EP0821731A1

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

Info

Publication number: EP0821731A1
Application number: EP96908957A
Authority: EP
Inventors: Arthur Roach; Andres Lozano; Monika Labes; John Roder
Original assignee: Mount Sinai Hospital Corp
Current assignee: Mount Sinai Hospital Corp
Priority date: 1995-04-13
Filing date: 1996-04-12
Publication date: 1998-02-04
Also published as: AU5264496A; CA2174025A1; JPH11503324A; CA2217731A1; WO1996032476A1

Abstract

A method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin. Neuronal cells which have a propensity for neurite growth are grown on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of affecting neurite outgrowth. The invention also relates to isolated nucleic acid molecules encoding a novel protein which plays a role in neurite outgrowth. The invention provides for various uses of the nucleic acid molecule and its protein product.

Description

Modul ator of neuronal cel l response to Inhibition by CNS myel in .

FIELP OF THE INVENTION 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. The same is not true however in the central nervous system (CNS). 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. The difference in the regenerative abilities of axons in the CNS and peripheral nervous system (PNS) has puzzled clinicians and neuroscientists and challenged them to explore the molecular basis of this phenomenon.

An increasing number of molecules regulating the growth of neuronal processes are being identified. Until recently, only environmental molecular cues exerting positive effects on neuronal growth were known. These positive signals include growth factors such as nerve growth factor, and brain derived neurotrophic factor (Barde YA., [Review] Neuron 2: 1525-1534, 1989; and Dechant et al. J. Journal of Neuroscience 13: 2610-2616, 1993), extracellular matrix components such as collagen, laminm and fibronectin (reviewed in Goodman and Schatz, 1993, Neuron 10 (Suppl.):77-98) and cell surface molecules such as NCAM (Goodman and Schatz, 1993, supra).

Of considerable interest recently, is the finding that 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) 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). Other molecules including 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. 106:1281-1, 1988) Downregulation of these molecules by the irradiation induced arrest of oligodendrocyte production in fetal rodents is associated with an increased propensity for axonal regeneration on the CNS (Savio and Schwab, PNAS 87:4130-4133, 1990). Further, neutralizing the activity of NI-35 or NI-250 using antibodies transforms the CNS environment from one that disallows to one that is permissive for axonal growth (Caroni and Schwab, J. Cell Biol. 106:1281-8, 1988). It has more recently been shown that in the presence of anti-NI-35 and NI-250 antibodies, a subset of axons interrupted in the spinal cord of adult rats were able to regenerate over long distances (Schnell and Schwab, 1990, Nature 343 (6255)269-72). Further, co-application of myelin neutralizing antibodies with local administration of the neurotrophin NT-3, resulted in even greater enhancement of adult rat spinal cord axonal regrowth (Schell et al , Nature 367:269-272, 1994).

The mechanism through which the adult CNS myelin proteins block neuπte outgrowth is unknown. When applied to the tips of dorsal root ganglion (DRG) neuronal growth cones in culture, 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.

From recent reports there are indications that the growth cone collapse induced by inhibitory molecules from adult rat CNS myelin may be preceded by a rise in mtracellular Ca⁺⁺ levels (Bandtlow et al , 1993, Science 259 80-83) and be dependent upon a G protem pathway (Igarashi et al., 1993, Science 259 77-79). The NI-35 induced collapse of DRG growth cones can be mimicked by mastoparan, a G protein activator and blocked by the G protein blocker, pertussis toxin. Further, growth cone collapse with the myelin derived inhibitors is specifically blocked by monoclonal antibodies to NI-35 (Bandtlow et al , 1993 Science 259:80-83 and Igarashi et al., 1993, Science 259:77-79)). Thus, 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. SUMMARY OF THE INVENTION A component of adult mammalian central nervous system (CNS) myelin causes collapse of neuronal growth cones and inhibits axonal growth. This activity may be responsible for the lack of regrowth of axons interrupted in the CNS. 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.

In accordance with a further aspect of the invention, hybridoma cell Imes are provided 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.

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.

In an embodiment of the invention, 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;

(b) nucleic acid sequences complementary to (a);

(c) 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,

SEQ. ID. NO. 7 and SEQ. ID. NO. 8;

(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 (d) in codon sequences due to the degeneracy of the genetic code.

The mvention also relates to a nucleic acid molecule comprismg

(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); (c) nucleic acid sequences which are at least 80%, preferably 90% identical to (a); or,

(d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.

Preferably, 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;

(b) nucleic acid sequences complementary to (a);

(c) nucleic acid sequences which are at least 80-90% identical, preferably 90% identical to (a); or,

(d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under strmgent hybridization conditions.

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 Accordingly, 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. In an embodiment of the invention, a method for preparmg a Petrin protein is provided 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.

In an embodiment of the invention a purified Petrin protem is provided 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.

The 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. Thus, 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.

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 ^cubstance that affects neuronal growth compπsmg 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.

Still further, 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 In an embodiment, 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 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 ιt\ 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; (d) inhibitors and activators of the activity of a protein of the invention; and (e) substances identified using the methods of the invention. The present invention also has diagnostic applications.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS The invention will be better understood with reference to the drawings in which:

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 20μg/cm² bovine serum albumin (BSA) (b), poly-L-lysine followed by 20μg/cm.2 CNS myelin (c), and (d) shows a single dbcAMPNG108 cell growing on a myelin-free patch;

Figure IB is a graph showing the proportion of ^dbcAMPNG108 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 μg/cm² 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 5μl 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 μg of protein from liver, cerebrum (both from 2 day old rats), ^dbcAMPNG108 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 (μg/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; and

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

The following standard abbreviations for the amino acid residues are used throughout the specification: 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; and p.Y., P.Tyr - phosphotyrosine.

For ease of explanation, the description of the invention is divided into the following sections: (A) assay for neurite growth inhibition by CNS myelin (B) hybridoma cell lines and monoclonal antibodies; (C) novel nucleic acid molecule and novel protein; and (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. A. ASSAY FOR NEURITE GROWTH INHIBITION

As discussed herein, 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.

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. Other neuronal cells which may be used in the method of the mvention include PC12 cells which have been induced to grow neuπtes by induction for 5-7 davs with 100 ng/ml NGF (Green), cerebellar neurons (Trenkner, E in Cultuπng 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).

"Mammalian CNS myelin" refers to extracts of mammalian central nervous system myelin containing myelin basic protein and myelin associated glycoprotein. In a preferred embodiment, 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.

In a preferred embodiment, 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.

The test substance may be added to the neuronal cells or the test substance may be introduced by genetically engineering the neuronal cells. For example, 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. In a preferred embodiment, 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 0.33cm².

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)

As a control for the method of the invention, 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

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 hybπdomas 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, Hybπdomas 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)

Generally, 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 oπgm 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 oπgm, 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. Within one embodiment of the present invention a subject animal such as a rat or mouse, for example a BALB/C mouse, is injected with 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. Between one and three weeks after the initial immunization 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. Once the animal has plateaued in its blockmg or bmdmg activity, it is sacrificed, and organs which contam large numbers of B cells such as the 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). Alternatively, within a preferred embodiment, 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)

Following the fusion, 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, ιe., 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.

Other techniques may also be utilized to construct monoclonal antibodies (see William D Huse et al , "Generation of a Large Combinational Library of the Immunoglobulm Repertoire m Phage Lambda," Science 246 1275-1281, December 1989, see also L Sastry et al , "Cloning of the Immunological Repertoire in Escheπchia coh for Generation of Monoclonal Catalytic Antibodies Construction of a Heavy Chain Variable Region-Specific cDNA Libran ," Proc Natl Acad Sci USA 86:5728-5732, August 1989, see also Michelle Alt g-Mees et al , "Monoclonal Antibody Expression Libraries A Rapid Alternative to Hybndomas,' Strategies in Molecular Biologs 3 1 -9, 1990, these references describe a commercial system available from Stratacyte, La Jolla, California, which enables the production of antibodies through recombinant techniques).

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 10μg/cm² 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. Further, this growth promoting effect of the 10D antibody was not observed with pure non-specific IgM antibodies nor with antibodies which bind to CNS myelin such as anti-galactose cerebroside, anti-myelin basic protein or anti-myelin associated glycoprotein, nor is it observed with antibodies which recognize neurons such as anti-NCAM and anti-THY-1; both of which are present on the cell surface of the neurons.

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 ^dbcAMP|s G108 cells and NIH 3T3 fibroblast cells using standard immunocytochemistry techniqu s 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, in particular the monoclonal antibody with the laboratory designation 10D, may be localized to specific neuronal cells in the brain, brains tern and cerebellum using conventional immunocytochemistry methods. In particular, 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. The methods of the mvention permit the identification of potential stimulators or inhibitors of neurite growth in the central nervous system environment which have various applications as discussed below. C. NOVEL NUCLEIC ACID MOLECULE AND PROTEIN The monoclonal antibody havmg the laboratory designation 10D was used to identify a clone hav g the laboratory designation "Dl" Transfectants contammg antisense constructs derived from the Dl clone showed significant enhancement of neurite growth on myelm The Dl mRNA appears to be an 7kb transcript present m bram and NG108-15 cells Sequence analysis of the partial Dl cDNA clone mdicated that it is a previously unreported gene

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.

Therefore, the present invention provides a isolated and purified nucleic acid molecule comprising

(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;

(b) nucleic acid sequences complementary to (a); (c) 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, SEQ. ID. NO. 7 and SEQ. ID. NO. 8;

(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

(d) in codon sequences due to the degeneracy of the genetic code.

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);

(c) nucleic acid sequences which are at least 80%, preferably 90% identical to (a); or,

(d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions. Preferably, 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:l l), wherein T can also be U;

(b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80-90% identical, preferably

90% identical to (a); or,

(d) a fragment of (a ) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions The term "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. The term "nucleic acid" is intended to include DNA and RNA and can be either double stranded or single stranded. Therefore, 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.

It will be appreciated that 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

Fragments of the nucleic acid molecules contemplated by the present mvention mclude 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.

Further, it will be appreciated that the invention mcludes 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 The term "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. 6, SEQ ID NO 7 and SEQ ID NO 8 or disclosed Figures 9, 11 to 14, and 21, or Figures 23 or SEQ.ID NO 11, I e the sequences function m substantially the same manner to produce substantially the same activity in the assays described in Section A here The variations may be attnbutable to local mutations or structural modifications

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 bπdizc 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.

Isolated and purified 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. Such 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. However, 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

An isolated and purified 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. ID NO 11) for use m PCR A 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. Petersburg, FL).

An isolated and purified nucleic acid molecule of the mvention which is RNA can be isolated by cloning a cDNA encoding a petrin protein into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protem which exhibits phosphatase activity. For example, 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 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

The 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. In an embodiment of the invention 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.

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). 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

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.

Therefore, 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

Figure 10, and as shown in the Sequence Listing as SEQ. ID. NO. 9; and sequences having

80-90% identity thereto. In an embodiment of the invention, 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.

NO. 12), 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 tπpeptide to a 1900 mer polypeptide For example, 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

At the ammo terminal end, 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. 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. Conserved ammo acid substitutions mvolve replacmg one or more amino acids with amino acids of similar charge, size, and/or hydrophobicity characteπsitics When only conserved substitutions are made the resultmg analog should be functionally equivalent to Petrm as described herem Non-conserved substitutions involve replacmg one or more ammo acids with one or more amino acids which possess dissimilar charge, size, and /or hydrophobicity characteristics

One or more 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 For example, 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

SEQ. ID. NO. 12) from the Petrin amino acid sequence as shown in Figure 24 (SEQ. ID. NO. 12).

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 transcπptional promoter and enhancer or RNA polymerase binding sequence, a πbosomal 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. Examples of reporter genes are genes encoding a protein such as β-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 β-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.

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. 4,399,216; Goeddel et al., U.S. Patent No. 4,766,075; and Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which are incorporated herein by reference and see the detailed discussion below). 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 β-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^β, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messmg, Gene 19:259-268, 1982), and pNH8A, pNHlόa, pNH18a, pCDM8, Bluescript M13 (Stratagene, La Jolla, Calif.), and pETIO (Studier et al, Meth. Enzymol. 185:60-89, 1990).

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 mclude, among others, YC_p50 (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 mclude, 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. 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).

Given the teachings provided herein, promoters, terminators, and methods for mtroducmg expression vectors of an appropriate type mto plant, avian, and msect cells may also be readily accomplished. For example, within one embodiment, 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 Agrobacteπum 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 mclude 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)

Alternativ cl , 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). The proteins of the invention, and parts thereof 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. Thus, 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. Examples of 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. P. APPLICATIONS The nucleic acid molecules of the invention or fragments thereof, allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences in biological materials including cells and tissues. Example of 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 ³²T, ³H, ¹ C or the like. Other detectable substances which may be used 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.

In accordance with one embodiment of the mvention, 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.

Within the context of the present invention, 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. As will be appreciated by one of ordinary skill in the art, 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. Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal. 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. Within one embodiment, 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 Vπa_> πb 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 ImmunoZAP™ 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. Thus, 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. In particular, 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. Generally, 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.

Where a radioactive label is used as a detectable substance, 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.

Still further, 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 In an embodiment, 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 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. For example, 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. Accordingly, 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.

The substances identified using the methods described herein or antibodies described herein, 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.

The pharmaceutical 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.

The pharmaceutical 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).

On this basis, 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. Freed et al., New Eng J Med 327(22):1549-1555, 1992, describe a method for mjectmg fetal cells into brams of Parkinson's patients. The methods described by Pace et al, Lancet 335(8691):738-747 for mtrathecally delivering substances mto the CNS may also be useful for administering pharmaceutical compositions contammg antisense nucleic acid molecules of the mvention Antisense nucleic acid molecules of the invention may also be mtroduced using mtracerebroventncular administration (See for example, C. Wahlestedt et al., Nature 363:260-263, 1993).

The utility of the substances, antibodies, antisense nucleic acid molecules, and compositions of the mvention may be confirmed m animal experimental model systems

For example, 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. 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.

Previous experiments with hybridoma implants into the brain have been used to deliver antibodies to the CNS. Using this approach, Schnell and Schwab were able to deliver antibodies agamst the CNS myelin inhibitors to promote the regeneration of CNS axons (Schnell L and Schwab ME. Nature 343(6255):269-72, 1990). More recently, this group has combined the use of inhibitor-neutralizing antibodies with co-application of neurotrophic factors to produce an even greater increase in the long distance regeneration of CNS fibres (Schnell et al, 1994, Nature 367: 170-173). FGF, BDNF and NT-3 have been shown to increase RGC survival after axotomy Johnson et al., 1986, J. Neurosci 6: 3031-3038; Lipton et al., 1988, Proc. Natl. Acad. Sci USA 85: 2388-2392; Mey and Thanos, 1993, Brain Research 602:304-317). The strategy of using antibodies with or without the addition of neurotrophins tl us has a precedent and the potential to yield a positive result.

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 In some experiments, 300-500 μg 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.

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)). Models for testmg the application of antisense nucleic acid molecules of the invention, and m particular, determining the physicological affects of the molecules, are described m C Wahlestedt et al., Nature 363 260-263, 1993

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:

EXAMPLES The following materials and methods were utilized in the investigations outlined in the examples: MATERIALS AND METHODS Cells

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. 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 μg/ml) were added to all media. Substrate Preparation 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. The sample was recentrifuged (28,000 g, 1 hour), the resultant interface was again washed, pelleted at 28,000 g for 4 hours and resuspended in a small volume of 30 mM Hepes pH 7.4. Western blot analysis using a monoclonal antibody against myelin basic protein confirmed a four fold enrichment for myelin in the bram extract versus total brain homogenate. Protein determinations were carried out using a protein assay kit (Bio-Rad) and bovine serum albumin (Type IV; Sigma) as a standard. Extracts of rat sciatic nerve, liver, muscle and human corpus callosum were prepared in a similar fashion. Antibody Production 3 X 10⁷ NGF treated PC-12 cells were homogenized in a glass homogenizer using 10 ml of 0.25 M sucrose, 0.1 mM MgCl, 10 mM Tris pH 7.4. The sample was centrifuged at 2000 g for 5 minutes. The supernatant was transferred to 5 ml tubes and spun at 90,000 g for 1 hour. The resultant pellet was resuspended in 10 mM Hepes pH 7.4. Balb/C mice were immunized 5 times with aliquots of this crude membrane fraction containing 50 μg of protem All mice produced sera recognizing PC-12 membranes in an ELISA assay. 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.

To collect antibody-containing supernatant from cloned hybridoma 10D, cells were grown m Cell/Perfect protem-free media supplement with Cell/Perfect Ab enhancer (Stratagene). The supernatant was collected and used for immunodetechon of membrane bound protein, either undiluted or diluted (up to 1:5), dependmg on the concentration of the Ab m the supernatant.

Ascites fluid was produced to generate high titre antibody solutions Balb/C mice were given 0.5 ml of incomplete Freund's adjuvant by intrapeπtoneal injection The next day, animals were irradiated with 350 mRads and injected with 10⁶ to 10⁷ 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

To test the biological activity of the hybridoma supematants, poly-L-lysine treated 96 well dishes (each well has a surface area of approximately 0 33 cm²) were plated with myelm protems. Briefly, 70 ml suspensions contammg 3.3 μg 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). Dishes were sterilized by exposure to uv light for 20 m utes 1000 to 2000 PC-12 or NG108-15 cells in 50 μl were plated onto each well in the presence of an equal volume of hvbπdoma supernatant Neurite Outgrowth Assay

Assays were done in 96 well dishes (NunC). Substrate testing wells were precoated with 100 μg/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 μl of hybridoma supernatant with an equal volume of cells suspension or using 1 to 10 μl of ascites in 100 μl of cells. 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. Ascites produced from the myeloma fusion partner Sp2 (Cedarlane) or directed against Thy-1 (New England Nuclear) were also used in control experiments. 1000 to 2000 cells were plated per well. After 24 to 72 hours of culture at 37 C and 5% C0₂, random fields were photographed. The percentage of cells bearing a process greater than 1 cell diameter in length was determined. In certain experiments the effect of substrate digestion with trypsin was studied. 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.

Cells were washed in PBS and reacted with ascites at a 1:1000 dilution in PBS with 1.5 % horse serum for 1 hour. Cells were fixed with 4% paraformaldehyde in PBS for 5 minutes. After washing for 10 minutes cells were processed for peroxidase linked immunocytochemistry using diaminobenzidine (ABC kit Vector Labs, Burlingame, CA 94010). In certain experiments cells were fixed before the application of the primary antibody. Western Blotting

Samples were separated and prepared for immunoblotting essentially as described in Ausubel et al., 1993, Current Protocols in Molecular Biology, Boston, Current Protocols, with transfer onto nitrocellulose membranes occurring overnight at 25 volts in 20% methanol transfer buffer. Membranes were preincubated in PBS containing 3% milk powder for 1 hour, then r sed and incubated in undiluted 10D supernatant for 2 hours, followed by three 10 min. washes in PBS with 0.1% Tween (PBS-T). The secondary Ab (goat anti-mouse IgM, horseradish peroxidase conjugated) was applied as a 1:1000 dilution in PBS-T. All steps were at room temperature. Detection of the Ag/Ab complexes was accomplished by using the enhanced chemiluminescence (ECL) kit (Amersham) following the manufacturers mstructions Library Screening

A λgtl l adult rat brain cDNA expression library (Clontech) was screened with 10D following the protocol handbook provided w ith the library In brief, the main steps were the following: E. coli Y1090r- cells were infected with 3xl0⁴ 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). Subsequently, 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 μg/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. For further analysis, 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.

EXAMPLE 1 NEURITE OUTGROWTH IS STRONGLY INHIBITED BY 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. Cells of this line grow in 10% serum-supplemented medium, and with reduction of serum and transfer to ImM dibutyrlyl cyclic AMP-containing medium, undergo a phenotypic change that includes acquiring competence for activity-dependent acetycholine release (Christian et al, Brain Res. 147:261-276, 1978) and enhanced extension of neurites. 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.

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 20μg/cm² bovine serum albumin (BSA) (b) and poly-L-lysine followed by 20μg/cm² 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 μg/cm². 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.

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 μg/cm² 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. In contrast, on adult rat CNS myelin, the elaboration of neural processes was strongly inhibited, with less than 2% of dbcAMPNG108 cells having significant neurites at 24 hours. Cells on CNS myelin were generally round 24 to 72 hours after plating. Cells on poly-L-lysine displayed more spreading, more neurites and longer processes (Figure 1A). In addition, whereas differentiated dbcAMPj jG108 cells could be maintained for several weeks on poly-L-lysine surfaces, there were few remaining viable cells after seven days on CNS myelin coated surfaces. The neurite growth inhibition by CNS myelin is contact dependent.

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.

Investigations were carried out to determine whether the inhibitory activity of CNS myelin was labile to trypsin digestion. Wells coated with 20 μg/cm2 of CNS myelin treated with 0.025% trypsin at room temperature for 10 minutes retained no significant inhibitory activity. On wells incubated with 0.00025% trypsin, approximately 10% of dbcAMPNG108 cells had processes at 24 hours.

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 μg of protein per cm². 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.

EXAMPLE ? AN ANTI-NEURONAL ANTIBODY INCREASES NEURITE OUTGROWTH ON INHIBITORY CNS DERIVED SUBSTRATE.

To study the neuronal molecules that mediate the inhibition of neurite growth by adult CNS myelin proteins, 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. To screen the hybridoma library, PC-12 or dbcAMPjsjG108 cells were grown on 10 μg/cm² 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 PNGlOδ 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^bcAMPNGlOδ cells grown on poly-L-lysine alone or 10 μg/cm2 of CNS myelin showing that 10D antibody reverses the growth inhibitory effect of CNS myelin. 5 μl of control ascites or 5 μl 10D antineuronal antibody ascites was added to the wells at the time of cell plating. Cells were photographed after 72 hrs in culture. The total volume in each assay was 105 μl. Bar = 100 μm.

Figure 3B shows the results of the quantitation of process-bearing cells grown on CNS myelin for 24 or 72 hours with 5μl per well of control ascites (filled bars) or 10D ascites (open bars). Whereas few dbcAMPj\JG108 cells were able to extend neurites on 10 μg/cm² of rat CNS myelin, antibody 10D was able to reverse this inhibition (Figure 3). Only 1-2% of dbcAMPNJGlOβ cells extended neurites at 24 or 72 hours on CNS myelin in the presence of control ascites. In contrast, in the presence of 10D ascites, the fraction of cells with neurites greater than one cell diameter in length after 24 hours increased to 32% (Figure 3A). By 72 hours, the fraction of process bearing cells on myelin with 10D was equal to that on poly-L-lysine indicating that the antibody completely overcame growth inhibition on this non-permissive substrate (Figure 3A and 3B). 10D ascites also neutralized the neurite growth inhibitory effects of myelin on PC-12 cells.

The biological activity of antibody 10D was also observed with primary neurons. Sympathetic superior cervical ganglion (SCG) neurons can be isolated from neonatal rats and maintained in culture in the presence of 100 ng/ml NGF and lOμM cytosine arabinofuranoside (AraC), under which conditions they survive and extend abundant neurites which fasciculate to form bundles connecting aggregates of cell bodies (Hawrot and Patterson, Meth. Enzymol. 58:574-585, 1979). When these cells are plated on 10μg/cm² CNS myelin however, the neurons survive as aggregates of cell bodies but neurite formation is inhibited, as shown in Figure 16A. Addition of antibody lOD-containing ascites to a sister culture of SCG neurons on a CNS myelin substrate causes reversal of the inhibition, allowing the formation of bundles of neurites as shown in Figure 16B. Therefore, 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. Similarly, 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. The interaction between these control antibodies and neural cells or myelin was confirmed using iπununocytochemistry and western blots.

EXAMPLE 3 10D RECOGNIZES dbcAMPNG108-15 AND PC12 CELLS

To confirm the interaction between antibody 10D and dbcAMPj G108 cells, these cells were processed for immunocytochemistry. In particular, 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.

EXAMPLE 4 CELLULAR PROTEINS RECOGNIZED BY THE 10D ANTIBODY

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. To determine the molecular species recognized by the 10D antibody, proteins from tissues and cell lines were separated on denaturing SDS-polyacrylamide gels, transferred to nitrocellulose and reacted with serum-free hybridoma supernatant. In particular, two identical denaturing 13% poyacrylamide-SDS gels were each loaded with marker proteins and 10 μg 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. One gel was stained for total proteins with Coomassie Brilliant B.ue (left) and the other transferred to nitrocellulose and reacted with the 10D monoclonal antibody. Also shown in Figure 5 is a lane from a gel run and blotted separately, loaded with 10 μg of protein from NTH3T3 cells.

Enhanced c_hemilurninescence detection revealed several immunoreactive species (Figure 5). Prominent in adult rat cerebrum, rat liver, dbcAMPNGlOδ cells and mouse NIH3T3 fibroblast samples was a band of M_r 35,000. This band did not correspond to any prominent Coomassie Blue stained species. All four samples also contained a slightly faster migrating band (M_r 33,000), reduced in intensity relative to the M_r 35,000 band to a similar degree in each case. Less intense immunoreactivity against some higher M_r species (M_r 60,000 to 100,000) was seen less consistently in some samples. It was not determined whether these bands represent aggregates of the faster migrating species, or immunologically cross-reactive but molecularly distinct proteins. Two bands at M_r 14,000 and lδ,000 were seen in many samples and correlated consistently in several independent experiments with the presence of two prominent Coomassie Blue bands. These may be the result of a lower-affinity interaction with a pair of abundant, widely-expressed proteins. Reaction of 10D with myelin proteins resulted in only a weak signal co-localizing with the highly abundant myelin basic proteins.

EXAMPLE S Dl: A cDNA CLONE CAPABLE OF MODIFYING NEURITE GROWTH INHIBITION ON CNS MYELIN SUBSTRATES.

In order to clone a gene encoding a neuronally-expressed protein required for sensitivity to CNS myelin inhibition, the 10D MAb was used to screen a rat brain cDNA (adult) library in the vector λgtll. Of 10⁶ 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.

1) the cDNA must be expressed in NGlOδ 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.

3) if previously described, 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 NGlOδ cells, stable transfectants were selected with G41δ, 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). In contrast, 15 out of 19 lines transfected with the antisense Dl construct showed significant enhancement of growth on myelin, with normal growth on several permissive substrates. The extent of neurite growth by antisense transformed lines varied. For the line presented in Figure 7 (D1/A3), 56% of cells grew processes on myelin, and addition of 10D antibody had little effect. Three lines transformed with the Dl antisense cDNA construct showed no significant effects on growth on a variety of permissive substrates (data not shown).

To test whether the enhanced ability of some Dl antisense transformants to extend neurites on myelin was due to antisense inhibition of Dl gene expression, the selected clones were analyzed in greater detail. The presence of stable integrated copies of the pBK-CMV-Dl construct was determined by probing gel blots of cellular DNA with the Dl cDNA insert. Intact copies would be expected to include a 1.0 kb EcoRI fragment representing the cDNA sequence in the construct, that would not be present in the genomic DNA of the parental NGlOδ cells. Figure 8 shows the Southern blot of EcoRI digested genomic DNA from NG108 cells (parental cells) and the transformed cell line A3 probed with the lkb Dl cDNA insert. 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 (SEQ. ID. NOS. 1 and 3) 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 (Figures 9 and 11 and SEQ. ID. NOS. 1 and 3) 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).

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 λgtlO 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 (both sense and antisense) 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

Smce the level of expression m NG108 cells is low 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.

EXAMTLE S REGENERATION OF OPTIC AXONS Adult female Sprague-Dawley rats are anaesthetized using ketamine

(40-80mg/kg) and Xylazine (5-lOmg/kg) IP or _M. In anaesthetized rats the left optic nerve is exposed with the aid of a microscope and crushed with a liquid nitrogen cooled jewellers forceps 3mm behind the globe. The supraorbital incision is closed and the animal is allowed to recover for 2-4 weeks. Animals are then reanaesthetized and 5 μl of anterograde tracer (3% Rhodamine Isothiocyanate or Horseradish Peroxidase) is injected intraoccularly with the Hamilton Syringe. After two days animals are given an anaestheitc overdose and perfused transcardiacly with 4% paraformaldehyde in phosphate buffered saline (PBS). The optic nerve is removed and sectioned with a cryostat. The sections are processed for neurofilament immuncytochemistry and to detect anterograde tracers to determine the position of the distal end of the optic axons. In experimental animals 10⁸ hybridoma cells in 100 μl secreting 10D antibody or an irrelevant sister control antibody are deposited at the site of optic nerve crush at the time of surgery. At the time of sacrifice, a sample of cerebrospinal fluid will be obtained for the detection of secreted mouse Ig to prove the production and delivery of 10D antibody within the spinal canal. In certain experiments the application of 10D secreting hybridoma cells will be combined with the intraoccular administration of 300-500 μg of NGF, BDBF, FGF or saline usmg a Hamilton syringe. Because these growth factors have been shown to enhance RGC survival after injury, it is expected that in combination with 10D antibody, they will lead to a greater enhancement of axonal growth in the injured CNS over the use of 10D antibody alone. EXAMPLE 7

REGROWTH OF CENTRAL PROCESSES OF DORSAL ROOT GANGLION NEURONS

Adult female Sprague-Dawley rats are anaesthetized using ketamine (40-80mg/kg) and Xylazine (5-10mg/kg) IP or IM. Using the microscope animals have a laminectomy to expose the lumbar spinal cord. The L5 dorsal root is crushed proximal to the dorsal root ganglia 10⁶ antibody secreting hybridoma cells are deposited at the surgical site. 10D secreting cells and sister clones producing irrelevant control antibodies will be used. Axons are allowed to regenerate towards the spinal cord for 2-4 weeks. 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.

EXAMPLE S

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. The Dl cDNA sequences from position bp230 to bpl,095 (as shown in the

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). Further, it has been shown using a panel of human-rodent hybrid cell lines

(NIGMS Human Genetic Mutant Cell Repository, Coriell Inst. for Medical Res., Camden, N.J. 08103, USA) that all the Dl gene sequences detected in the human genome reside on chromosome 12 (Figures 17 and 18). The success of this mapping indicates that it will be possible to use the Dl probes described herein to determine whether human disorders are genetically linked to the Dl gene.

In particular, Figures 17 and 18 shows that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12. lOμg 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.

It has also been demonstrated that the 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.

In particular, Figure 19 shows that the Dl cDNA recognizes human RNA transcripts. 12μg 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.

EXAMPLE 9 To study the relevance of the CNS myel /neurite outgrowth assav of the mvention to the human, a bioassay was developed which uses human brain-derived mvc n as a substrate. The results indicate 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 inhibition increases with increasing myelin concentration (Figure 20). It is dependent on the direct contact between neurites and the myelin substrate. 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.

EXAMPLE 10 Sequence Analysis

Based on overlapping partial cDNA clones (isolated from a ratbrain cDNA expression library) 4515bp were sequenced which contain an open reading frame (ORF) of 1941bp encoding 647aa. Three methionine codons are located at positions 436, 475 and 486, the third of which is preceded by a Kozak consensus sequence. Based on this finding, this ATG is the most likely site for translational initiation and the predicted molecular weight for the encoded protein would be approximately 60kD. The protein, designated as Petrin, contains two putative tyrosine phosphorylation sites at the C-terminus. No transmembrane domain or other known motifs could be found in the sequence. The most related known protein to date is protein phosphatase 2C (PP2C), a ser/thr phoshatase isolated from a number of species and different tissues ( Mann, D.J. Et al., Biochim Biophys Acta 1130:100-4, 1992; Hou, E.W. Et al., Biochem Mol Biol Int 32:773-780, 1994; Terasawa, T. Et al., Arch Biochem Biophys 307:342-9, 1993; Kato, S., et al., Arch Biochem Biophys 318:3δ7-393, 1995; andT. Kuromori & Yamamoto, Nucleic Acids Res 22: 5296-5301, 1994). Distinct regions of petrin exhibit amino acid identities /similarities of up to 63%, the over all homology is below 20%. DNA and RNA hybridization experiments confirmed the presence of highly related genes in mouse, hamster and human. The human petrin homologue could be localized on chromosome 12. Expression: Northern blot and in situ Analyses

Expression of petrin appears to be brain specific. 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.

In situ hybridizations using a DIG-UTP-labeled RNA probe (3') revealed that petrin is specifically expressed in neurons. Staining is found all over the brain to different degrees, with some regions containing intensely stained neurons (e.g.: cerebellum: Purkinje cells; cortex: cells in the 3rd and 4th layer; hippocampus). Functional analysis

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 ³⁵S-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. When transfecting COS cells with an expression construct containing the complete ORF, specific 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.

Peptide sequences containing an HA (hemagglutinine) epitope were regenerated and cloned into the N'- and C-terminal regions of the protein (into the BsiWI and the EcoRV sites, respectively). When introducing expression constructs containing the HA- tagged derivatives of Petrin into COS cells and using an anti-HA monoclonal antibody for immunoprecipitation of COS cell lysates, Pase activity could be detected with the 3'HA- tagged petrin, and to a lesser extent (1/3) with the 5'HA-tagged petrin (control negative). In Western blots the anti-HA Ab specifically detects a band at appr. 60kD in lysates from ORF- HA transfected COS cells. Application of antisense oligonucleotides on NG108 cells and growth on myelin substrate

An oligonucleotide (GCT GCC AGC CAT GAT GCC GCC CAT) overlapping the two distal ATGs, i.e. the assumed translational start site, was applied for 4 days to cAMP- treated NGlOδ cells (final concentration lμmol, for 3 days every 24th and on the fourth day every 12h), and those cells where subsequently plated on myelin. 45% of the antisense treated cells extended neurites on myelin, whereas 7% of cells treated with a scrambled version and 2% of the untreated cells showed neurite growth. On permissive substrate poly-L-lysine δO-90% of the cells extended neurites. Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The following sequence listings form part of the application. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Roach, Arthur Lozano, Andres Labes, Monika Roder, John

(ii) TITLE OF INVENTION: Novel Agents Modulating the Response of Neuronal Cells to Inhibition by Mammalian Central Nervous System Myelin

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: BERESKIN & PARR

(B) STREET: 40 King Street West

(C) CITY: Toronto

(D) STATE: Ontario

(E) COUNTRY: Canada

(F) ZIP: M5H 3Y2

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kurdydyk, Linda M.

(B) REGISTRATION NUMBER: 34,971

(C) REFERENCE/DOCKET NUMBER: 3153-169

(lx) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (416) 364-7311

(B) TELEFAX: (416) 361-1398

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus

(F) TISSUE TYPE: Central Nervous System

(G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression

Library

(B) CLONE: Dl T7 ( ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 10..276

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCCGG GGT AAA TGT TAC ACA ACA AAG ACA GAC CGT CAC CTT AGA 48

Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg 1 5 10

CAT TGC TCT GGA CAA AAC TAT GGG GCA CAG AAC ATG GGA CTA GTC AGA 96 His Cys Ser Gly Gin Asn Tyr Gly Ala Gin Asn Met Gly Leu Val Arg 15 20 25

ATG GGC TGG TTT CTG ATC TGG AAA CTG TCC AGT GAC AAT TTG GAA AGT 144 Met Gly Trp Phe Leu lie Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser 30 35 40 45

CCC GGT GGA GGG AAA TGG GAA AGA TGG GAG AAA TGT CAA AAA AAC AAA 192 Pro Gly Gly Gly Lys Trp Glu Arg Trp Glu Lys Cys Gin Lys Asn Lys 50 55 60

AAC AAA ACA AAA AAA AAA CCC AAA AAA ACT CTG CCT CAC CCC ACC ATC 240 Asn Lys Thr Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr lie 65 70 75

ACC ACA AAA GAA TAT AGA GAA ACG GAG GGG GCA GGA AATGGGGGCA 286

Thr Thr Lys Glu Tyr Arg Glu Thr Glu Gly Ala Gly 80 85

GGAAGGATCC CTGAAATAAC TGGAACACAC AATGAGATGA CTGCTCGTAC TTT 339

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 89 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg His Cys Ser 1 5 10 15

Gly Gin Asn Tyr Gly Ala Gin Asn Met Gly Leu Val Arg Met Gly Trp 20 25 30

Phe Leu He Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser Pro Gly Gly 35 40 45

Gly Lys Trp Glu Arg Trp Glu Lys Cys Gin Lys Asn Lys Asn Lys Thr 50 55 60

Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr He Thr Thr Lys 65 70 75 80

Glu Tyr Arg Glu Thr Glu Gly Ala Gly 85

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 434 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus

(F) TISSUE TYPE: Central Nervous System

(G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression

Library

(B) CLONE: Dl T3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAATTCCGGC CTGAAGGTTC ATGACTCCAA CATCTACATA AAACCATTCC TGTCTTCAGC 60

TCCAGAGGTC AGAGTCTACG ATCTCTCCAA ATACGAGCAC GGAGCGGATG ACGTGCTGAT 120

CCTGGCTACT GATGGACTCT GGGATGTCTT ATCAAATGAA GAAGTAGCGG AAGCAATCAC 180

TCAGTTTCTT CCTAACTGTG ATCCAGATGA CCCTCACAGG TACACACTGG CAGCTCAGGA 240

CCTGGTGATG CGTGCCCGAG GCGTCCTGAA GGACCGAGGA TGGCGGATAT CAAATGACCG 300

ACTGGGCTCA GGAGATGACA TTTCTGTATA CGTCATTCCT TTAATACACG GAAACAAACT 360

GTCATGAAAG TGACCCAGGG GACTAGGAAG ACAGAAGAAG GGAAGAAAAC TGGGGTGCCT 420

CCAAGCAGGG CGGC 434 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 449 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus

(F) TISSUE TYPE: Central Nervous System

(G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression

Library

(B) CLONE: Dl-1 T3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : TGCAGGTCGA CACTAGTGGA TCCCTGAAAT AACTGGAACA CACAATGAGA TGACTGCTCG 60 TACTTTCTGA GGTATGGTCC CCCAACTTTT CAATGTGGCT CTTCTCCTGG AGAGATGCCT 120 GGCAGCTTGA CAGGCTGAGA AGTCTTTTAC CAGTTACACC CGGAATGGCT GTCCCTGCTA 180

TTCTAGGTAA AATAAATAAA CGTGTGCCTG CGCCCCAAAT AGGCTGCTGC CGCCCTAGGA 240

GCCCTACTGT AGAAACCAAG AGAGATGTAA CCCTCAGTAG CAGGCTGTGC GTTTTGCCTC 300

CTTATTCAGT GTCCAGACCC TGTTTACCCA AAGAAGAAAC GGAAACCCAA GGGTCTCCCT 360

TGAAGCCAAA GCCCAGAGGC CCTTTTGGCC ACAAAATTCC CAACCTGGGC TGGGGGAAAA 420

ATTGTTCCGT ACCCTGCGAA TTCCACCCA 449 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 479 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus

(F) TISSUE TYPE: Central Nervous System

(G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression

(B) CLONE: Dl 4T3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TGCAGGTCGA CACTAGTGGA TCCAAAGAAT TCACCCCTGA GACTGAGCGT CAGAGACTTC 60

AGTACCTGGC GTTCATGCAG CCTCACTTGC TGGGAAATGA GTTCACACAC TTGGAGTTTC 120

CAAGGAGAGT ACAGAGGAAA GAACTCGGGA AGAAGATGCT GTACCGGGAC TTTAACATGA 180

CAGGCTGGGC ATACAAAACC ATTGAGGATG ATGACTTGAA GTTTCCCCTT ATATATGGAG 240

AAGGCAAGAA GGCCCGGGTA ATGGCAACTA TTGGAGTGAC AAGGGGACTT GGGGACCATG 300

ACCTGAAGGT TCATGACTCC AACATCTACA TAAAACCATT CCTGTCTTCA GCTCCAGAGG 360

TCAGAGTCTA CGATCTCTCC AAATACGAGC ACGGAGCGGA TGACGTGCTG ATCCTGGCTA 420

CTGATGGACT CTGGGATGTC TTATCAAATG AAGAAGTAGC GGAAGCAATC ACTCAGTTT 479 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 487 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus

(F) TISSUE TYPE: Central Nervous System

(G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression

Library

(B) CLONE: Dl 5T7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCCAAAAGCC AGCCCGGCCG CGCAGAAGAC CTCGAGAAGC TTTTTGAATT CTGAGTGTGT 60

TGGCCTTTGG CCTTCCCAGG AGTTCCTCCA AGGGCCAACT TGCTACACAG GTCCATCCTG 120

TTCTGTGAAG GGGTGACTTA GAGTCTCAGG GAACAACGAG GCCCACACAG AGTAAGCCCA 180

AAGCACAGAG CAATATCAGT GCTGGGCAGA TCTGCCATGG GCATTAACAA ATCACTTTCC 240

CCACTTTGGT TTATTCACTT CATTTGTAGT CTTCTCCCCT GAGCCCCACC CCTGCAACAC 300

AAACTAAGAT CTTTCCACAC AGCGGCGGCC TCACGGAGAG AACTCCTTTC CAACTAAAAC 360

CAGCAAGGAC TCCGGGTTTT AGGGTAATTT GAATTTGGGT TTTCGGGTTT GGTTTGGTTT 420

GGTTGACGGT ACATGAACTG GGGAGAATGT TGTCATGACA CCTACAGGAT ATTCACACTC 480

CAATTCG 487 (2) INFORMATION FOR SEQ ID NO:7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4262 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GAATTCCGGG CCCGTGCAGC CTGATCATCA CATGACTCGA CGGCGGTAGC GTTGGGCAAG 60

CAAGGAGCGG CGGGTCCGCG GGCTCGCCGG GTGGGCTCAG CTCCGCGCAC GCAGAAACGG 120

GGCGCAGGGG GGCGGGGAAG AGACCTGCGG CAGCCGCCGC CCGCCAGCCG TTCCCGGGAC 180

TCTCGCTGTC CCTCCTCAGC CGCCCGCTCG CTCCATGTCG CCCGGTTGCG GAAGTGTCGC 240

TGGGAGAGGT GGCGGCGTCG TCGTCAGGGA GCACGGGAGG CCGGGGCTCG GCCCCTGCAG 300

CACTGAGCTC CCGGAGCCGC GCGTCCAGCG TCCCCACCGC CCGTGCGCCC CGCGCCCGCC 360

GCAGCCTGCA TGCCCCGCGC TGCACCTCGG CCGGCCGCCG CCTCCTGCTC GCTCAGCGGC 420

TGCCCGGCGC CGGAGTAATA TGCTCACTCG GGTGAAATCT GCCGTGGCCA ATTTCATGGG 480

CGGCATCATG GCTGGCAGCT CCGGCTCCGA GCACGGCGGC AGCGGCTGCG GAGGCTCGGA 540

CCTGCCCCTG CGCTTCCCGT ACGGGCGGCC AGAGTTCCTC GGGCTGTCTC AGGATGAGGT 600

GGAGTGCAGC GCAGACCACA TCGCCCGCCC CATCCTCATC CTCAAGGAGA CCCNNNNNNN 660 NNNNNNNNNN NCGGCCTGCT GCAGCACCAA TCACACAACA GCTGCAGGAC ATCGTGGAGA 720 TCCTGAAGAA CTCCGCCATC CTTCCCCCGA CCTGCCTAGG GGAGGAGCCA GAGAGCACGC 780 CGGCCCATGG CAGGACCCTA ACCCGCGCAG CCTACTGCGT GGAGGGGTGG GTGCCCCCGG 840 CTCCCCCAGC ACACCACCCA CGCGCTTCTT CACGGAGAAG AAGATTCCTC ATGAGTGTTT 900 GGTCATCGGG GCCCTGGAGA GCGCCTTCAA GGAAATGGAC CTTCAAATTG AACGGGAGAG 960

GAGTGCATAT AATATATCCG GTGGCTGCAC AGCCCTCATC GTGGTTTGCC TTCTGGGGAA 1020

GCTCTACGTG GCAAATGCAG GTGACAGCAG GGCCATAATC ATCCGAAATG GAGAAATCAT 1080

CCCCATGTCT TCCGAATTCA CCCCTGAGAC TGAGCGTCAG AGACTTCAGT ACCTGGCGTT 1140

CATGCAGCCT CACTTGCTGG GAAATGAGTT CACACACTTG GAGTTTCCAA GGAGAGTACA 1200

GAGGAAAGAA CTCGGGAAGA AGATGCTGTA CCGGGACTTT AACATGACAG GCTGGGCATA 1260

CAAAACCATT GAGGATGATG ACTTGAAGTT TCCCCTTATA TATGGAGAAG GCAAGAAGGC 1320

CCGGGTAATG GCAACTATTG GAGTGACAAG GGGACTTGGG GACCATGACC TGAAGGTTCA 1380

TGACTCCAAC ATCTACATAA AACCATTCCT GTCTTCAGCT CCAGAGGTCA GAGTCTACGA 1440

TCTCTCCAAA TACGAGCACG GAGCGGATGA CGTGCTGATC CTGGCTACTG ATGGACTCTG 1500

GGATGTCTTA TCAAATGAAG AAGTAGCGGA AGCAATCACT CAGTTTCTTC CTAACTGTGA 1560

TCCAGATGAC CCTCACAGGT ACACACTGGC AGCTCAGGAC CTGGTGATGC GTGCCCGAGG 1620

CGTCCTGAAG GACCGAGGAT GGCGGATATC AAATGACCGA CTGGGCTCAG GAGATGACAT 1680

TTCTGTATAC GTCATTCCTT TAATACACGG AAACAAACTG TCATGAAAGT GACCCAGGGG 1740

ACTAGGAAGA CAGAAGAAGG GAAGAAAACT GGGGTGCCTC CAAGCAGGGC GGGACTGGGG 1800

AGTAAGTACC TGGGCTGGAT TCCAGGTGAC GCACATTTTC CCCAGCCCAG GTGGGGAATT 1860

TGCTGCCAAA AGGCCTCCTG GCTTTGCTTC AAGGAGACCC TTGGGTTTCC GTTTCTTCTT 1920

TGGTAACAGT CTGGACACTG AATAAGAGCA AAACGCACAG CCTGCTACTG AGGGTTACAT 1980

CTCTCTTGGT TTCTACAGTA GGGCTCCTAG GCGGCAGCAG CCTATTTGGG GCGCAGGCAC 2040

ACGTTTATTT ATTTTACCTA GAATAGCAGG GACAGCCATT CCGGGTGTAA CTGGTAAAAG 2100

ACTTCTCAGC CTGTCAAGCT GCCAGCATCT CTCCAGGAGA AGAGCCACAT TGAAAAGTTG 2160

GGGGACCATA CCTCAGAAAG TACGAGCAGT CATCTCATTG TGTGTTCCAG TTATTTCAGG 2220

GATCCTTCCT GCCCCCCATT TCCTGCCCCC TCCGTTTCTC TATATTCTTT TGTGGTGATG 2280

GTGGGGTGAG GCAGAGTTTT TTTGGGTTTT TTTTTGTTTT GTTTTTGTTT TTTTGACATT 2340

TCTCCCATCT TTCCCATTTC CCTCCACCGG GACTTTCCAA ATTGTCACTG GACAGTTTCC 2400

AGATCAGAAA CCAGCCCATT CTGACTAGTC CCATGTTCTG TGCCCCATAG TTTTGTCCAG 2460

AGCAATGTCT AAGGTGACGG TCTGTCTTTG TTGTGTAACA TTTACCCCGG GGTTCTGTTT 2520

TTCTCCCCAA ATAGATATGT TTGCTTCAAA ACATGGGTGT TTCATTGGAC CAGTGGTTCC 2580

TGGGGTTATC TTTAAGGCCC CTCTGTGTGT CTGGAGGCTC TGCCACGAGA GGCTGGGTTT 2640 GCGGTTCTGG ATTGGCGATA CTCCCCGCCT TCTGTGTCCT GGAGAGGCAT AGGAAGCAGC 2700

GTTCAGGACC ACGGTCTAAG CCAGGCTCTT GTTATCCAGC ACTTGACCAT GTTCGCGTTG 2760

AGGGAAGGGG GTGTGGGATT CAGGCTCCTT GGTCGCTGAC TGTTCTCCAG GGCACAGGAG 2820

GATCGAGTGT CACAGCTAGC TAAGCAGCAG CTCTTCCTGA CACCTTTGTG CAAGGATAAC 2880

TAGGATGACA CTTGAATAAA AGTGAATTTG AATTGCAGTT GGTCATTGTG ATGCCCCCCT 2940

CCCCTTTCAC ATTGCTGAGA TCTCCTTCCT TTTATGCATC CACTGGTGTG TGTGCCTCAG 3000

TGGGCACACA CGGGCACATG AGCACCTGAG CACAGTATCT GTCCCCTGCT TCCTTGCTGG 3060

GAGGACCAGC AAAGTCCAGT TTAAAAATCA GCCGTCTCTT GGGCAGACTG CTGCTCTGCC 3120

CAGGGGCCTT CAGAGTAGCA TCCGGTTGCC TATTAGTCCT GTTCCTGTTG TCTTCCAGGA 3180

TATCAGCTTT CTGACAACTG GGTATAAATC AGACATTTCC AACCCAAGAA TGGATCCAAT .240

GGTGTCATTT CCCTAAAATG CTTGAGGAGA AGGCAGTTCC AACCTCCCAG GGCAGCGGGG 3300

CATTCCCTCC CCGCCGGAAA GCCGTCCACA TTCCTAGAAT TGTAGATATT TTCTTAGGGG 3360

AAGGCCTGGT GCCATCCCAC TCAGGAACAA AGTCACCCCA CTGTGTAGAG CCAGGGCCCA 3420

GCCCGGCAGG TGACATACTG TGAGTGTGTG CCAACTCGCT GCCTGAGGAC TGAGCTGTGG 3480

CCATGCTGGG GGCACCTACG TGTGCCTCTT TTTCAGGATG CTTCCCACTC CTGACACCGA 3540

TGCTGGAAGT GTTCTGTGGC AACCATTGCT TBCCTGACAG AATACAATGC TGTGGGAAAC 3600

TGTTCAGGCA CGCTACAGCA GCGTAGTCCT CTTCCAGCCC GTGCCCCGTT CTCAAAGTCA 3660

CACACAAACG GGAAACTTGA GAAGGTCTTG AACTCTGCGG AAGACCTGAG CTGCCTTCCA 3720

TAGGGAGTAT TTCTGGGTTC CCCGTGCCTT TCATATTTTT GCTTTTCTGA CCTCCCGAGT 3780

CTCACTTTGA CCTTCTTCAA TCACATTCAA GCCTCCTGTC GAATTGGAGT GTGAATATCC 3840

TGTAGGTGTC ATGACAACAT TCTCCCAGTT CATGTACCGT CAAACCAAAC CAAACCAAAC 3900

CCGAAAACCC AAATTCAAAT TACCCTAAAA CCCGGAGTCC TTGCTGGTTT TAGTTGGAAA 3960

GGAGTTCTCT CCGTGAGGCC GCCGCTGTGT GGAAAGATCT TAGTTTGTGT TGCAGGGGGT 4020

GGGGCTCAGG GGAGAAGACT ACAAATGAAG TGAATAAATC AAAAGTGGGG AAAGTGATTT 4080

GTTAATGCCC ATGGCAGATC TGCCCAGCAC TGATATTGCT CTGTGCTTTG GGCTTACTCT 4140

GTGTGGGCCT CGTTGTTCCC TGAGACTCTA AGTCACCCCT TCACAGAACA GGATGGACCT 4200

GTGTAGCAAG TTGGCCCTTG GAGGAACTCC TGGGAAGCCA AAGGCCAACA CACTCAGAAT 4260

TC 4262 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3590 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..3590

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 1..3590

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

CGG CCT GCT GCA GCA CCA ATC ACA CAA CAG CTG CAG GAC ATC GTG GAG 48 Arg Pro Ala Ala Ala Pro He Thr Gin Gin Leu Gin Asp He Val Glu 1 5 10 15

ATC CTG AAG AAC TCC GCC ATC CTT CCC CCG ACC TGC CTA GGG GAG GAG 96 He Leu Lys Asn Ser Ala He Leu Pro Pro Thr Cys Leu Gly Glu Glu 20 25 30

CCA GAG AGC ACG CCG GCC CAT GGC AGG ACC CTA ACC CGC GCA GCC TAC 144 Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr 35 40 45

TGC GTG GAG GGG TGG GTG CCC CCG GCT CCC CCA GCA CAC CAC CCA CGC 192 Cys Val Glu Gly Trp Val Pro Pro Ala Pro Pro Ala His His Pro Arg 50 55 60

GCT TCT TCA CGG AGA AGA AGA TTC CTC ATG AGT GTT TGG TCA TCG GGG 240 Ala Ser Ser Arg Arg Arg Arg Phe Leu Met Ser Val Trp Ser Ser Gly 65 70 75 80

CCC TGG AGA GCG CCT TCA AGG AAA TGG ACC TTC AAA TTG AAC GGG AGA 288 Pro Trp Arg Ala Pro Ser Arg Lys Trp Thr Phe Lys Leu Asn Gly Arg 85 90 95

GGA GTG CAT ATA ATA TAT CCG GTG GCT GCA CAG CCC TCA TCG TGG TTT 336 Gly Val His He He Tyr Pro Val Ala Ala Gin Pro Ser Ser Trp Phe 100 105 110

GCC TTC TGG GGA AGC TCT ACG TGG CAA ATG CAG GTG ACA GCA GGG CCA 384 Ala Phe Trp Gly Ser Ser Thr Trp Gin Met Gin Val Thr Ala Gly Pro 115 120 125

TAA TCA TCC GAA ATG GAG AAA TCA TCC CCA TGT CTT CCG AAT TCA CCC 432 * Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro 130 135 140

CTG AGA CTG AGC GTC AGA GAC TTC AGT ACC TGG CGT TCA TGC AGC CTC 480 Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu 145 150 155 160

ACT TGC TGG GAA ATG AGT TCA CAC ACT TGG AGT TTC CAA GGA GAG TAC 528 Thr Cys Trp Glu Met Ser Ser His Thr Trp Ser Phe Gin Gly Glu Tyr 165 170 175

AGA GGA AAG AAC TCG GGA AGA AGA TGC TGT ACC GGG ACT TTA ACA TGA 576 Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr * 180 185 190

CAG GCT GGG CAT ACA AAA CCA TTG AGG ATG ATG ACT TGA AGT TTC CCC 624 Gin Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro 195 200 205

TTA TAT ATG GAG AAG GCA AGA AGG CCC GGG TAA TGG CAA CTA TTG GAG 672 Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gin Leu Leu Glu 210 215 220 TGA CAA GGG GAC TTG GGG ACC ATG ACC TGA AGG TTC ATG ACT CCA ACA 720

* Gin Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr 225 230 235 240

TCT ACA TAA AAC CAT TCC TGT CTT CAG CTC CAG AGG TCA GAG TCT ACG 768

Ser Thr * Asn His Ser Cys Leu Gin Leu Gin Arg Ser Glu Ser Thr 245 250 255

ATC TCT CCA AAT ACG AGC ACG GAG CGG ATG ACG TGC TGA TCC TGG CTA 816

He Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu 260 265 270

CTG ATG GAC TCT GGG ATG TCT TAT CAA ATG AAG AAG TAG CGG AAG CAA 864

Leu Met Asp Ser Gly Met Ser Tyr Gin Met Lys Lys * Arg Lys Gin 275 280 285

TCA CTC AGT TTC TTC CTA ACT GTG ATC CAG ATG ACC CTC ACA GGT ACA 912

Ser Leu Ser Phe Phe Leu Thr Val He Gin Met Thr Leu Thr Gly Thr

290 295 300

CAC TGG CAG CTC AGG ACC TGG TGA TGC GTG CCC GAG GCG TCC TGA AGG 960

His Trp Gin Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg

305 310 315 320

ACC GAG GAT GGC GGA TAT CAA ATG ACC GAC TGG GCT CAG GAG ATG ACA 1008

Thr Glu Asp Gly Gly Tyr Gin Met Thr Asp Trp Ala Gin Glu Met Thr 325 330 335

TTT CTG TAT ACG TCA TTC CTT TAA TAC ACG GAA ACA AAC TGT CAT GAA 1056

Phe Leu Tyr Thr Ser Phe Leu * Tyr Thr Glu Thr Asn Cys His Glu 340 345 350

AGT GAC CCA GGG GAC TAG GAA GAC AGA AGA AGG GAA GAA AAC TGG GGT 1104

Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly 355 360 365

GCC TCC AAG CAG GGC GGG ACT GGG GAG TAA GTA CCT GGG CTG GAT TCC 1152

Ala Ser Lys Gin Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser

370 375 380

AGG TGA CGC ACA TTT TCC CCA GCC CAG GTG GGG AAT TTG CTG CCA AAA 1200

Arg * Arg Thr Phe Ser Pro Ala Gin Val Gly Asn Leu Leu Pro Lys

385 390 395 400

GGC CTC CTG GCT TTG CTT CAA GGA GAC CCT TGG GTT TCC GTT TCT TCT 1248

Gly Leu Leu Ala Leu Leu Gin Gly Asp Pro Trp Val Ser Val Ser Ser 405 410 415

TTG GTA ACA GTC TGG ACA CTG AAT AAG AGC AAA ACG CAC AGC CTG CTA 1296

Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu 420 425 430

CTG AGG GTT ACA TCT CTC TTG GTT TCT ACA GTA GGG CTC CTA GGC GGC 1344

Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly 435 440 445

AGC AGC CTA TTT GGG GCG CAG GCA CAC GTT TAT TTA TTT TAC CTA GAA 1392

Ser Ser Leu Phe Gly Ala Gin Ala His Val Tyr Leu Phe Tyr Leu Glu

450 455 460

TAG CAG GGA CAG CCA TTC CGG GTG TAA CTG GTA AAA GAC TTC TCA GCC 1440

* Gin Gly Gin Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala 465 470 475 480

TGT CAA GCT GCC AGC ATC TCT CCA GGA GAA GAG CCA CAT TGA AAA GTT 1488

Cys Gin Ala Ala Ser He Ser Pro Gly Glu Glu Pro His * Lys Val 485 490 495

GGG GGA CCA TAC CTC AGA AAG TAC GAG CAG TCA TCT CAT TGT GTG TTC 1536 Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gin Ser Ser His Cys Val Phe 500 505 510

CAG TTA TTT CAG GGA TCC TTC CTG CCC CCC ATT TCC TGC CCC CTC CGT 1584 Gin Leu Phe Gin Gly Ser Phe Leu Pro Pro He Ser Cys Pro Leu Arg 515 520 525

TTC TCT ATA TTC TTT TGT GGT GAT GGT GGG GTG AGG CAG AGT TTT TTT 1632 Phe Ser He Phe Phe Cys Gly Asp Gly Gly Val Arg Gin Ser Phe Phe 530 535 540

GGG TTT TTT TTT GTT TTG TTT TTG TTT TTT TGA CAT TTC TCC CAT CTT 1680 Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu 545 550 555 560

TCC CAT TTC CCT CCA CCG GGA CTT TCC AAA TTG TCA CTG GAC AGT TTC 1728 Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe 565 570 575

CAG ATC AGA AAC CAG CCC ATT CTG ACT AGT CCC ATG TTC TGT GCC CCA 1776 Gin He Arg Asn Gin Pro He Leu Thr Ser Pro Met Phe Cys Ala Pro 580 585 590

TAG TTT TGT CCA GAG CAA TGT CTA AGG TGA CGG TCT GTC TTT GTT GTG 1824

* Phe Cys Pro Glu Gin Cys Leu Arg * Arg Ser Val Phe Val Val

595 600 605

TAA CAT TTA CCC CGG GGT TCT GTT TTT CTC CCC AAA TAG ATA TGT TTG 1872

* His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * He Cys Leu 610 615 620

CTT CAA AAC ATG GGT GTT TCA TTG GAC CAG TGG TTC CTG GGG TTA TCT 1920 Leu Gin Asn Met Gly Val Ser Leu Asp Gin Trp Phe Leu Gly Leu Ser 625 630 635 640

TTA AGG CCC CTC TGT GTG TCT GGA GGC TCT GCC ACG AGA GGC TGG GTT 1968 Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val 645 650 655

TGC GGT TCT GGA TTG GCG ATA CTC CCC GCC TTC TGT GTC CTG GAG AGG 2016 Cys Gly Ser Gly Leu Ala He Leu Pro Ala Phe Cys Val Leu Glu Arg 660 665 670

CAT AGG AAG CAG CGT TCA GGA CCA CGG TCT AAG CCA GGC TCT TGT TAT 2064 His Arg Lys Gin Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr 675 680 685

CCA GCA CTT GAC CAT GTT CGC GTT GAG GGA AGG GGG TGT GGG ATT CAG 2112 Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly He Gin 690 695 700

GCT CCT TGG TCG CTG ACT GTT CTC CAG GGC ACA GGA GGA TCG AGT GTC 2160 Ala Pro Trp Ser Leu Thr Val Leu Gin Gly Thr Gly Gly Ser Ser Val 705 710 715 720

ACA GCT AGC TAA GCA GCA GCT CTT CCT GAC ACC TTT GTG CAA GGA TAA 2208 Thr Ala Ser ^• Ala Ala Ala Leu Pro Asp Thr Phe Val Gin Gly * 725 730 735

CTA GGA TGA CAC TTG AAT AAA AGT GAA TTT GAA TTG CAG TTG GTC ATT 2256 Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gin Leu Val He 740 745 750 GTG ATG CCC CCC TCC CCT TTC ACA TTG CTG AGA TCT CCT TCC TTT TAT 2304

Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr 755 760 765

GCA TCC ACT GGT GTG TGT GCC TCA GTG GGC ACA CAC GGG CAC ATG AGC 2352

Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser 770 775 780

ACC TGA GCA CAG TAT CTG TCC CCT GCT TCC TTG CTG GGA GGA CCA GCA 2400

Thr * Ala Gin Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala

785 790 795 800

AAG TCC AGT TTA AAA ATC AGC CGT CTC TTG GGC AGA CTG CTG CTC TGC 2448

Lys Ser Ser Leu Lys He Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys 805 810 815

CCA GGG GCC TTC AGA GTA GCA TCC GGT TGC CTA TTA GTC CTG TTC CTG 2496

Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu 820 825 830

TTG TCT TCC AGG ATA TCA GCT TTC TGA CAA CTG GGT ATA AAT CAG ACA .544

Leu Ser Ser Arg He Ser Ala Phe * Gin Leu Gly He Asn Gin Thr 835 840 845

TTT CCA ACC CAA GAA TGG ATC CAA TGG TGT CAT TTC CCT AAA ATG CTT 2592

Phe Pro Thr Gin Glu Trp He Gin Trp Cys His Phe Pro Lys Met Leu 850 855 860

GAG GAG AAG GCA GTT CCA ACC TCC CAG GGC AGC GGG GCA TTC CCT CCC 2640

Glu Glu Lys Ala Val Pro Thr Ser Gin Gly Ser Gly Ala Phe Pro Pro

865 870 875 880

CGC CGG AAA GCC GTC CAC ATT CCT AGA ATT GTA GAT ATT TTC TTA GGG 2688

Arg Arg Lys Ala Val His He Pro Arg He Val Asp He Phe Leu Gly 885 890 895

GAA GGC CTG GTG CCA TCC CAC TCA GGA ACA AAG TCA CCC CAC TGT GTA 2736

Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val 900 905 910

GAG CCA GGG CCC AGC CCG GCA GGT GAC ATA CTG TGA GTG TGT GCC AAC 2784

Glu Pro Gly Pro Ser Pro Ala Gly Asp He Leu * Val Cys Ala Asn 915 920 925

TCG CTG CCT GAG GAC TGA GCT GTG GCC ATG CTG GGG GCA CCT ACG TGT 2832 Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys 930 935 940

GCC TCT TTT TCA GGA TGC TTC CCA CTC CTG ACA CCG ATG CTG GAA GTG 2880

Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val

945 950 955 960

TTC TGT GGC AAC CAT TGC TTC CTG ACA GAA TAC AAT GCT GTG GGA AAC 2928

Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn 965 970 975

TGT TCA GGC ACG CTA CAG CAG CGT AGT CCT CTT CCA GCC CGT GCC CCG 2976

Cys Ser Gly Thr Leu Gin Gin Arg Ser Pro Leu Pro Ala Arg Ala Pro 980 985 990

TTC TCA AAG TCA CAC ACA AAC GGG AAA CTT GAG AAG GTC TTG AAC TCT 3024

Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser 995 1000 1005

GCG GAA GAC CTG AGC TGC CTT CCA TAG GGA GTA TTT CTG GGT TCC CCG 3072

Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro 1010 1015 1020

TGC CTT TCA TAT TTT TGC TTT TCT GAC CTC CCG AGT CTC ACT TTG ACC 3120 Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr 1025 1030 1035 1040

TTC TTC AAT CAC ATT CAA GCC TCC TGT CGA ATT GGA GTG TGA ATA TCC 3168 Phe Phe Asn His He Gin Ala Ser Cys Arg He Gly Val * He Ser 1045 1050 1055

TGT AGG TGT CAT GAC AAC ATT CTC CCA GTT CAT GTA CCG TCA AAC CAA 3216 Cys Arg Cys His Asp Asn He Leu Pro Val His Val Pro Ser Asn Gin 1060 1065 1070

ACC AAA CCA AAC CCG AAA ACC CAA ATT CAA ATT ACC CTA AAA CCC GGA 3264 Thr Lys Pro Asn Pro Lys Thr Gin He Gin He Thr Leu Lys Pro Gly 1075 1080 1085

GTC CTT GCT GGT TTT AGT TGG AAA GGA GTT CTC TCC GTG AGG CCG CCG 3312 Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro 1090 1095 1100

CTG TGT GGA AAG ATC TTA GTT TGT GTT GCA GGG GGT GGG GCT CAG GGG 3360 Leu Cys Gly Lys He Leu Val Cys Val Ala Gly Gly Gly Ala Gin Gly 1105 1110 1115 1120

AGA AGA CTA CAA ATG AAG TGA ATA AAT CAA AAG TGG GGA AAG TGA TTT 3408 Arg Arg Leu Gin Met Lys * He Asn Gin Lys Trp Gly Lys * Phe 1125 1130 1135

GTT AAT GCC CAT GGC AGA TCT GCC CAG CAC TGA TAT TGC TCT GTG CTT 3456 Val Asn Ala His Gly Arg Ser Ala Gin His * Tyr Cys Ser Val Leu 1140 1145 1150

TGG GCT TAC TCT GTG TGG GCC TCG TTG TTC CCT GAG ACT CTA AGT CAC 3504 Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His 1155 1160 1165

CCC TTC ACA GAA CAG GAT GGA CCT GTG TAG CAA GTT GGC CCT TGG AGG 3552 Pro Phe Thr Glu Gin Asp Gly Pro Val * Gin Val Gly Pro Trp Arg 1170 1175 1180

AAC TCC TGG GAA GCC AAA GGC CAA CAC ACT CAG AAT TC 3590

Asn Ser Trp Glu Ala Lys Gly Gin His Thr Gin Asn 1185 1190 1195

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1196 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Arg Pro Ala Ala Ala Pro He Thr Gin Gin Leu Gin Asp He Val Glu 1 5 10 15

He Leu Lys Asn Ser Ala He Leu Pro Pro Thr Cys Leu Gly Glu Glu 20 25 30

Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr 35 40 45 Cys Val Glu Gly Trp Val Pro Pro Ala Pro Pro Ala His His Pro Arg 50 55 60

Ala Ser Ser Arg Arg Arg Arg Phe Leu Met Ser Val Trp Ser Ser Gly 65 70 75 80

Pro Trp Arg Ala Pro Ser Arg Lys Trp Thr Phe Lys Leu Asn Gly Arg 85 90 95

Gly Val His He He Tyr Pro Val Ala Ala Gin Pro Ser Ser Trp Phe 100 105 110

Ala Phe Trp Gly Ser Ser Thr Trp Gin Met Gin Val Thr Ala Gly Pro 115 120 125

Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro 130 135 140

Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu 145 150 155 160

Thr Cys Trp Glu Met Ser Ser His Thr Trp Ser Phe Gin Gly Glu Tyr 165 170 175

Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr 180 185 190

Gin Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro 195 200 205

Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gin Leu Leu Glu 210 215 220

* Gin Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr 225 230 235 240

Ser Thr * Asn His Ser Cys Leu Gin Leu Gin Arg Ser Glu Ser Thr 245 250 255

He Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu 260 265 270

Leu Met Asp Ser Gly Met Ser Tyr Gin Met Lys Lys * Arg Lys Gin 275 280 285

Ser Leu Ser Phe Phe Leu Thr Val He Gin Met Thr Leu Thr Gly Thr 290 295 300

His Trp Gin Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg 305 310 315 320

Thr Glu Asp Gly Gly Tyr Gin Met Thr Asp Trp Ala Gin Glu Met Thr 325 330 335

Phe Leu Tyr Thr Ser Phe Leu Tyr Thr Glu Thr Asn Cys His Glu 340 345 350

Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly 355 360 365

Ala Ser Lys Gin Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser 370 375 380

Arg * Arg Thr Phe Ser Pro Ala Gin Val Gly Asn Leu Leu Pro Lys 385 390 395 400 Gly Leu Leu Ala Leu Leu Gin Gly Asp Pro Trp Val Ser Val Ser Ser 405 410 415

Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu 420 425 430

Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly 435 440 445

Ser Ser Leu Phe Gly Ala Gin Ala His Val Tyr Leu Phe Tyr Leu Glu 450 455 460

* Gin Gly Gin Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala 465 470 475 480

Cys Gin Ala Ala Ser He Ser Pro Gly Glu Glu Pro His * Lys Val 485 490 495

Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gin Ser Ser His Cys Val Phe 500 505 510

Gin Leu Phe Gin Gly Ser Phe Leu Pro Pro He Ser Cys Pro Leu Arg

515 520 525

Phe Ser He Phe Phe Cys Gly Asp Gly Gly Val Arg Gin Ser Phe Phe 530 535 540

Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu 545 550 555 560

Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe 565 570 575

Gin He Arg Asn Gin Pro He Leu Thr Ser Pro Met Phe Cys Ala Pro 580 585 590

* Phe Cys Pro Glu Gin Cys Leu Arg * Arg Ser Val Phe Val Val

595 600 605

* His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * He Cys Leu 610 615 620

Leu Gin Asn Met Gly Val Ser Leu Asp Gin Trp Phe Leu Gly Leu Ser 625 630 635 640

Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val 645 650 655

Cys Gly Ser Gly Leu Ala He Leu Pro Ala Phe Cys Val Leu Glu Arg 660 665 670

His Arg Lys Gin Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr 675 680 685

Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly He Gin 690 695 700

Ala Pro Trp Ser Leu Thr Val Leu Gin Gly Thr Gly Gly Ser Ser Val 705 710 715 720

Thr Ala Ser * Ala Ala Ala Leu Pro Asp Thr Phe Val Gin Gly * 725 730 735

Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gin Leu Val He

740 745 750 Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr 755 760 765

Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser 770 775 780

Thr * Ala Gin Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala 785 790 795 800

Lys Ser Ser Leu Lys He Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys 805 810 815

Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu 820 825 830

Leu Ser Ser Arg He Ser Ala Phe * Gin Leu Gly He Asn Gin Thr 835 840 845

Phe Pro Thr Gin Glu Trp He Gin Trp Cys His Phe Pro Lys Met Leu 850 855 860

Glu Glu Lys Ala Val Pro Thr Ser Gin Gly Ser Gly Ala Phe Pro Pro 865 870 875 880

Arg Arg Lys Ala Val His He Pro Arg He Val Asp He Phe Leu Gly 885 890 895

Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val 900 905 910

Glu Pro Gly Pro Ser Pro Ala Gly Asp He Leu * Val Cys Ala Asn 915 920 925

Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys 930 935 940

Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val 945 950 955 960

Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn 965 970 975

Cys Ser Gly Thr Leu Gin Gin Arg Ser Pro Leu Pro Ala Arg Ala Pro 980 985 990

Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser 995 1000 1005

Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro 1010 1015 1020

Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr 1025 1030 1035 1040

Phe Phe Asn His He Gin Ala Ser Cys Arg He Gly Val * He Ser 1045 1050 1055

Cys Arg Cys His Asp Asn He Leu Pro Val His Val Pro Ser Asn Gin 1060 1065 1070

Thr Lys Pro Asn Pro Lys Thr Gin He Gin He Thr Leu Lys Pro Gly 1075 1080 1085

Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro 1090 1095 1100 Leu Cys Gly Lys He Leu Val Cys Val Ala Gly Gly Gly Ala Gin Gly 1105 1110 1115 1120

Arg Arg Leu Gin Met Lys * He Asn Gin Lys Trp Gly Lys * Phe 1125 1130 1135

Val Asn Ala His Gly Arg Ser Ala Gin His * Tyr Cys Ser Val Leu 1140 1145 1150

Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His 1155 1160 1165

Pro Phe Thr Glu Gin Asp Gly Pro Val * Gin Val Gly Pro Trp Arg 1170 1175 1180

Asn Ser Trp Glu Ala Lys Gly Gin His Thr Gin Asn 1185 1190 1195

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 239 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg His Cys Ser 1 5 10 15

Gly Gin Asn Tyr Gly Ala Gin Asn Met Gly Leu Val Arg Met Gly Trp 20 25 30

Phe Leu He Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser Pro Gly Gly 35 40 45

Gly Lys Trp Glu Arg Trp Glu Lys Cys Gin Lys Asn Lys Asn Lys Thr 50 55 60

Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr He Thr Thr Lys 65 70 75 80

Glu Tyr Arg Glu Thr Glu Gly Ala Gly Asn Xaa Gly Gin Glu Gly Ser 85 90 95

Leu Lys Xaa Leu Glu His Thr Met Arg Xaa Leu Leu Val Leu Ser Glu 100 105 110

Val Trp Ser Pro Asn Phe Ser Met Trp Leu Phe Ser Trp Arg Asp Ala 115 120 125

Trp Gin Leu Asp Arg Leu Arg Ser Leu Leu Pro Val Thr Pro Gly Met 130 135 140

Ala Val Pro Ala He Leu Gly Lys He Asn Lys Arg Val Pro Ala Pro 145 150 155 160

Gin He Gly Cys Cys Arg Pro Arg Ser Pro Thr Val Glu Thr Lys Arg

165 170 175 Asp Val Thr Leu Ser Ser Arg Leu Cys Val Leu Pro Pro Tyr Ser Val 180 185 190

Ser Arg Pro Cys Leu Pro Lys Glu Glu Thr Glu Thr Gin Gly Ser Pro 195 200 205

Leu Lys Pro Lys Pro Arg Gly Pro Phe Xaa Pro Xaa Lys Phe Pro Thr 210 215 220

Trp Ala Gly Gly Lys He Val Pro Tyr Pro Ala Asn Ser Xaa Pro 225 230 235

Claims

WE CLAIM:

1. A method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin comprising growing neuronal cells which have a propensity for neurite growth, on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of affecting neurite outgrowth, and assaying for neurite outgrowth.

2. 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 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 /or SEQ ID. NO. δ.

3. The isolated nucleic acid molecule as claimed in claim 2 which 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. δ, or in Figures 9, 11 to 14, and 21 wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least δO-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, SEQ. ID. NO. 7 and SEQ. ID. NO. δ; (d) a fragment of (a) to (c) 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 (d) in codon sequences due to the degeneracy of the genetic code.

4. An isolated and purified 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); (c) nucleic acid sequences which are at least δ0%, preferably 90% identical to (a); or, (d) a fragment of (a) or

(b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.

5. A nucleic acid sequence as claimed in claim 4 in an antisense orientation.

6. A recombinant molecule adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in claim 4.

7. A transformed host cell containing a recombinant molecule as claimed in claim 6.

β. A method for preparing a protein comprismg (a) transferring a recombinant expression vector as claimed in claim 6; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.

9. A isolated and purified protein encoded by the nucleic acid molecule as claimed in claim 3 including the amino acid sequence as shown in the Sequence Listing as SEQ. ID. NO. 2 or

SEQ ID. NO. 9 and sequences having at least δO-90% identity thereto, and which is expressed in brain, NGlOδ, PC12, and fibroblast cells.

10. An isolated and purified protem comprising an ammo acid sequence as shown in Figure 24 or SEQ ID NO: 12, and truncations, analogs, homologs, and isoforms of the protein and truncations thereof.

11. A method for assaymg for the presence of an activator or inhibitor of the protem as claimed in claim 10, comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelin and which express the protem m the presence of a suspected activator or inhibitor substance, and assaying for neurite outgrowth

12 A method for idenhfymg a substance which is capable of binding to a protein as claimed in claim 10, comprising reacting the protein with at least one substance which potentially can bind with the protem, or part of the protem, under conditions which permit the formation of substance-protem complexes, and assaymg for substance-protem complexes, for free substance, and/or for non-complexed protem.

13. A method for assaymg for a substance that affects the phosphatase activity of a protein as claimed in claim 10 comprismg reactmg the protein with a substrate which is capable of bemg dephosphorylated by the protem to produce a dephosphorylated product, m the presence of a substance which is suspected of affectmg the phosphatase activity of the protem, and under conditions which permit dephosphorylation of the substrate, assaying for dephosphorylated product; and, comparmg to product obtamed m the absence of the substance to determine the affect of the substance on the phosphatase activity of the protem

14 Antibodies havmg specificity against an epitope of a protem as claimed in claim 10

15 Monoclonal antibodies which (a) immunoreact with neuronal membrane protems, (b) neutralize the inhibition of neurite growth by mammalian central nervous system mvehn, and, (c) recognize bands of M_r 35,000 and M_r 33,000 expressed in neuronal and fibroblast cell lines and m rat cerebrum and rat liver