D antibody;
  • Figure 8 shows a Southern blot of EcoRI digested genomic DNA from NG108 cells and the transformed cell line A3 probed with the lkb Dl cDNA insert;
  • Figure 9 shows the nucleotide sequence of a fragment of the cDNA clone Dl which is designated D1T7;
  • Figure 10 shows the amino acid sequence of a portion of the protein encoded by the nucleic acid molecule of the invention.
  • Figure 11 shows the nucleotide sequence of a fragment of the cDNA clone Dl which is designated D1T3;
  • Figure 12 shows the nucleotide sequence of a fragment of the cDNA clone which is designated ML07T3;
  • Figure 13 shows the nucleotide sequence of a fragment of the cDNA clone which is designated S4T3;
  • Figure 14 shows the nucleotide sequence of a fragment of the cDNA clone which is designated S5T7;
  • Figure 15 is a schematic diagram showing the positions of the sequenced fragments of the Dl cDNA clone
  • Figure 16 are photographs showing a control (A) and (B) the neutralization of the neurite growth inhibitory effects of myelin on newborn rat superior cervical ganglion primary neurons by 10D ascites;
  • Figure 17 is an immunoblot showing that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12;
  • Figure 18 is a blot showing that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12;
  • Figure 19 is a blot showing that the Dl cDNA recognizes human RNA transcripts;
  • Figure 20 is a graph showing % of NG-108-15 cells with neurite extension versus myelm concentration ( ⁇ g/cm >-
  • Figure 21 shows a nucleotide sequence of a fragment of the Dl cDNA clone
  • Figure 22 is a schematic diagram having the sequenced regions of the Dl cDNA;
  • Figure 23 shows the nucleotide sequence of a Petrin protein of the invention.
  • Figure 24 shows the amino acid sequence of a Petrin protein of the invention and the amino acid sequences of other members of the protein phosphatase 2C family. DETAILED DESCRIPTION OF THE INVENTION
  • A Ala - alanine
  • C Cys - cysteine
  • D Asp- aspartic acid
  • E Glu - glutamic acid
  • F Phe - phenylalanine
  • G Gly - glycine
  • H His - histidine
  • I He - isoleucine
  • K Lys - lysine
  • L Leu - leucine
  • M Met - methionine
  • N Asn - asparagine
  • P Pro - proline
  • Q Gin - glutamine
  • R Arg - arginine
  • S Ser - serine
  • T Thr - threonine
  • V Val - valine
  • W Trp- tryptophan
  • Y Tyr - tyrosine
  • p.Y. P.Tyr - phosphotyrosine.
  • A assay for neurite growth inhibition by CNS myelin
  • B hybridoma cell lines and monoclonal antibodies
  • C novel nucleic acid molecule and novel protein
  • D applications for which the hybridoma cell lines, monoclonal antibodies, nucleic acid molecules, protein, and the substances identified using the methods described herein are suited.
  • the present inventors have developed an in vitro method where the limited neurite outgrowth on CNS myelin in vitro resembles the limited axonal outgrowth in the CNS in vivo .
  • the method may be used to assay for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelm.
  • the method involves preparing neuronal cells which have a propensity for neurite growth, growing the neuronal cells on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of affectmg neurite growth, and assaying for neurite growth.
  • CNS central nervous system
  • Neuronal cells which have a propensity for neurite growth which may be used in the method of the invention include the NG108-15 rat neuroblastoma and glioma hybrid cell lme induced with dibutyryl cyclic AMP and fetal calf serum, preferably cells treated with 0.5 to 1 mM, preferably ImM of dbcAMP, and 5% fetal calf serum, for 1 to 7 days, preferablv 2 days.
  • neuronal cells which may be used in the method of the mvention include PC12 cells which have been induced to grow neu ⁇ tes by induction for 5-7 davs with 100 ng/ml NGF (Green), cerebellar neurons (Trenkner, E in Cultu ⁇ ng Nerve Cells, Banker, G , and Goslin, K. (eds) (Cambridge, USA: MIT Press) 1991), and cortical neurons (Baughman et al., in Culturing Nerve cells, Banker, G. and Goslin, K.(eds) (Cambridge, USA: MIT Press) 1991).
  • NGF Green
  • cerebellar neurons Terenkner, E in Cultu ⁇ ng Nerve Cells, Banker, G , and Goslin, K. (eds) (Cambridge, USA: MIT Press) 1991
  • cortical neurons Baughman et al., in Culturing Nerve cells, Banker, G. and Gosl
  • mammalian CNS myelin refers to extracts of mammalian central nervous system myelin containing myelin basic protein and myelin associated glycoprotein.
  • the mammalian CNS myelin is a preparation enriched approximatley four fold for the myelin-specific markers, myelin basic protein and myelin associated glycoprotein. This preparation may be obtained from adult rat brains following standard procedures for myelin isolation as described in Norton and Poduslo, J. Neurochem 21:749-758, 1973.
  • the amount of myelin basic protein and myelin associated glycoprotein may be determined by standard Western blotting techniques (Li et al., Nature 369:747-750, 1994).
  • the myelin may be obtained from any mammals, preferably humans, bovines and tats, and preferably adult mammals.
  • the assay uses human brain-derived myelin as a substrate.
  • the inventors have found that powerful neurite outgrowth inhibitory activity is present in human CNS myelin.
  • Human CNS myelin strongly inhibits neuritic outgrowth from newborn rat dorsal root ganglion neurons and NG-108-15 cells.
  • the inhibitory activity in human CNS myelin closely resembles the myelin inhibition of neurite growth that is observed with adult rodent CNS myelin.
  • the inhibition of neurite outgrowth by human CNS myelin can be used as a model to develop strategies to enhance neural recovery and repair in the injured Human CNS.
  • the mammalian CNS myelin is dried as a suspension on a support.
  • the support may be a solid support such as glass or plastic and it may be in the shape of for example, a tube, test plate, disc, wells etc.
  • the support is preferably coated with a substance which promotes neuronal outgrowth, for example, poly-L-lysine (PLL), fibronectin, and or laminin.
  • PLL poly-L-lysine
  • fibronectin fibronectin
  • laminin laminin
  • the test substance may be added to the neuronal cells or the test substance may be introduced by genetically engineering the neuronal cells.
  • the neuronal cells may be transfected with recombinant molecules containing sequences encoding the test substance, or sequences encoding a test substance suspected of being required for inhibition of neurite growth in an antisense orientation.
  • Conditions for carrying out the above described method of the invention may be selected having regard to factors such as the nature and amounts of the neuronal cells, test substance and mammalian CNS myelin.
  • the neuronal cells on CNS myelin are grown in the presence of the test substance for about 18 to 72 hours, preferably 24 and 72 hours at about 37°C and 5% C0 .
  • the concentration of the neuronal cells which may be used in the assay is between 100 and 3000 cells per square cm, preferably 1000 cells per 0.33cm 2 .
  • Neurite outgrowth is assayed by determining the number of neuronal cells with neural processes. This may be determined by counting both the number of cells with processes greater than 1 cell diameter in length and the total number of cells Neurite outgrowth may also be assayed by measuring neurite morphology (Lochter et al , J Cell Biol 113:1159-1171, 1991), measuring biochemical correlates of neurite growth (Goslin and Banker, J Cell Biol 108:1507-1515, 1989) and usmg image analysis systems such as the system known as Leica QuantiMet 500 Plus (Leica, Deerfield, 111)
  • the method can be carried out by growing the neuronal cells on a non-inhibitmg substrate usmg larrurun or PLL, or on a neutral substrate usmg bovine serum albumin B.
  • HYBRIDOMAS AND MONOCLONAL ANTIBODIES are non-inhibitmg substrate usmg larrurun or PLL, or on a neutral substrate usmg bovine serum albumin B.
  • the present mvention contemplates a hybridoma cell lme which produces monoclonal antibodies which (a) immunoreact with neuronal membrane protems, (b) neutralize the inhibition of neurite growth by adult mammalian central nervous system myelin, and, (c) recognize bands of M r 35,000 and M r 33,000 expressed m neuronal and fibroblast cell Imes and m rat cerebrum and rat liver Preferred hybridoma cell Imes are those having the laboratory designation D10
  • the hyb ⁇ domas of the present invention may be formed using conventional methods such as those described by Kohler and Milstein, Nature 256, 495 (1975) and m U S Patent Nos RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference (See also Monoclonal Antibodies, Hyb ⁇ domas A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds ), 1980, and Antibodies A Laboratory Manual, Harlow and Lane (eds ), Cold Sprmg Harbor Laboratory Press, 1988, which are also incorporated herem by reference)
  • hybridoma cell lines are prepared by a process mvolvmg the fusion under appropriate conditions of an immortalizmg cell lme and spleen cells from an animal appropriately immunized to produce the desired antibody
  • Immortalizmg cell Imes may be murme m o ⁇ gm however, cell lines of other mammalian species may be employed including those of rat, bovine, cannine, human origin, and the like
  • the lmmora zing cell Imes are most often of tumor o ⁇ gm, particularly myeloma cells but may also mclude normal cells transformed with, for example, Epstem Barr Virus Any immortalizmg cell may be used to prepare the hybndomas of the present mvention
  • Antibody producing cells may be employed as fusion partners such as spleen cells or peripheral blood lymphocytes
  • the animal from w hich the cells are to be derived may be immunized at intervals with a membrane fraction obtained from neuronal cells such as rat pheochromocytoma PC-12 (ATCC NO CRL 1721)
  • the immortalizing cells and lymphoid cells mav be fused to form hybndomas according to standard and w ell-known techniques mg polvethvlene glvcol as a fusing agent Alternatn eh , fusion mav be accomplished b ⁇ eletrofusion Hybridomas are screened for appropriate monoclonal antibody secretion by assaying the supernatant or protein purified from the ascites for reactivity using the method described in Section A herein. The hybridomas are screened for antibodies which would modulate the inhibition of neurite growth by adult mammalian CNS myelin.
  • a subject animal such as a rat or mouse, for example a BALB/C mouse
  • a membrane fraction obtamed from neuronal cells such as rat pheochromocytoma PC-12.
  • the membrane fraction may be admixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response.
  • an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response.
  • the animal may be reimmunized with another booster immunization, and its serum tested for antibodies which react with neuronal proteins, or for the ability to block neurite inhibition using the assays described herein.
  • spleen and lymph nodes are harvested.
  • Cells which are obtained from the immunized animal may be immortalized by transfection with a virus such as the Epstein Barr virus (EBV) (see Glasky and Readmg, Hybridoma 8(4):377-389, 1989).
  • EBV Epstein Barr virus
  • the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a hybridoma which secretes monoclonal antibody.
  • suitable myeloma lines include, for example, Sp2 myeloma cells (Shulman et al. Nature 276:269-270, 1978)
  • the cells may be placed into culture plates containmg a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional mgredients, such as Fetal Bovme Serum (FBS, ⁇ e., from Hyclone, Logan, Utah, or JRH Biosciences) Additionally, the medium should contam a reagent which selectively allows for the growth of fused spleen and myeloma cells such as HAT (hypoxanthine, ammopterm, and thymidme) (Sigma Chemical Co., St. Louis, Missouri). After about seven days, the medium m which the resulting fused cells or hybridomas have been growing may be screened in order to determine the presence of antibodies which modulate the inhibitory activity of CNS myelin in the assays described herein.
  • a suitable medium such as RPMI 1640, or DMEM (Dulbec
  • the monoclonal antibodies produced by the hybridoma cell lines of the invention are also part of the present invention.
  • the monoclonal antibodies produced by the hybridoma cell lines of the present invention immunoreact with neuronal membrane proteins and belong to the immunoglobulin M protein class.
  • Monoclonal antibodies which immunoreact with neuronal membrane proteins includes homogeneous populations of immunoglobulins which are capable of immunoreaction with antigens expressed on neuronal cells. It is understood that immunoglobulins may exist in acidic, basic, or neutral form depending on their amino acid composition and environment, and they may be found in association with other molecules such as saccharides or lipids. It is also understood that there may be a number of antigens present on the surface of any cell and, alternatively, that certain antigens on neuronal cells may also occur on other cell types. Moreover, such antigens may, in fact, have a number of antigenic determinants. The monoclonal antibodies produced by hybridoma cell lines of the invention may be directed against one or more of these determinants.
  • any characteristic antigen associated with neuronal membranes may provide the requisite antigenic determinant. It is contemplated that monoclonal antibodies produced by the hybridoma cell lines fall within the scope of the present invention so long as they remain capable of selectively reacting with neuronal membrane proteins, particularly neuronal membrane proteins obtained from neuronal cells such as rat pheochromocytoma PC-12.
  • Monoclonal antibodies produced by hybridoma cell lines according to the invention were found to neutralize the inhibition of neurite growth by adult mammalian central nervous system myelin.
  • the monoclonal antibody having the laboratory designation 10D was shown to reverse the near-complete suppression of neurite growth exerted by a substrate of 10 ⁇ g/cm 2 of CNS myelin, on NG108-15 cells, PC12NGF cells and primary SCG neurons.
  • the 10D monoclonal antibody did not increase neurite growth on non-inhibitory (laminin, PLL) or neutral (BSA) substrates.
  • the antigens recognized by the monoclonal antibodies described herein are also a part of the present invention.
  • the present inventors investigated the immunoreactivity of the monoclonal antibodies of the present mvention with proteins from tissues, for example adult rat cerebrum and rat liver, and cell Imes, for example dbc AMP
  • the monoclonal antibodies were found to be immunoreactive against bands of M r 35,000 and 33,000 expressed in neuronal and fibroblast cell lines, and in rat brain and liver.
  • An antigen recognized by a monoclonal antibody produced by a hybridoma cell line of the invention may be localized to specific neuronal cells in the brain, brains tern and cerebellum using conventional immunocytochemistry methods.
  • embryonic, newborn and adult Sprague-Dawley rats may be used.
  • Cryostat sections of fixed brain, cerebellum, brainstem or spinal cord may be mcubated with 10D ascites at 1:50 to 1:500 dilutions and processed by the avidin-biotin-peroxidase technique (ABC Vectastam). This will determine which class of cells in the CNS express the 10D antigen.
  • Both neurons and glia may express this molecule. Regions in the CNS that express the 10D antigen may be 5 urveyed. The possible localization of the antigen to a subset of neural paths and the pattern of acquisition of the 10D antigen will provide important msights on the function oi the 10D antigen and establish the optimal neuronal population for determmmg the effects of 10D antigen blocking or overexpression. If 10D monoclonal antibody binds to the putative neuronal receptor to the inhibitory myelm proteins, then neurons early in development which appear to be msensitive to the myelm inhibitors (Wictorm et al., 1990; Nature, Vol. 347: 556 and Davies et al., 1994) may be negative for 10D staining. The acquisition of the susceptibility to the myelin inhibitors should coincide with the developmental appearance of neuronal 10D immunoreactivity.
  • the invention also provides a method for assaymg for the presence of an activator or inhibitor of a monoclonal antibody produced by hybridoma cell Imes of the mvention comprising growmg neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelm m the presence of a known concentration of the monoclonal antibody, and in the presence of a suspected activator or mhibitor of the monoclonal antibody, and assaymg for neurite outgrowth.
  • CNS central nervous system
  • the present inventors sequenced the Dl clone and found that it includes the nucleic acid sequences set out m SEQ ID No 1, SEQ ID No 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7,and SEQ ID NO 8, and in Figures 9, 1 1 to 14 and 21
  • the partial sequences show no sequence identity with previously-reported genes.
  • the location of the nucleic acid sequences shown in the Sequence Listing in the Dl gene is shown in Figure 15.
  • a diagram of the sequenced regions of Dl is shown in Figure 16.
  • Probes derived from sequences in the partial cDNA clone were used to screen a cDNA library, and a gene designated "petrin" encoding a protein which plays a role in neurite growth inhibition was identified.
  • the sequence of the Petrin gene is shown in Figure 23.
  • the putative initiation codon is at nucleotide 486 to an in-frame stop codon at nucleotide 1977.
  • the petrin locus was localized to chromosome 12.
  • the present invention provides a isolated and purified nucleic acid molecule comprising
  • nucleic acid molecule (d) a fragment of the nucleic acid molecule that is at least 15 bases and that will hybridize to (a) or (b) under stringent hybridization conditions, or (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to
  • the invention also relates to a nucleic acid molecule comprising (a) a nucleic acid sequence encoding a protein having the amino acid sequence as shown in Figure 24 (or SEQ. ID. NO. 12); (b) nucleic acid sequences complementary to (a);
  • the isolated and purified nucleic acid molecule comprises
  • isolated and purified refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • An "isolated and purified" nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • the invention contemplates a do ubl e stranded nucleotide sequence comprising a nucleic acid molecule of the mvention or a fragment thereof, hydrogen bonded to a complementary nucleotide base sequence, and an RNA made by transcription of this double stranded nucleotide sequence.
  • the mvention mcludes nucleic acid molecules encodmg truncations of the protein encoded by the Petrin gene, and analogs and homologs of the protem and truncations thereof, as described herem. It will also be appreciated that variant forms of the nucleic acid molecules of the mvention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention
  • nucleic acid molecules contemplated by the present mvention include the fragments of the nucleic acid molecule are the nucleotide sequences shown m SEQ. ID. NO 1, SEQ. ID. No 3, SEQ. ID. NO. 4, SEQ ID. NO 5, SEQ.ID. NO 6, SEQ ID NO. 7 and SEQ ID. NO 8 and Figures 9, 11 to 14, and 21 It is also contemplated that nucleic acid molecules of the mvention will be prepared havmg mutations such as msertion or deletion mutations, e g nucleic acid molecules encodmg analogs of the Petrm protem.
  • nucleic acid molecules comprising nucleic acid sequences havmg substantial sequence identity with the nucleic acid sequences shown m SEQ ID NO:l, SEQ ID No 3, SEQ ID NO 4, SEQ ID NO 5, SEQ.ID. NO 6, SEQ.ID. NO 7 and SEQ ID. NO. 8 or m Figures 9, 11 to 14, and 21, or shown m Figures 23 or SEQ ID NO 11, and fragments thereof
  • sequences havmg substantial sequence identity means those nucleic acid sequences which have slight or mconsequential sequence variations from the sequences disclosed m SEQ ID NO 1, SEQ ID No 3, SEQ ID NO 4, SEQ ID. NO 5, SEQ ID NO.
  • Nucleic acid sequences having substantial identity include nucleic acid sequences havmg at least 80-90%, preferably 90% identity with the nucleic acid sequences as shown in SEQ ID NO 1, SEQ ID No 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 or in Figures 9, 11 to 14, and 21, or as shown in Figures 23 or SEQ ID NO 11 and fragments thereof hav mg at least 15 bases w hich w ill hv b ⁇ dizc to thesi sequences under stringent hybridization conditions.
  • Stringent hybridization conditions are those which are stringent enough to provide specificity, reduce the number of mismatches and yet are sufficiently flexible to allow formation of stable hybrids at an acceptable rate. Such conditions are known to those skilled in the art and are described, for example, in Sambrook, et al, (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor). By way of example only, stringent hybridization with short nucleotides may be carried out at 5-10° below the T m using high concentrations of probe such as 0.01-1.Opmole/ml.
  • nucleic acid molecules encoding a protein havmg the activity of Petrin as described herein, and having a sequence which differs from the nucleic acid sequence shown in Figure 23 (or SEQ ID NO:ll) due to degeneracy in the genetic code are also within the scope of the invention.
  • nucleic acids encode functionally equivalent proteins (e.g., a protein having Petrin phosphatase activity) but differ m sequence from the sequence of Figure 23 (or SEQ ID NO: 11) due to degeneracy m the genetic code.
  • DNA sequence polymorphisms withm the nucleotide sequence of Petrm may result in "silent" mutations in the DNA which do not affect the ammo acid encoded.
  • DNA sequence polymorphisms may lead to changes in the ammo acid sequences of Petrm within a population.
  • These variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding proteins havmg the activity of Petrm may exist among mdividuals within a population due to natural allelic variation
  • Such nucleotide variations and resultmg ammo acid polymorphisms are within the scope of the mvention
  • nucleic acid molecule of the invention which comprises DNA can be isolated by preparmg a labelled nucleic acid probe based on all or part of the nucleic acid sequence shown in Figure 23 or SEQ.ID. NO. 11, or shown m Figures 9, 11 to 14, and 21 (SEQ ID NO 1, SEQ. ID. No. 3, SEQ ID. NO 4, SEQ.ID NO 5, SEQ ID NO 6, SEQ.ID. NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 11), and usmg this labelled nucleic acid probe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library) Nucleic acids isolated by screenmg of a cDNA or genomic DNA library can be sequenced by standard techniques. An isolated and purified nucleic acid molecule of the mvention which is
  • DNA can also be isolated by selectively amplifying a nucleic acid encodmg a petrm protein usmg the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence shown in Figures 9, 11 to 14, 21 or 23, (SEQ ID NO.l, SEQ. ID No 3, SEQ ID NO 4, SEQ.ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, or SEQ.
  • PCR polymerase chain reaction
  • nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques
  • the amplified nucleic acid can be cloned mto an appropriate vector and characterized by DNA sequence analysis
  • cDNA may be prepared from mRN'A bv isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwm et al., Biochemistry, 18, 5294-5299 (1979).
  • cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • RNA nucleic acid molecule of the mvention which is RNA
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.
  • a bacteriophage promoter e.g. a T7 promoter
  • a nucleic acid molecule of the invention mcludmg fragments, m ly also be chemically synthesized using standard techniques Various methods of chemically synthesizing polydeoxynucleotides are known, mcludmg solid-phase synthesis which, like peptide synthesis, has been fully automated m commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049, Caruthers et al. U.S Patent No 4,458,066, and Itakura U.S Patent Nos 4,401,796 and 4,373,071).
  • Determination of whether a particular nucleic acid molecule encodes a protem havmg Petrm activity can be accomplished by expressmg the cDNA m an appropriate host cell by standard techniques, and testing the phosphatase activity of the expressed protem or the ability of the expressed protem to inhibit neurite outgrowth as described herem
  • a cDNA havmg the biological activity of Petrm so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencmg, to determine the nucleic acid sequence and the predicted ammo acid sequence of the encoded protem.
  • the initiation codon and untranslated sequences of Petrin may be determined usmg currently available computer software designed for the purpose, such as PC/Gene (IntelliGenetics Inc., Calif.).
  • the mtron-exon structure and the transcription regulatory sequences of the gene encodmg Petrm may be identified by usmg a nucleic acid molecule of the mvention encodmg Petrm to probe a genomic DNA clone library Regulatory elements can be identified usmg conventional techniques
  • the function of the elements can be confirmed by usmg them to express a reporter gene such as the bacterial gene lacZ which is operatively lmked to the elements
  • These constructs may be mtroduced mto cultured cells usmg standard procedures or mto non-human transgenic animal models Such constructs may also be used to identify nuclear protems interacting with the elements, usmg techniques known in the art
  • nucleic acid sequences contained in the nucleic acid molecules of the mvention or a fragment thereof, preferablv one or more of the nucleic acid sequences shown in the Sequence Listing as SEQ. ID. NO. 1 SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ. ID. NO. 8 and in Figures 9, 11 to 14, and 21, or in Figure 23 (or SEQ.ID. NO. 11) may be inverted relative to their normal presentation for transcription to produce antisense nucleic acid molecules.
  • the antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the antisense nucleic acid molecule comprises the following sequence: GCT GCC AGC CAT GAT GCC GCC CAT (SEQ. ID. NO: 13). This antisense sequence enhanced neurite growth in a functional in vitro assay.
  • Petrin cDNA Translation of the Petrin cDNA revealed a single large open reading frame from a putative initiation codon at nucleotide 486 to an in-frame stop codon at nucleotide 1977.
  • the inventors have determined the primary structure of the deduced protem and have determined that it has predicted molecular weight of 60 to 64 kDa.
  • the protein has 3 to 4 distinct regions with up to 60% identity with members of the protem phosphatase 2C family (“PP2C”) (See Figure 24).
  • P2C protem phosphatase 2C family
  • Members of the protein phosphatase 2C family dephosphorylate serine and threonine residues in proteins. See review articleby Wera, S., and B.A. Hemmmgs, Biochem. J. (1995) 311, 17-29).
  • the novel protein has been designated "petrin”.
  • the present inventors have also shown by in situ hybridization that the petrin gene is expressed in neurons in brain tissue and in particular, m the Purkinje cells of the cerebellum; in the 3rd and 4th layers of the cerebral cortex; and, dispersed neurons in the hippocampus. Expression of petrin occurred after embryonic day 13 and increased constantly with the highest expression found in adults. Northern and DNA analysis also showed that the protein is present in different mammalian species such as mouse, rat, hamster, and human
  • Petrin The biological function of Petrin was investigated using phosphatase assays on immunoprecipitated material and like other members of the PP2C family, it exhibited magnesium-dependent serine/ threonine phosphatase activity.
  • the protein was also shown to have magnesium-dependent tyrosine phosphatase activity. Serine/ threonine- phosphatase and tyrosine phosphatase activities were inhibited by okadaic acid or ortho- vanadate, respectively.
  • the present inventors also prepared antisense oligonucleotides and found that they enhanced neunte growth in a functional in vitro assay. Phosphatase activity was also shown to be highest while NG108 cells are proliferating and growing neurites, and was not detected in late growth stages.
  • the present invention also includes a protem containing the amino acid sequences as shown in the Sequence Listing as SEQ. ID. NO. 2 and 10 and as shown in
  • the protein comprises the amino acid sequence as shown in Figure 24 (or SEQ. ID. NO. 12).
  • the protein of the invention may be found in brain, NG108, and PC12 cells In addition to the full length amino acid sequence ( Figure 24 or SEQ ID.
  • the protems of the present invention include truncations and analogs, and homologs of the protem and truncations thereof as described herein
  • Truncated proteins may comprise pephdes of between 3 and 1900 amino acid residues, ranging m size from a t ⁇ peptide to a 1900 mer polypeptide
  • a truncated protem may comprise the regions highly conserved among the PP2C protems (e.g. ammo acids 281 to 324, 411 to 451, 516 to 557, or 630 to 640 m Figure 24 or SEQ. ID NO. 12).
  • Truncated protems also include the proteins having the sequences shown in the Sequence Listing as SEQ. ID. Nos. 2, 9, 10, or as shown m Figure 10
  • the truncated protems may have an ammo group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T- butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule mcludmg but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
  • -NH2 ammo group
  • a hydrophobic group for example, carbobenzoxyl, dansyl, or T- butyloxycarbonyl
  • PMOC 9-fluorenylmethoxy-carbonyl
  • a macromolecule mcludmg but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
  • the truncated proteins may have a carboxyl group, an amido group, a T- butyloxycarbonyl group, or a macromolecule including lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
  • the protems of the mvention may also mclude analogs of Petrm as shown m Figure 24 (SEQ. ID. NO. 12) and/or truncations thereof as described herem, containing one or more ammo acid substitutions, mserhons, and /or deletions Ammo acid substitutions may be of a conserved or non-conserved nature.
  • ammo acid msertions may be mtroduced mto the ammo acid sequence as shown m Figure 24 (SEQ ID NO 12)
  • Ammo acid msertions may consist of single ammo acid residues or sequential ammo acids ranging from 2 to 15 ammo acids in length
  • amino acid insertions may be used to destrov the phosphatase activitv of the protein
  • Deletions may consist of the removal of one or more amino acids, or discrete portions (e.g.amino acids 281 to 324, 411 to 451, 516 to 557, or 630 to 640 in Figure 24 or
  • the deleted amino acids may or may not be contiguous.
  • the lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.
  • the proteins of the invention also include homologs of Petrin as shown in Figure 24 or SEQ. ID. NO. 12 and/or truncations thereof as described herein.
  • Such homlogs are proteins whose amino acid sequences are comprised of the amino acid sequences of Petrin regions from other species that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain Petrin as shown in Figure 24 or SEQ. ID. NO. 12.
  • Homologs will have the same regions characteristic of Petrin and PP2C proteins. It is anticipated that, outside of these regions of Petrin a protein comprising an amino acid sequence which is about 50% similar, preferably 80 to 90% similar, with the amino acid sequence shown in Figure 24 or SEQ. ID. NO. 12 will exhibit phosphatase activity and inhibit neurite outgrowth.
  • the invention also contemplates isoforms of the Petrin protein of the invention.
  • An isoform contains the same number and kinds of amino acids as the protein of the invention, but the isoform has a different molecular structure.
  • the isoforms contemplated by the present invention are those having the same properties as the protein of the invention as described herein.
  • the present invention also includes a Petrin protein conjugated with a selected protein, or a selectable marker protein (see below) to produce fusion proteins. Additionally, immunogenic portions of Petrin proteins are within the scope of the invention.
  • the protein encoded by nucleic acid molecules of the mvention may be prepared using recombinant DNA methods. Accordingly, the nucleic acid molecules of the present mvention or a fragment thereof may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses, so long as the vector is compatible with the host cell used.
  • the invention therefore contemplates a recombinant molecule of the invention containing a nucleic acid molecule of the mvention, or a fragment thereof, and the necessary elements for the transcription and translation of the inserted sequence.
  • Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or msect genes. Selection of appropriate transcription and translation elements is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordmary skill in the art Examples of such elements include: a transc ⁇ ptional promoter and enhancer or RNA polymerase binding sequence, a ⁇ bosomal binding sequence, mcludmg a translation initiation signal Additionally, depending on the host cell chosen and the vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary transcription and translation elements may be supplied by the native gene and/or its flanking regions.
  • the recombinant molecules of the invention may also contain a reporter gene encoding a selectable marker protein which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • reporter genes are genes encoding a protein such as ⁇ -galactosidase (e.g.lac Z), chloramphenicol, acetyl-transferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immimoglobulin preferably IgG.
  • Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. This makes it possible to visualize and assay for expression of recombinant molecules of the invention and in particular to determine the effect of a mutation on expression and phenotype.
  • the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation etc.
  • Methods for transforming transfecting, etc. host cells to express foreign DNA are well known in the art (see, e.g., Itakura et al, U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950; Axel et al., U.S. Patent No.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells, preferably neuronal cells such as NG108-derived lines and PC12.
  • Bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.
  • Representative examples of bacterial host cells include E.coli BL21, DE3, Streptomyces lividans strain 66.
  • Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication.
  • Representative promoters include the ⁇ -lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth in Enzymology 101 :155, 1983), the tac promoter (Russell et al., Gene 20: 231, 1982), and the phage T3 promoter (Studier and Moffat, J Mol. Biol. 189:113-130, 1986).
  • Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes.
  • Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bolivar et al.. Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19, pUCll ⁇ , pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messmg, Gene 19:259-268, 1982), and pNH8A, pNHl ⁇ a, pNH18a, pCDM8, Bluescript M13 (Stratagene, La Jolla, Calif.), and pETIO (Studier et al, Meth. Enzymol. 185:60-89, 1990).
  • bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bolivar et al.. Gene 2:9S, 1977)
  • Yeast and fungi host cells suitable for carrying out the present invention include, among others Saccharomyces cerevisae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus.
  • Suitable expression vectors for yeast and fungi m include, among others, YC p 50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989). Protocols for the transformation of yeast are also well known to those of ordinary skill in the art (See for example, Hinnen et al., PNAS USA 75:1929, 1978; Itoh et al., J. Bacteriology 153:163, 1983;and Cullen et al. Bio /Technology 5:369, 1987)
  • Mammalian cells suitable for carrying out the present mvention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells.
  • COS e.g., ATCC No. CRL 1650 or 1651
  • BHK e.g., ATCC No CRL 6281
  • CHO ATCC No. CCL 61
  • HeLa e.g., ATCC No. CCL 293
  • CHOP e.g., ATCC No. 1573
  • NS-1 cells e.g., NS-1 cells.
  • Suitable expression vectors for directing expression in mammalian cells generally mclude a promoter, as well as other transcnphon and translation control sequences.
  • Common promoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR Protocols for the transfection of mammalian cells are well known in the art and include calcium phosphate mediated electroporahon, retroviral, and protoplast fusion-mediated transfection (see Sambrook et al., supra).
  • promoters, terminators, and methods for mtroducmg expression vectors of an appropriate type mto plant, avian, and msect cells may also be readily accomplished.
  • the nucleic acid molecule of the invention may be expressed from plant cells (see Sinkar et al , J Biosci (Bangalore) 11:47-58, 1987, which reviews the use of Agrobacte ⁇ um rhizogenes vectors, see also Zambryski et al., Genetic Engineering, Principles and Methods, Hollaender and Setlow (eds ), Vol VI, pp. 253-278, Plenum Press, New York, 1984, which describes the use of expression vectors for plant cells, mcludmg, among others, pAS2022, pAS2023, and pAS2034)
  • Insect cells suitable for carrying out the present mvention m clude cells and cell lines from Bombyx or Spodotera species Suitable expression vectors for directing expression in insect cells include Baculoviruses such as the Autographa California nuclear polyhedrosis, virus (Miller et al 1987, m Genetic Engineering, Vol 8 ed Setler, J K et al , Plenum Press, New York) and the Bombyx mori nuclear polyhedrosis v lrus (Maeda et al , 1985, Nature 315.592)
  • Baculoviruses such as the Autographa California nuclear polyhedrosis, virus (Miller et al 1987, m Genetic Engineering, Vol 8 ed Setler, J K et al , Plenum Press, New York) and the Bombyx mori nuclear polyhedrosis v lrus (Maeda et al , 1985, Nature 315.592)
  • the protein encoded bv the nucleic acid molecule of the invention may be expressed in non-human transgenic animals such as, mice, rats, rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al. (Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiter and Brinster (Cell. 41:343-345, 1985) and U.S. Patent No. 4,736,866).
  • non-human transgenic animals such as, mice, rats, rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al. (Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiter and Brinster (Cell. 41:34
  • the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • the proteins of the invention may be conjugated with other molecules, such as proteins or polypeptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • fusion proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the protein, and a selected protein with a desired biological function.
  • the resultant fusion proteins contain the protein or a portion thereof fused to the selected protein.
  • proteins which may be used to prepare fusion proteins include neurotrophic factors, such as nerve growth factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), and NT-3.
  • NNF nerve growth factor
  • BDNF Brain-Derived Neurotrophic Factor
  • CNTF ciliary neurotrophic factor
  • FGF fibroblast growth factor
  • NT-3 fibroblast growth factor
  • probes include the fragments shown in the Sequence Listing as SEQ. ID. NO. 1 and NOS. 3 to 6, 7, 8 and 9.
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32 T, 3 H, 1 C or the like.
  • detectable substances include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the amount of nucleic acid available for hybridization.
  • Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule of the invention under strmgent hybridization conditions as described herein.
  • the Dl or Petrin cDNA ( Figure 23 or SEQ. ID. NO 11 ) may be used to identify, study and isolate the corresponding human gene.
  • the present inventors have shown that the Dl cDNA sequences from position bp230 to bpl,095 (as shown in the Sequence Listing as SEQ. ID. NO. 7) specifically recognize human genomic DNA fragments similar in number to those recognized in rat and mouse DNA.
  • the present inventors have also shown using a panel of human-rodent hybrid cell lines that all the Dl gene sequences detected in the human genome reside on chromosome 12. The Dl probes can thus be used to determine whether human disorders are genetically linked to the petrin or Dl gene.
  • the present inventors have also demonstrated that the rat Dl cDNA can be used to detect human Dl mRNA and study its expression in normal tissue and in disease.
  • the proteins of the invention or parts thereof, may be used to prepare antibodies.
  • Antibodies having specificity for the protein may also be raised from fusion proteins created by expressing fusion proteins in host cells as described above.
  • antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab') 2 and recombinantly produced binding partners.
  • Antibodies are understood to be reactive against the protein encoded by the nucleic acid molecule of the invention if they bind with a K a of greater than or equal to 10- 7 M.
  • K a of greater than or equal to 10- 7 M.
  • antibodies may be developed which not only bind to the protein, but which bind to a regulator of the protein, and which also block the biological activity of the protein.
  • Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Briefly, a protein of the invention is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to the protein. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background.
  • Monoclonal antibodies may also be readily generated using conventional techniques as described above.
  • Binding partners may be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody.
  • the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the v ariable region. These primers may be synthesized by one of ordinary skill in the art, or mav be purchased from commercially available sources. Primers for mouse and human variable regions including, among others, primers for V ⁇ a > ⁇ b VH O ⁇ Hd» HI / VL and CL regions are available from Stratacyte (La Jolla, Calif).
  • These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTM H or ImmunoZAP TM L (Stratacyte), respectively. These vectors may then be introduced into £. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced (See Bird et al., Science 242:423-426, 1988). In addition, such techniques may be utilized to change a "murine" antibody to a "human” antibody, without altering the binding specificity of the antibody.
  • the polyclonal or monoclonal antibodies and binding partners may be used to detect a protein of the invention for example, in various biological materials, for example they may be used in an Elisa, radioimmunoassay or histochemical tests.
  • the an tibodies may be used to quantify the amount of the protein in a sample in order to determine its role in particular cellular events or pathological states and to diagnose and treat such pathological states.
  • the polyclonal and monoclonal antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a protein of the invention, to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a protein of the invention.
  • an antibody specific for the protein may be labelled with a detectable substance as described herein and the protein may be localised in tissue based upon the presence of the detectable substance.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the protein encoded by the nucleic acid molecule of the invention.
  • the protein encoded by the nucleic acid molecule of the invention may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • the above described methods for detecting nucleic acid molecules and fragments thereof and protein can be used to monitor neurite growth by detecting and localizing the nucleic acid molecule and /or protein of the invention in organisms, tissues, and embryos. It would also be apparent to one skilled in the art that the above described methods may be used to study the developmental expression of a protein of the invention and, accordingly, will provide further insight into the role of the protein in neuronal growth in the CNS.
  • the invention provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising growing neuronal cells which have a propensity for neurite growth in the presence of a protein of the invention and a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
  • the invention also provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelin and which express the protein of the invention, in the presence of a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
  • the activator or inhibitor may be an endogenous physiological substance or it may be a natural or synthetic drug. Conditions for carrying out these methods of the invention are selected to favour neurite outgrowth and having regard to factors such as the nature and amounts of the neuronal cells and test substance.
  • the methods permit the identification of potential activators or inhibitors of neurite growth m the central nervous system environment which have various applications as discussed below. Substances which affect cell neurite growth may also be identified by comparing the pattern and level of expression of the novel nucleic acid of the mvention or its protein product, in tissues and cells in the presence and in the absence of a test substance.
  • the invention also contemplates a method for assaying for a substance that affects neuronal growth comprismg administering to a non-human animal or to a tissue of an animal, a substance suspected of affecting neuronal growth, and detectmg, and optionally quantitatmg, the nucleic acid molecule of the mvention or a protem of the invention in the non-human animal or tissue.
  • the invention also contemplates a method for identifying a substance which is capable of bmdmg to a protem of the mvention, or a part of the protem, comprismg reactmg the protem, or part of the protem, with at least one substance which potentially can bmd with the protem, or part of the protem, under conditions which permit the formation of substance-protein complexes, and assaymg for substance-protein complexes, and /or for free substance, and for non-complexed protem.
  • the mvention provides a method for assaymg a medium for the presence of an activator or inhibitor of the mteraction of the protem of the mvention or part thereof, and a substance which bmds to the protein
  • the method comprises providmg a known concentration of a protem of the mvention, or part of the protem, mcubatmg the protem, or part of the protem with a substance which bmds to the protem, or part of the protein, and a suspected activator or mhibitor substance, under conditions which permit the formation of substance-protem complexes, and assaymg for substance complexes
  • the mvention also contemplates a method for assaymg for a substance that affects the phosphatase activity of a protein of the invention comprising reacting a protem of the mvention with a substrate which is capable of being dephosphorv lated bv the protein to produce a dephosphorylated product, in the presence of a substance which is suspected of affecting the phosphatase activity of the protein, under conditions which permit dephosphorylation of the substrate, assaying for dephosphorylated product, and comparing to a product obtained in the absence of the substance to determine the affect of the substance on the phosphatase activity of the protein.
  • Suitable substrates include serine, threonine, or tyrosine phospho-peptides. Conditions which permit the dephosphorylation of the substrate, may be selected having regard to factors such as the nature and amounts of the substance, substrate, and the amount of protein.
  • Substances which modulate neurite growth identified using the methods of the invention including the monoclonal antibody produced by a hybridoma cell line of the invention, the nucleic acid molecule and protein of the invention, and the antisense nucleic acid molecules of the invention, may be useful in regulating neurite outgrowth in vivo and may form the basis for a strategy to enhance or inhibit neurite growth/axonal regeneration in the mammalian CNS.
  • the substances may be used to enhance (1) axonal regrowth in the CNS following traumatic CNS lesions; (2) formation of neuronal connections in neural transplantation therapies; and 3) the ability of surviving neurons to form new connections and thereby take over some of the functions of neurons lost in CNS neurodegenerative diseases such as Alzheimer's and Parkinson's Disease.
  • the substances identified herein may be used to stimulate or inhibit neuronal regeneration associated with conditions involving nerve damage resulting from traumatic injury, stroke, or degenerative disorders of the central nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demyelinating diseases, progressive spinal amyotrophy, trauma and ischemia resulting from stroke, and tumors of nerve tissue, epilepsy, glaucoma, and neurofibromatosis.
  • neuronal regeneration associated with conditions involving nerve damage resulting from traumatic injury, stroke, or degenerative disorders of the central nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demyelinating diseases, progressive spinal amyotrophy, trauma and ischemia resulting from stroke, and tumors of nerve tissue, epilepsy, glaucoma, and neurofibromatosis.
  • compositions may be incorporated into a pharmaceutical composition containing the substance or antibodies, alone or together with other active substances.
  • Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets.
  • the methods described by Perm et al, Lancet 335(8691):738-747,1990 for intrathecally delivering substances into the CNS may be particularly useful for administering the pharmaceutical compositions of the invention.
  • compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
  • compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the pharmaceutical compositons may additionally contain other agents such as neurotrophic factors, in particular NGF, BDNF, CNTF, T-3 and FGF.
  • the antisense nucleic acid molecules of the invention may be used m gene therapy to enhance axonal regeneration. For a discussion of the regulation of gene expression using anh-sense genes see Wemtraub, H. et al., Antisense RNA as a molecular tool for genetic analysis. Reviews - Trends m Genetics, Vol. 1(1) 1986.
  • Recombinant molecules comprismg an antisense sequence or oligonucleotide fragment thereof, may be directly mtroduced mto cells or tissues in vivo usmg delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be mtroduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA mto liposomes. Recombinant molecules may also be delivered m the form of an aerosol or by lavage.
  • the antisense nucleic acid molecules of the mvention may also be applied extracellularly such as by direct injection mto cells.
  • the effect of 10D antibody and substances identified usmg the methods of the mvention can be tested in vivo on the regeneration of interrupted neural pathways by CNS neurons m the rat optic nerve (See Thanos S., and von Boxderg, i , Metabolic Bram Disease 4 67-72, 1989) Axons from retmal ganglion cells (RGC) project mto the CNS environment of the optic nerve.
  • CNS neurons m the rat optic nerve
  • RRC retmal ganglion cells
  • This projection does not normally regenerate after mjury, but the axons will grow mto a non-inhibitory PNS graft, implicating environmental factors
  • the model can be used to determine whether RGC axons interrupted with the optic nerve will mcrease their propensity for regeneration m the presence of a test substance and optionally neurotrophic factors
  • the regeneration of retmal ganglion cells m the optic nerve is a useful model since this discrete axonal projection, entirely within the CNS, is readih accessible and surgical techniques using the optic nerve are known.
  • a specific protocol for the optic nerve model is described in Example 6.
  • a second model involves examining the regeneration of central processes of dorsal root ganglion neurons (See Carlstedt et al., Bram Res. Bulletin, Vol.22:93-102, 1989).
  • the ability of test substances to modulate regrowth of peripheral axons within the spmal cord can be tested usmg this model.
  • the model also permits an assessment of the effect of a test substance on the active phase of axonal regrowth in the face of CNS inhibitors
  • 300-500 ⁇ g of NGF or vehicle can also be mjected mto the spmal cord at the time of initial surgery.
  • the administration of other neurotrophms (NT-3, BDNF, CNTF and FGF) m combination with a test substance identified m accordance with the present mvention can also be studied.
  • Example 7 A specific protocol for the regrowth of dorsal root ganglion neurons is described m Example 7.
  • Other examples of non-human animal models for testmg the application of substances identified m accordance with the present invention are models of neurodegenerative conditions, for example, the MPTP model as described in Langston J W et al., Symposium of Current Concepts and Controversies in Parkmson's Disease, Montebello, Quebec, Canada, 1983 and Tatton W.G. et al., Can J Neurol Sci 1992, 19, and traumatic nerve damage for example, animal stroke models such as the one 1 described m MacMillan et al Bram Research 151:353-368 (1978)).
  • the mvention also provides methods for exammmg the function of the protem encoded by the nucleic acid molecule of the mvention
  • Cells, tissues, and non-human animals lacking in expression or partially lacking in expression of the protein may be dev eloped usmg recombmant molecules of the invention having specific deletion or insertion mutations in the nucleic acid molecule of the mv ention
  • a recombmant molecule mav be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue or animal.
  • Such a mutant cell, tissue or animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on the protein encoded by the nucleic acid molecule of the invention.
  • the following non-limiting examples are illustrative of the present invention:
  • Rat pheochromocytoma PC-12 were obtained from the American Type Culture Collection (ATTC NO. CRL 1721, Rockville, Maryland). Cells were grown in RPMI-1640 media (Gibco) with 15% fetal calf serum (FCS). PC-12 cells were differentiated with 100 ng/ml of nerve growth factor (NGF) for 7 days. Cells of the NG-108-15 line were obtained from Dr. G. Cheng (University of Manitoba, Manitoba, Canada ). The preparation of the cells is described in Nelson et al., Proc. Nat. Acad. Sci. USA 73:123-127, 1976.
  • NGF nerve growth factor
  • NG108-15 cells were grown in DME medium with 10% FCS, 1XHAT medium (Gibco) and were induced to differentiate to the neuronal phenotype by reducing the serum to 5% and by the addition of 1 mM dibutyryl cyclic adenosine-monophosphate (dbcAMP) (Sigma) for 2 to 4 days.
  • Primary superior cervical ganglion neurons were obtained from newborn rats and cultured as described in Paterson and Chun, Dev. Biol. 56:263-280, 1977. Penicillin (25 U/ml) and Streptomycin (25 ⁇ g/ml) were added to all media.
  • CNS myelin was prepared from brains of Sprague-Dawley rats (250-300g) using modifications of previously described procedures (Caroni and Schwab, J. Cell. Biol 106:1281-1298, 1988). Homogenization was carried out using 10 mis per gram of tissue of 0.25 M sucrose, 5 mM EDTA, 5 mM iodoacetamide (homogenization buffer) using a glass homogenizer. The homogenate was centrifuged at 2000 rpm in a Sorval HB-4 rotor for 3 minutes to pellet cell debris and nuclei.
  • the supernatant was layered atop 20 mis of 0.85 M sucrose, 5 mM EDTA, 5 mM iodoacetamide in 38 ml SW-28 tubes (Beckman) and centrifuged at 4°C and 28,000 g for 1 hour. The interface was collected, kept on ice and washed in 20 volumes of 30 mM Hepes pH 7.4, 5 mM EDTA, 5 mM iodoacetamide. After centrifugation at 28,000 g for 4 hours, the pellet was resuspended in homogenization buffer and layered onto 0.85M sucrose with protease inhibitor PMSF.
  • Splenic lymphocytes were fused with Sp2 myeloma cells (Shulman et al., 1978, Nature 367:170-173) following established procedures (Harlow and Lane, Antibodies, A Laboratory Manual (CSH:CSHL, New York) 1988). All fusion products from a single mouse were plated m 96 wells and supematants tested for reaction with PC-12 membranes in an ELISA assay. Positive supematants were tested in the in vitro bioassay described below. Hybridoma pools giving positive bioassay results were plated at limiting dilutions to obtam clonal cell Imes.
  • Ascites fluid was produced to generate high titre antibody solutions Balb/C mice were given 0.5 ml of incomplete Freund's adjuvant by intrape ⁇ toneal injection The next day, animals were irradiated with 350 mRads and injected with 10 6 to 10 7 hybridoma cells. After 2 to 3 weeks, ascites were collected by paracentesis. Ascites fluid was mcubated at 37°C for 1 hour and centrifuged at 2000 g for 5 minutes The supernatant was a quoted and stored at 4°C
  • Hybridoma supematants and ascites were isotyped using a commercial kit (Gibco, N.Y.). Antibody concentration was measured using an ELISA assay with commercially available immunoglobulins used as standards (Cedarlane, R.R#1, Hornby, Ontario, Canada) Screening
  • poly-L-lysine treated 96 well dishes were plated with myelm protems. Briefly, 70 ml suspensions contammg 3.3 ⁇ g of CNS extract protem were plated onto test wells. After overnight drying, wells were washed twice with 10 mM sodium phosphate 140 mM NaCl sal e (PBS).
  • Assays were done in 96 well dishes (NunC). Substrate testing wells were precoated with 100 ⁇ g/ml of poly-L-lysine (Sigma). Test wells were in addition coated with bovine serum albumin (BSA type IV;Sigma) or adult rat brain, sciatic nerve, muscle or liver homogenates or extracts. Substrate coated wells were UV light treated and washed with PBS twice. Assays were carried out using 50 ⁇ l of hybridoma supernatant with an equal volume of cells suspension or using 1 to 10 ⁇ l of ascites in 100 ⁇ l of cells.
  • BSA type IV bovine serum albumin
  • Control hybridoma supematants from the same fusion as well as hybridoma supematants producing antibodies to myelin basic protein (MBP), galactose cerebroside (Gal C), tyrosine hydroxylase (TH), and neural cell adhesion molecule (NCAM) served as controls.
  • MBP myelin basic protein
  • Gal C galactose cerebroside
  • TH tyrosine hydroxylase
  • NCAM neural cell adhesion molecule
  • Substrate coated wells were treated with 0.25 to 0.00025% trypsin (Sigma T-2904) in PBS for 10 minutes at room temperature. Wells were washed twice with 10% FCS containing cell culture medium. Neurite outgrowth was determined as described above. Immunocytochemistry NG108-15 cells were grown on poly-L-lysine coated multi-chamber slides.
  • a ⁇ gtl l adult rat brain cDNA expression library (Clontech) was screened with 10D following the protocol handbook provided w ith the library
  • the main steps were the following: E. coli Y1090r- cells were infected with 3xl0 4 pfu per plate and after 3h incubation at 42°C covered with IPTG-treated NC-filters and incubated for another 3.5h at 37°C Filters were removed, rinsed in PBS with 0.1% Tween 20 (PBS-T) and blocked in PBS with 20% fetal calf serum for 2h at room temperature (RT).
  • the filters were incubated in 10D hybridoma supernatant (1:5 dilution) for 2h at RT (hybridoma cells were grown in "Cell perfect protein-free" tissue culture medium supplemented with Ab-enhancer (Stratagene); obtained Ab concentrations were 10 to 20 ⁇ g/ml).
  • the secondary Ab (goat anti mouse IgM-HRP conjugate, Biorad) was applied 1:1000 in PBS for lh at RT. Positive plaques were detected by using the ECL chemiluminescence kit from Amersham. Phage Preparation, Subcloning and Sequence Analysis.
  • Phage lysates and DNA extracts were obtained following the pre tocols in the library handbook.
  • cDNA inserts were cloned into the vector pBS-KS+ (Stratagene), and sequence analysis was performed using the AutoRead Sequencing kit and the ALF sequencing system (Pharmacia). Primers were fluorescein-labeled T3- and T7 primers. Sequence analysis and data base search were performed using the GCG package.
  • CNS myelin To test the influence of CNS myelin on neurite outgrowth, an in vitro assay was developed. A sucrose density fraction was prepared from adult rat brains following standard procedures for myelin isolation, and was estimated by Western blotting to be enriched four fold for the myelin-specific markers myelin basic protein and myelin associated glycoprotein (data not shown). This material is referred to as "CNS myelin” below. The inhibitory properties of this material as a substrate for neurite growth were studied primarily with the NG108-15 rat neuroblastoma and glioma hybrid cell line.
  • NG108-15 cells that had been induced with ImM dbcAMP for two days (herein called dbcAMPNG108 cells) were plated in tissue culture wells that had been treated with poly-L-lysine (PLL) alone, or PLL followed by an extract of CNS myelin proteins.
  • PLL poly-L-lysine
  • Figure 1A shows photomicrographs of representative fields of cultures of dbcAMPjN G108 cells plated onto poly-L-lysine alone (a), poly-L-lysine followed by 20 ⁇ g/cm 2 bovine serum albumin (BSA) (b) and poly-L-lysine followed by 20 ⁇ g/cm 2 CNS myelin (c).
  • Panel (d) shows a single dbcAMPNG108 cell growing on a myelin-free patch. The border between the myelin-coated (“ens”) and uncoated (“pL" for poly-L-lysine) surfaces is emphasized with small arrowheads.
  • Figure I B shows the studies where bovine serum albumin or extracts from muscle, sciatic nerve and brain were dried onto poly-L-lysine coated wells at 20 ⁇ g/cm 2 . Equal numbers of dbcAMPMG108 cells were plated in each well. After 24 hours, random fields were photographed and the proportion of cells with a process greater than 1 cell diameter was determined. 10 to 16 independent wells were scored for each substrate.
  • the error bars in Figure IB represent the standard error of the mean. * denotes statistically different from poly-L-lysine at p ⁇ 0.025; ** denotes significant at p ⁇ 0.01; *** p ⁇ 0.0005 using t-test.
  • dbcAMPNG108 cells As shown in Figure 1 the propensity for neurite extension by dbcAMPNG108 cells was significantly influenced by their substrate. 24 hours after plating onto a poly-L-lysine surface, 64% ⁇ 5% (mean and standard error) of dbcAMPjs G108 cells had neuritic processes greater than 1 cell diameter in length. The fraction of cells with processes was slightly reduced on wells coated with 20 ⁇ g/cm 2 of bovine serum albumin (BSA) or with extracts of muscle proteins or peripheral nerve myelin that had been prepared in the same manner as the CNS myelin.
  • BSA bovine serum albumin
  • Myelin coated wells often contained a peripheral rim containing patches that were bare of myelin proteins. Cells in such areas displayed interesting properties but they were not scored in the assays. As shown in one such area in Figure 1A, the arrest of neurite outgrowth is often limited to those neurites in contact with the inhibitory substrate. Other processes on the same cell are seemingly not affected. This suggests that arrest of neurite advance occurs through a contact dependent mechanism restricted to the process in contact with inhibitor.
  • Figure 2 shows the results of studies where CNS myelin was plated onto poly-L-lysine coated wells. The fraction of process bearing cells was determined at 24 hrs. Error bars in Figure 2 represent the standard error of the mean. Each point represents data from 2 to 10 wells.
  • the neurite growth inhibition by myelin protem enriched CNS extract was concentration dependent. As shown in Figure 2, the fraction of process bearing dbcA M P G108 cells decreased as the amount of plated myelin increased . The half maximal-inhibition of neurite outgrowth was observed at approximately 5 ⁇ g of protein per cm 2 . This observation indicates that poor neurite outgrowth on CNS myelin is due to a concentration dependent inhibition rather than a lack of trophic factor support. A similar concentration dependent inhibition of neurite growth on CNS myelin was observed with PC12 cells and primary newborn superior cervical ganglion (SCG) neurons (not shown). As discussed below, these results indicate that the in vitro assay detects an inhibitory activity that parallels the previously-described CNS myelin inhibitor of neurite growth.
  • SCG superior cervical ganglion
  • monoclonal antibodies were generated to neural cell membranes and these antibodies were screened in vitro for their ability to promote outgrowth on this inhibitory substrate.
  • a panel of monoclonal antineuronal antibodies was produced by immunizing mice with a crude membrane preparation from NGF treated PC-12 cells. Those hybridoma pools which were positive against PC-12 membranes on an ELISA were tested for their ability to promote neurite outgrowth on CNS myelin.
  • PC-12 or dbcAMPjsjG108 cells were grown on 10 ⁇ g/cm 2 of CNS myelin in microtitre wells, with a 1:1 mixture of medium supplemented with NGF or dbcAMP, and antibody-containing hybridoma supematants.
  • Those hybridoma pools yielding supematants that increased neurite production over control levels were plated at limiting dilutions to generate clonal lines.
  • Ascites fluid was produced with one line called 10D, with the highest neurite promoting activity.
  • bcA PNGlO ⁇ cells were used predominantly in subsequent experiments because of their rapid growth and readily-induced neuronal differentiation with a reliable proliferation of neurites.
  • Figure 3A shows photomicrographs of d b cAMPNGlO ⁇ cells grown on poly-L-lysine alone or 10 ⁇ g/cm2 of CNS myelin showing that 10D antibody reverses the growth inhibitory effect of CNS myelin.
  • Figure 3B shows the results of the quantitation of process-bearing cells grown on CNS myelin for 24 or 72 hours with 5 ⁇ l per well of control ascites (filled bars) or 10D ascites (open bars). Whereas few dbcAMPj ⁇ JG108 cells were able to extend neurites on 10 ⁇ g/cm 2 of rat CNS myelin, antibody 10D was able to reverse this inhibition (Figure 3). Only 1-2% of dbcAMPNJGlO ⁇ cells extended neurites at 24 or 72 hours on CNS myelin in the presence of control ascites.
  • SCG smpathetic superior cervical ganglion
  • AraC cytosine arabinofuranoside
  • antibody 10D may be useful to promote the growth of neurites by primary neurons in an inhibitory CNS environment.
  • the improved outgrowth with 10D is not due to a non-specific immunoglobulin effect since sister hybridoma supematants and ascites derived from the same fusion, and control ascites derived from Sp2 cells (the hybridoma fusion partner), did not overcome neurite growth inhibition.
  • the improved outgrowth with 10D antibody on this inhibitory substrate is unlikely to be due to non-specific blocking of myelin components in the substrate since myelin-specific antibodies recognizing galactose cerebroside (GalC), myelin basic protein (MBP) and myelin associated glycoprotein (MAG) did not promote neurite outgrowth.
  • immunoglobulin binding to neuronal cells is not sufficient to overcome myelin inhibition of neurite outgrowth since antibodies to tyrosine hydroxylase (TH), neural cell adhesion molecule (NCAM) and Thy-1 did not block the growth inhibitory properties of the CNS myelin.
  • TH tyrosine hydroxylase
  • NCAM neural cell adhesion molecule
  • Thy-1 did not block the growth inhibitory properties of the CNS myelin.
  • the interaction between these control antibodies and neural cells or myelin was confirmed using i ⁇ ununocytochemistry and western blots.
  • db_AMPNG108-15cells were grown on poly-L-lysine coated glass slides for 48 hours, fixed and processed for immunocytochemistry with 10D ascites (b) or control ascites (a) each diluted 1:1000.
  • a secondary antibody linked to a biotin-avidin and diaminobenzidine system was used. As shown in Figure 4, the antibody reacted with the cellular soma, processes and growth cones. In certain cells, staining is heaviest at the cell surface but there was often a more diffuse staining throughout the cell body ( Figure 4). Using peroxidase conjugated fluorescent labelled secondary antibodies gave similar results.
  • the 10D antibody blocks the effect on neurons in culture of a CNS myelin inhibitor that has been reported to affect the interactions of both neurons and fibroblasts with substrates.
  • proteins from tissues and cell lines were separated on denaturing SDS-polyacrylamide gels, transferred to nitrocellulose and reacted with serum-free hybridoma supernatant.
  • two identical denaturing 13% poyacrylamide-SDS gels were each loaded with marker proteins and 10 ⁇ g of protein from liver, cerebrum (both from 2 day old rats), dbcAMPNG108 cells and adult CNS myelin (same preparation as was used as an inhibitory growth substrate), and run simultaneously.
  • EXAMPLE S Dl A cDNA CLONE CAPABLE OF MODIFYING NEURITE GROWTH INHIBITION ON CNS MYELIN SUBSTRATES.
  • the 10D MAb was used to screen a rat brain cDNA (adult) library in the vector ⁇ gtll. Of 10 6 plaques, 11 rescreened positive and partial sequence data was obtained to permit preliminary identification. In addition, each insert was used to probe blots of RNA from NG108 cells and brains of postnatal day 1 and adult rats. Six of the eleven represented known brain-expressed sequences, while five were previously unreported. The following set of criteria were used to determine which of these eleven to pursue with further studies.
  • the cDNA must be expressed in NGlO ⁇ cells, since these cells are inhibited by CNS myelin and this inhibition is modulated by MAb 10D. 2) the cDNA must be expressed in the CNS.
  • the gene product must be one that can accommodate a role in the regulation of growth on inhibitory substrates.
  • Figure 6 is a schematic representing the strategy used to characterize clones selected with the 10D monoclonal antibody. Clones Dl, D5, Dll and D12 met these criteria most clearly. To determine which if any of these might represent a gene regulating neurite growth on CNS inhibitory substrates, a functional strategy was pursued. This was to down-regulate, y cellular expression of antisense RNA, the gene products corresponding to specific cDNAs and then assay neurite growth characteristics on CNS myelin and non-inhibitory substrates (see Figure 7).
  • Th e cDNA inserts were subcloned from three novel clones, in antisense orientation, into the vector pBK-CMV, in which the strong HCMV promoter can drive transcription in mammalian cells. These constructs were electroporated into NGlO ⁇ cells, stable transfectants were selected with G41 ⁇ , and assayed for neurite growth on a permissive (PLL) and an inhibitory CNS myelin substrate. Ten individual lines derived from antisense D5 and D12 constructs all showed normal growth on PLL (approximately 60-90% cells with identifiable processes) and normal inhibition on CNS myelin (1-4% cells with processes).
  • the control lane contains 10pg of Dl insert (EcoRI-fragment).
  • Figure 8 shows that clone A3 has, in addition to numerous bands also present in NG108 cells and presumed to arise from the endogenous Dl genes, the expected 1 kb hybridizing fragment. Thus, it w as shown that some clones of NG108 cells transfected with the Dl antisense construct, but not other cDNA antisense constructs, have acquired the ability to grow neurites on an inhibitory CNS myelin substrate.
  • Figures 9 and 11 show the sequence of two fragments from each end of the cDNA clone Dl.
  • the first nucleotides of each fragment is the EcoRI site added in the linker used in the library construction. There is an unsequenced gap of apporximately 200 bp separating the two sequences.
  • Computerized database searching of the portion sequenced indicated no previous reports of substantially similar sequences from any species (Genbank release 84.0; EMBL release 39.0). Sequences of other fragments of the cDNA clone are shown in Figures 12 to 14 (SEQ. ID. NOS.4 to 6).
  • SEQ. ID. NOS.7-9, Figure 21 Additional cDNA sequence data was obtained (SEQ. ID. NOS.7-9, Figure 21). The salient features of the data are, 1) that it extends the open reading frame significantly m the 5' direction, and 2) that there is a gap of about 100-300 bp near the 5' end. It is likely that the open reading frame continues on the 5' side of this gap (see ORFs in this region).
  • SEQ ID. NOS. 8 and 9 show the nucleotide and ammo acid sequence of the downstream portion of the gene. A new diagram of the cloned regions of Dl is shown in Figure 22.
  • the partial cDNA sequence of Dl will be extended by rescreening to isolate overlapping cDNAs. Both commercially obtamed libraries, as well as a ⁇ gtlO library rigorously selected for long inserts, will be screened. The inhibition-reversmg properties of Dl will be tested independently by transfecting NG 108 cells with antisense constructs of non-overlapping fragments derived from the Dl gene. Additional sequence data will be determined to predict the primary structure of the encoded protem
  • Additional independently-isolated clonal Dl transfectants are bemg grown up for functional and molecular characterization Cells will be grown on both inhibitory CNS and permissive substrates, and neurite growth quanititated by the standard procedures described herem. Lmes with different levels of neurite growth will be identified for correlation with molecular data It is expected that antisense (AS) RNA derived from other regions of the Dl gene will be effective; therefore, AS constructs will be made with newly-isolated portions of the Dl cDNA as they are obtamed PC12 cells will also be transfected with the antisense Dl construct. The resulting lmes will provide an independent test of the inhibition-blocking activity of Dl antisense, and will also be used to probe the lntracellular pathways mediatmg inhibition and its reversal
  • an RNase protection assay may be used which is capable of measuring individually sense and antisense Dl transcripts (Melton, D A , 1984, Nucleic Acids Res 12 7035-7056) This assay will be used to correlate steady-state mRNA levels with myelin growth characteristics If the mechanism of reversal of inhibition is interference with mRNA processing or promotion of degradation due to duplex formation, mRNA levels will be reduced. Alternatively mRNA levels could remain constant if the mechanism involves a specific inhibition of translation.
  • Animals are re-anaesthetized, the L5 dorsal root ganglion is re-exposed and injected with an anterograde tracer as described above. After 48 hours annuals are sacrificed by anaesthetic overdose and they are perfused with 4% paraformaldehyde in PBS. The spinal cord is collected and processed for GAP-43 immunocytochemistry to visualize axons m a phase of growth and to visualize the anterograde tracers. At the time of sacrifice, a sample of cerebrospinal fluid will be obtained tor the detection of secreted mouse immunoglobulin by Western blotting to demonstrate the production and delivery of 10D antibody within the spinal canal. These techniques will determine the extent of axonal regrowth within the spinal cord.
  • the Dl cDNA can be used to identify, study and isolate the corresponding human gene. This will make possible the study of the suspected role of the Dl gene and its protein product in development of the nervous system and in regeneration in the adult, as well as other more general roles in cell-substrate interactions, as suggested by in vitro data. It will allow the cloning of the human gene and its expression for use in drug discovery applications to find potential therapeutic agents that enhance regrowth of injured nerve fibres in the CNS.
  • Sequence Listing as SEQ. ID. NO. 7 and in Figure 21) were shown to specifically recognize human genomic DNA fragments similar in number to those recognized in rat and mouse DNA
  • Figures 17 and 18 shows that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12.
  • lO ⁇ g of DNA was digested with EcoRI, electrophoresed, transferred to nylon membrane, and hybridized with the Dl (bp230 to bpl,095) probe.
  • Samples were hamster, human or mouse genomic DNA (as marked), or DNA from hybrid cell lines containing mostly mouse or hamster chromosomes and the human chromosome marked.
  • the human specific bands appear only in the hybrid DNA from the "Hamster/Chl2" line.
  • rat Dl cDNA described herem can be used to detect human Dl mRNA and study its expression in normal tissue and in disease.
  • Figure 19 shows that the rat Dl seqences bp230 to bpl,095 can detect the correspondmg human RNA, in this case isolated from a surgically removed lung metastatic tumour.
  • the transcripts detected are of the same gel migration and similar abundance to those detected in RNA from rat brain tissue and cell lines.
  • Figure 19 shows that the Dl cDNA recognizes human RNA transcripts. 12 ⁇ g of total RNA from a human metastatic tumour of lung origin, and adult rat bram, was denatured, electrophoresed, transferred to nylon and hybridized with Dl cDNA (bp230 to bpl095) probe.
  • P2C protein phosphatase 2C
  • ser/thr phoshatase isolated from a number of species and different tissues
  • Petrin mRNA is not detectable in liver, spleen, muscle, or fibroblasts. In rat brain, its expression is developmentally regulated. It is first detectable after embryonic day E13, increases steadily with age and is highest in the adult rat.
  • Polyclonal antibodies were generated in rabbits against a GST-fusion protein containing the C-terminal 210aa of Petrin.
  • One of two antisera (#11 ) specifically precipitates a 60-64kD protein (from 35 S-labelled NG108 cell lysates).
  • the antibody (Ab) is not functional in western blots, nor does it block neurite growth inhibition on myelin substrate.
  • the immunoprecipitates from rat brain and NG108 cells exhibit Mg + dependent phosphatase (Pase) activity. This activity is ca. 5-fold higher when performed with a ser/thr phospho-peptide than with a tyr phospho-peptide.
  • Pase activity can be precipitated using Ab #11. The majority of the activity is found in the cytosolic fraction of cell lysatc after crude fractionation into membranes and soluble protein.
  • CAGCAAGGAC TCCGGGTTTT AGGGTAATTT GAATTTGGGT TTTCGGGTTT GGTTTGGTTT 420
  • TCTCCCATCT TTCCCATTTC CCTCCACCGG GACTTTCCAA ATTGTCACTG GACAGTTTCC 2400
  • GATCGAGTGT CACAGCTAGC TAAGCAGCAG CTCTTCCTGA CACCTTTGTG CAAGGATAAC 2880
  • ATC TCT CCA AAT ACG AGC ACG GAG CGG ATG ACG TGC TGA TCC TGG CTA 816
  • GCC TCC AAG CAG GGC GGG ACT GGG GAG TAA GTA CCT GGG CTG GAT TCC 1152

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