WO1995035373A2 - Molecules d'acide nucleique codant la contactine humaine - Google Patents

Molecules d'acide nucleique codant la contactine humaine Download PDF

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WO1995035373A2
WO1995035373A2 PCT/US1995/007408 US9507408W WO9535373A2 WO 1995035373 A2 WO1995035373 A2 WO 1995035373A2 US 9507408 W US9507408 W US 9507408W WO 9535373 A2 WO9535373 A2 WO 9535373A2
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contactin
seq
human contactin
recombinant
human
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PCT/US1995/007408
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WO1995035373A3 (fr
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Barbara Ranscht
Erik O. Berglund
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La Jolla Cancer Research Foundation
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Publication of WO1995035373A3 publication Critical patent/WO1995035373A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the field of molecular biology and, in particular, to nucleic acid molecules encoding human contactin.
  • Contactin (known as Fll in chick and F3 in the mouse) is a neural cell adhesion molecule of the immunoglobulin gene superfamily (IgSF) that is attached to the cell surface through a glycosyl phosphatidyl- inositol (GPI) membrane anchor. It binds to extracellular matrix molecules of the tenascin family, including tenascin-C and tenascin-R, and to the IgSF molecules Ng-CAM and Nr-CAM/Bravo. Contactin has been suggested to guide the establishment of neuronal circuitry in developing embryos by facilitating axon extension, fasciculation and synapse formation.
  • IgSF immunoglobulin gene superfamily
  • Contactin expression is restricted to neuronal cell surfaces, including the growth cone, and is particularly prominent as neurons extend axons and establish synaptic contacts.
  • Cells transfected with mouse or chicken contactin or soluble mouse contactin have been shown to promote the extension of dorsal root ganglion neurites. Therefore, contactin supports the extension of neurites both as an immobilized substrate and as a soluble molecule.
  • Recombinant human contactin and soluble human contactin would be useful for promoting growth of neurons both in vivo and in vi tro.
  • This invention satisfies these needs and provides related advantages as well by providing nucleic acid molecules encoding human contactin, recombinant human contactin and recombinant soluble human contactin.
  • This invention provides isolated nucleic acid molecules coding on expression for human contactin 2, a soluble human contactin, or a minor variant of either of them. It also provides expression vectors having an expression control sequence operatively linked to a nucleic acid sequence coding on expression for human contactin 2, a soluble human contactin, or a minor variant of either of them. It further provides unicellular hosts transfected with these expression vectors and methods of making recombinant contactin 2, recombinant soluble contactin or a recombinant minor variant of either by culturing these unicellular hosts. This invention also provides recombinant human contactin 2, soluble recombinant human contactin, and a recombinant minor variant of either of them. It further provides pharmaceutical compositions having recombinant human contactin, recombinant soluble human contactin or a recombinant minor variant of either of them in a pharmaceutically acceptable carrier.
  • This invention also provides methods useful for promoting neurite extension in a neuron in culture by contacting the neuron with recombinant human contactin, recombinant soluble human contactin or a recombinant minor variant of either of them.
  • This invention provides methods useful for promoting neuron regeneration in a person with neuron damage by administering a therapeutically effective amount of a pharmaceutical composition having recombinant human contactin, recombinant soluble human contactin or a recombinant minor variant of either of them in a pharmaceutically acceptable carrier.
  • This invention also provides methods useful for modulating neurite outgrowth by binding contactin with an effective amount of a neurite modulating contactin- binding ligand.
  • This invention also provides methods useful for promoting neuron regeneration in a person by providing the person at the site of neuronal damage with mammalian cells transfected with an expression vector of this invention.
  • This invention provides methods useful for identifying contactin-binding ligands by screening samples suspected of containing contactin-binding ligands with a human contactin for ligands which specifically bind contactin.
  • Figures la to lc depict the nucleotide sequence and deduced amino acid sequence of human contactin.
  • the amino acid sequence is numbered starting with the first amino acid of mature human contactin 1.
  • Human contactin 2 lacks the boxed 11 amino acid residues at the NH 2 -terminus of the mature protein. Hydrophobic domains at the NH 2 - and the COOH-terminals are underscored by a single line. Putative sites for N-linked glycosylation are indicated by triangles. Cys conserved in the immunoglobulin (Igr) domains are boxed, and the characteristic Trp and Tyr/Phe residues of the FNIII domains are encircled. The stop codon is indicated with three stars.
  • Residue 973 (Ser) [SEQ ID NO:2] , marked with a single star, represents the probable attachment site for the GPI-anchor.
  • the Gly-Pro rich region, possibly functioning as a hinge, is indicated by wavy lines.
  • Figure 2 depicts a schematic representation of the human contactin protein.
  • Human contactin consists of six NH 2 -terminal Ig domains, (represented as loops) and four FNIII repeats (unfilled boxes) that are separated by a hinge-region (saw-tooth) .
  • Two hydrophobic domains are indicated as filled boxes at the NH 2 -and the COOH-termini.
  • Figures 3a to 3b depict an aligned comparison of human contactin isoforms [SEQ ID NOS: 1 and 2] , mouse F3 [SEQ ID NO: 3] , and chicken contactin/Fll [SEQ ID NO: 4] .
  • Amino acid sequences were aligned using the Alignment function of the Geneworks program (Intelligenetics) , followed by visual verification and correction to achieve the best match. Gaps were introduced to maximize identities in pairwise comparisons. In the alignment of Ig domains and FNIII repeats, high priority was assigned to the correct positioning of characteristic Cys, Trp and Tyr/Phe residues, respectively. Amino acids identical in all four sequences are boxed. Human contactin shares 94% amino acid identity with mouse F3 and 78% with chick contactin/Fll.
  • Figures 4a to 4c show the effect of contactin on neurite growth.
  • Rat cerebellar neurons were grown on a bed of B28 cells and, after fixation, stained with the anti-neurofilament antibody, Nel4, followed by fluorescein-conjugated secondary antibody.
  • the cells in Figure 4a were mock-transfected.
  • the neurons grown on the mock transfected cells have short neurites.
  • the B28 cells in Figures 4b and 4c were transfected with contactin cDNA and express h-contactin.
  • the neurons grown on these cells have long neurites.
  • Figure 5 compares length of neurites from rat cerebellar neurons grown on contactin and mock- transfected B28 cells.
  • This invention provides cDNA molecules encoding human contactin.
  • the isolation of these cDNAs allows the production of recombinant human contactin and other forms of the molecule.
  • the recombinant proteins of this invention are useful in methods of promoting neurite extension of neurons both in vivo and in vi tro.
  • human contactin refers to human contactin in either of its mature isoforms, optionally including a signal sequence. Human contactin binds to the extracellular matrix proteins tenascin-C and tenascin-R, and the immunoglobulin-like adhesion molecules Ng-CAM and Nr-CAM/Bravo. Human contactin functions as a neuronal cell adhesion protein and promotes neurite extension in neurons. It has six Ig-like C2 domains, four fibronectin type III repeats and a glycosyl phosphatidylinositol membrane anchor.
  • Human contactin 1 has the sequence of amino acids 1 to 998 of Figure 1 [SEQ ID NO:2] .
  • Human contactin 2 has the sequence of amino acids 12-998 of Figure 1 [SEQ ID NO:2] .
  • the native signal sequence of human contactin 1 and 2 has the sequence of amino acids -20 to -1 of Figure 1 [SEQ ID NO:2] . It is recognized that in the recombinant production of human contactin, the native signal sequence can be replaced with another signal sequence that will guide the polypeptide through the membrane. In most eukaryotic expression systems, the signal sequence will be cleaved off during processing, yielding a mature protein.
  • Human contactin also refers to allelic variants of human contactin and any variants resulting from alternative splicing of contactin mRNA.
  • human contactin can be purified from endogenous sources, such as brain, for example.
  • soluble human contactin refers to human contactin which has been altered so that, upon expression, it is not anchored into the cell membrane but is secreted into the medium. It is understood that soluble human contactin retains the ability to promote neurite growth. Soluble human contactin can be engineered by removing a sufficient part of the amino acid sequence that anchors the protein to the cell membrane.
  • the fi-.il glycosyl phosphatidyl- inositol membrane anchor corresponds to the amino acid sequence 973-998 of Figure 1 [SEQ ID NO:2] .
  • soluble forms of mature contactin 1 and 2 include those having the amino acid sequence 1-972 and 12-972 of Figure 1 [SEQ ID NO:2] , respectively.
  • soluble human contactin can be produced by removal of residues C-terminal to serine 981. Soluble human contactin also can be engineered by eliminating the GPI anchor attachment site. GPI is attached through serine at position 973. Therefore, soluble human contactin can be produced by substituting serine for an amino acid that cannot serve as an anchor attachment site.
  • the amino acids glycine, alanine, cysteine, aspartic acid and asparagine can serve as GPI anchor attachment sites. Therefore, these amino acids cannot be substituted for serine in soluble human contactin.
  • a "minor variant" of human contactin or soluble human contactin means a protein having an amino acid sequence that corresponds except for minor modifications to the sequence of a human contactin or soluble human .contactin. Minor variants include conservative substitutions, additions or deletions to the amino acid sequence that do not eliminate the ability of contactin to bind tenascin-C, tenascin-R, Ng-CAM or Nr- CAM/Bravo, or to promote neurite growth. These modifications may be deliberate, as through site-directed mutagenesis to a nucleic acid molecule encoding human contactin, or may be accidental, such as through mutation in hosts having DNA encoding these polypeptides.
  • Conservative substitutions include the substitution of an amino acid for another having a side chain off the alpha carbon of the same class, i.e., non-polar (hydrophobic), neutral, positively charged or negatively charged (hydrophilic) .
  • Variants of human contactin that retain the ability of contactin to bind tenascin-C, tenascin-R, Ng-CAM, or Nr-CAM/Bravo, or to promote neurite outgrowth include, for example, fragments of contactin. Such fragments can comprise the tenascin-R binding domain or the neurite promoting domain of contactin.
  • Such fragments can be recombinantly expressed by standard molecular methods and identified, for example, using a tenascin-R binding assay or the assay for neurite promoting activity described below.
  • Such functional fragments of the polypeptide sequence are also encompassed within minor variants of human contactin or soluble human contactin.
  • This invention is directed to isolated nucleic acid molecules that code on expression for a human contactin, a soluble human contactin or a minor variant of either of them. These molecules are useful for producing recombinant human contactin and for expression of human contactin in the gene therapy methods of this invention.
  • Nucleic acid molecules of this invention include those coding on expression for human contactin 1 having any signal sequence and comprising nucleotides 142 to 3135 of Figure 1 [SEQ ID NO:l] , or having the native signal sequence and comprising nucleotides 82 to 3135 of Figure 1 [SEQ ID NO:l] ; those coding on expression for contactin 2 having any signal sequence and comprising nucleotides 175 to 3135 of Figure 1 [SEQ ID N0:1]; or having the native signal sequence and comprising nucleotides 82 to 141 and 175 to 3135 of Figure 1 [SEQ ID NO:l] .
  • nucleic acid molecules of this invention include those coding on expression for soluble human contactin 1 comprising nucleotides 142 to 3057 of Figure 1 [SEQ ID NO:l] , or having the native signal sequence and comprising nucleotides 82 to 3057 of Figure 1 [SEQ ID N0:1] ; those coding on expression for human contactin 2 having any signal sequence and comprising nucleotides 175 to 3057 of Figure 1 [SEQ ID NO:l] ; or having the native signal sequence and comprising nucleotides 82 to 141 and 175 to 3057 of Figure 1 [SEQ ID N0:1] .
  • nucleic acid molecule includes both DNA and RNA molecules. It will be understood that when a nucleic acid molecule is described as having a DNA sequence, this also includes RNA molecules having the corresponding RNA sequence in which "U" replaces "T.”
  • nucleic acid molecules of this invention include those whose nucleotide sequence encodes a polypeptide directly, such as synthetic or cDNA, or those whose nucleotide sequence includes introns that are spliced out upon transcription into mRNA, such as genomic DNA. It also includes nucleic acid molecules having sequences which are degenerate versions of any of the aforementioned nucleotide sequences.
  • the nucleic acids of this invention can be produced by PCR on cDNA libraries from human brain cortex using primers based on the 5' and 3 ' regions of the human contactin nucleotide sequence of Figure 1 [SEQ ID N0:1] ; by probing cDNA libraries with probes derived from the nucleotide sequence of Figure 1 [SEQ ID N0:1] ; and by analyzing cDNA expression libraries with antibodies against human or mouse contactin.
  • Nucleic acid sequences encoding human contactin or allelic variants can be identified by probing cDNA or genomic libraries with the aforementioned probes using standard hybridization protocols. Standard hybridization protocols are described, for example, in Sambrook et al. and Ausubel et al.
  • nucleic acid molecules of this invention can also be made by organic synthesis on a commercial nucleic acid synthesizer. Other methods for identifying and isolating genes are also known and can be used.
  • nucleic acid probes comprising a reporter group and a nucleic acid molecule identical to or complementary to a unique sequence of human contactin of at least 15 nucleotides. These probes are useful in hybridization assays, such as Southern and Northern blots, for identifying nucleic acids having a nucleotide sequence of a human contactin gene.
  • Appropriate reporter groups for nucleic acid probes are well known in the art and include fluorescent, and radioactive and enzymatic labels.
  • This invention is further directed to expression vectors having an expression control sequence operatively linked to a nucleic acid sequence of this 12 invention.
  • An expression control sequence is operatively linked to a nucleic acid sequence when it directs the transcription and translation of that sequence in an appropriate host cell. This includes provision of appropriate start and stop codons.
  • Expression vectors useful in this invention depends on their intended use. Expression vectors must, of course, contain expression and replication signals compatible with the host cell. Expression vectors useful for expressing contactin include viral vectors, such as retroviruses, adenoviruses and adeno-associated viruses; plasmid vectors; cosmids; liposomes and the like. Viral and plasmid vectors are preferred for transfecting mammalian cells.
  • the expression vector pcDNAl in which the expression control sequence comprises the CMV promoter, provides good rates of transfection and expression.
  • Contactin- expressing clones are selectable upon co-transfection of a plasmid carrying a selectable marker gene, such as neomycin. Adeno-associated viral vectors are most useful in the therapeutic methods of this invention.
  • Appropriate expression control sequences for mammalian cells include, for example, the SV40 promoter, the RSV (Rous sarcoma virus) promoter and, especially, the CMV (cytomegalovirus) promoter.
  • This invention is further directed to unicellular hosts transfected with an expression vector of this invention.
  • Such hosts are useful for expressing large amounts of human contactin for purification and for transplantation.
  • the unicellular hosts can be prokaryotic or eukaryotic.
  • Cells such as E. coli , yeast, CHO and COS are especially useful for producing recombinant contactin.
  • Mammalian cells that can integrate into the nervous system environment, such as fibroblasts, glial cells and neuronal precursor cells, are especially useful in therapeutic methods of this invention.
  • B28 cells (Schubert et al., iVature, 249:224- 227 (1974) ) transfected with DNA encoding human contactin promote neurite growth, however, any cell line that can be produced as a bed and that does not normally produce contactin can be used.
  • a "recombinant" protein means a composition of substantially purified protein made by the expression of recombinant DNA encoding the protein. These proteins are useful for promoting neurite extension of neurons in vi tro and in vivo.
  • This invention is also directed to methods of making recombinant human contactin, recombinant soluble human contactin, or a recombinant minor variant of either of them by culturing a unicellular host of this invention.
  • the protein is then isolated from the culture by standard isolation techniques.
  • recombinant techniques allow the commercial production of pure proteins not possible by isolation from natural sources.
  • This invention provides methods useful for promoting neurite growth of neurons in culture.
  • the methods involve contacting the neuron with recombinant human contactin, recombinant soluble human contactin or recombinant minor variant of either of them.
  • compositions comprising recombinant human contactin, recombinant soluble human contactin or a recombinant minor variant of either of them in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier encompasses any of tie standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • compositions will, in general, contain an effective amount of the compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the individual.
  • This invention is also directed to therapeutic methods useful for promoting neuron regeneration in an individual.
  • the methods involve administering a therapeutically effective amount of a pharmaceutical composition of this invention to a person with neuron damage and, in particular, administering at the site of neuronal damage.
  • This method is especially useful for regeneration of cells located in an environment in which contactin is normally found, including, but not limited to, cells of the spinal cord, retina, cerebellum and cerebral cortex.
  • a pharmaceutical composition of this invention as described above is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound of the invention.
  • the pharmaceutical composition can be usefully administered at the site of neuron damage.
  • the particular dosage of pharmaceutical composition to be administered to the subject will depend on a variety of considerations including the nature of the disease, the severity thereof, the schedule of administration, the age and physical characteristics of the subject, and so forth. Proper dosages may be established using clinical approaches familiar to the medicinal arts.
  • This invention is also directed to methods of modulating neurite outgrowth by binding contactin with an effective amount of a neurite modulating contactin- binding ligand.
  • a “neurite modulating contactin-binding ligand” is a molecule which is capable of selectively binding contactin and which promotes or inhibits the neurite outgrowth function of contactin.
  • a ligand which selectively binds contactin includes ligands exhibiting selective binding to contactin and contactin-like molecules, but do not substantially bind other unrelated proteins.
  • An "effective amount" of a neurite modulating contactin-binding ligand is that amount which is sufficient to modulate neurite outgrowth. As discussed above, for therapeutic applications, proper dosages to be administered depend on a variety of considerations which are known to one skilled in the medicinal arts.
  • the modulation of neurite outgrowth by binding contactin with an effective amount of a neurite modulating contactin-binding ligand can be either positive or negative.
  • ligands which are negative modulators of neurite outgrowth can inhibit binding of natural ligands which act to promote neurite outgrowth.
  • Such ligands include, for example, antibodies, or other molecules, which inhibit the neurite promoting function of contactin.
  • ligands which are positive regulators of neurite outgrowth include, for example, those ligands which may inhibit the interaction of contactin with negative modulators. Examples of this class of contactin ligands include inhibitors of contactin binding to tenascin-R.
  • This invention is also directed to methods useful for promoting neuron regeneration in a person by providing the person at the site of neuronal damage with mammalian cells transfected with an expression vector of this invention.
  • the cells can be transfected outside the body and introduced.
  • the cells can be any cells that produce contactin recombinantly and that are immunologically compatible with the individual or that can be protected from rejection by, for example, micro- encapsulation.
  • the cells originate from the person being treated. Bjorklund, TINS, 14:319-322 (1991) describes considerations for neural transplantation.
  • the cells are human cells, and, in particular, fibroblasts, glial cells or neuronal precursor cells. Neuronal precursor cells are attractive in therapeutic methods because they can be grown in culture, transfected with the vector and re- introduced back into the individual, where they are integrated.
  • CMF-HBSS Hanks' balanced salt solution
  • the cells are dispersed by 10 minute incubation in 0.04% trypsin in CMF-HBSS, and, following the addition of DMEM and 10% fetal calf serum, gently triturated.
  • the cell suspension is placed onto coated tissue culture dishes at concentrations of 50,000 to 200,000 cells/cm 2 and incubated at 33°C. These cells are capable of proliferating and differentiating into neurons and glial cells.
  • This invention is also directed to methods of identifying contactin-binding ligands.
  • the method includes contacting human contactin, soluble human contactin or minor variant thereof such as a functional fragment of human contactin with a sample suspected of containing contactin-binding ligands and determining the ligands which specifically bind contactin.
  • a contactin-binding ligand is a molecule which can selectively bind contactin and contactin-like molecules, but does not substantially bind other unrelated proteins.
  • the ligands identified can be used in the methods described herein to modulate contactin-mediated neurite outgrowth.
  • Samples to be screened in this method include, for example, crude and partially purified protein extracts, proteins such as those produced by cDNA expression libraries, polypeptide fragments, organic compounds, and other small molecules. Screens for the determination of specific contactin- binding activity can take any of several forms and include, for example, both in vitro and cell-based assays.
  • biochemical protein-ligand binding assays are well known in the art, and are amenable to high through-put screening of protein or compound libraries.
  • affinity chromatography using human contactin of this invention coupled to a solid support can be used to determine binding of, for example, crude or partially purified protein extracts.
  • varying conditions such as ionic strength, for example, will discriminate selective from non-selective interactions.
  • Screens for the determination of contactin-binding ligands also include immunoassays, for example, utilizing anti-human contactin monoclonal antibodies such as CF3 (Berglund et al., Brain Res . , 549:292-296 (1991)) .
  • cDNA libraries are also well known in the art.
  • the screening of cDNA libraries with a labeled protein probe is one such molecular method which can be used to identify contactin binding ligands (see, for example, Sambrook et al. , Molecular Cloning -- A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) ) .
  • Appropriate cDNA expression libraries include, for example, those isolated from neuronal sources and are readily commercially available.
  • the probe for identification of human contactin ligands can be human recombinant contactin, soluble human contactin, or a functional fragment of contactin. Such probes can be recombinantly produced, for example, or isolated from natural sources.
  • a particularly useful functional fragment for the purposes of this method is one which retains the ability to bind known contactin ligands such as tenascin-R.
  • the conditions suitable for detecting selective protein-ligand interactions are well known in the art.
  • screens for human contactin ligands may be cell- or functionally-based. Assays utilizing the ability of contactin to modulate the neurite extension of a neuron in culture is one such example of a cell-based assay. Cell-based assays may be used as primary screens or may be used in conjunction with an in vi tro assay such as those described above as a secondary screen for contactin-binding ligands.
  • Two cDNAs encoding isoforms of human contactin were isolated and sequenced as follows.
  • a custom made adult human brain cortex Lambda ZAP cDNA library (Stratagene, La Jolla, CA, 1.6 x 10 G independent plaques) was screened with a 1 kb fragment (nucleotides 1141-2241) of chicken contactin cDNA (Ranscht, B., J " . Cell Biol . , 107:1561-1573 (1988) .
  • the probe was labeled with - 32 P-dCTP (Amersham Corp., Arlington Heights, IL) by nick-translation (GIBCO/Bethesda Research Laboratories, Gaithersberg, MD) and hybridized under low stringency conditions (5x SSPE, 37° C, 16 hours) to nylon replica filters (Hybond-N, Amersham Corp., Arlington Heights, IL) . Washes with increasing stringency identified one positive clone at the highest stringency (O.lx SSPE, 65°C) . This clone was characterized by partial sequencing and revealed a 1.5 kb insert with homology to chick and mouse contactin.
  • the isolated cDNA clone was used to screen both a Lambda gtll library from 21 week old human fetal brain (CLONTECH Laboratories Inc., Palo Alto, CA; 8 x 10 4 individual plaques) , and Lambda ZAP II cDNA library (Stratagene, La Jolla, CA; 9 x 10 5 individual plaques) from 17-18 week old human fetal brain for larger inserts.
  • Five clones were isolated from the Lambda ZAP II library and transformed into pBluescript plasmids by in vivo excision according to the manufacturer's instructions (Stratagene, La Jolla, CA) .
  • Sequencing templates were generated as nested deletions using ExoIII/Mung Bean nuclease (Stratagene, La Jolla, CA) . Both strands of each clone were sequenced using either T3, T7 or synthetic oligonucleotide primers. Protein and nucleotide sequences were analyzed and compared using Microgenie (Beckman Instruments Inc. ) or GeneWorks software (IntelliGenetics, Mountain View, CA) .
  • Clone 157A1 (h-contactin 2) contains a 5 kb insert with an open reading frame (ORF) that starts at the ATG translation initiation codon at nt 82 and ends with a stop codon at nt 3135.
  • Clone 11A1 (h-contactin 1) is 3.5 kb in length and lacks the translation initiation start codon and 21 nt of the ORF present at the 5' end of clone 157A1.
  • mRNAs from human fetal and adult brain were amplified by RT-PCR using oligonucleotides corresponding to nt 83-123 and 353-334 in Figure 1 [SEQ ID NO:l] as forward and reverse primers, respectively.
  • Two DNA fragments, a highly abundant fragment of 271 nt, and a low-abundance fragment of 238 nt were amplified.
  • the sequence of the larger fragment was identical to that of h-contactin 1 (11A1) , while that of the smaller fragment w ⁇ - identical to that of h- contactin 2 (157A) .
  • the NH 2 terminus of h-contactin 1 (clone 11A1) starts with glutamic acid (E) and is 100% identical to the NH 2 -terminal amino acid sequence of the purified Gpl35 glycoprotein isolated from human brain cortex (Berglund et al. , Eur. J. Biochem. , 197:549-554 (1991)) .
  • the putative h-contactin 2 (clone 157A) sequence is 33 nucleotides shorter than that of h- contactin 1 and predicts a protein with a shortened amino terminus in which valine (V) is the Nonterminal amino acid ( Figure 1) . No other sequence differences between the two clones were detected.
  • the ORFs of both h- contactin 1 and 2 cDNAs terminate with a hydrophobic region of 17 amino acids (underlined in Figure 1) .
  • human contactin can be divided into two main domains separated by a proline-glycine rich region.
  • the amino terminal portion contains 12 evenly spaced cysteines (boxed in Figure 1) surrounded by amino acids typical for Ig-domains (Hood et al., Cell , 40:225 (1985); (Williams, Immunol . Today, 8: 298-303 (1987) ; (Williams and Barclay, Ann. .Rev. Immunol . , 6:381-405 (1988) .
  • the average length of these domains is 76-84 amino acids and the conserved cysteines indicate folding of C2-type Ig-domains.
  • the carboxy-terminal half of the polypeptide contains four repeats of the FNIII-type with characteristic tryptophan and tyrosine residues (encircled in Figure 1; Kornblihtt et al., EMBO J. , 4:1755-1759 (1985) .
  • human contactin 1 and 2 are aligned, domain by domain, with chick contactin and mouse F3, to illustrate the homology between these proteins.
  • the sequence for chick contactin (Ranscht, J. Cell Biol . , 107:1561-1573 (1988) was reevaluated and revealed 100% identity with GPI-linked chick Fll (Br ⁇ mmendorf, Neuron, 2:1351-1361., (1989) . The corrected sequence is shown.
  • Human contactin shows 78% amino acid identity with chick contactin and 94% with mouse F3.
  • Blots were prehybridized for 1 hour in 1% BSA, 1 mM EDTA, 0.5 M NaHP04, pH 7.2 and 7% SDS at 65° C.
  • a 1 kb fragment of human contactin (nucleotides 1032-2112 [SEQ ID N0:1]) was labeled with ⁇ - 32 P-dCTP by random priming (Amersham, Corp., Arlington Heights, IL) and hybridized to the blot at 65° C for 18 hours.
  • telomere 157A1 eukaryotic expression vector pcDNAl/neo (Invitrogen, San Diego, CA) .
  • COS-7 cells were transfected with 12-15 ⁇ g DNA/ml medium using DEAE dextran and chloroquine (Ausubel et al. , supra . , 1993 supplement) . Transfection with the empty vector served as a control. COS-7 cells were maintained in DME medium containing 10% fetal calf serum (FCS) , glutamine, and antibiotics.
  • FCS fetal calf serum
  • h-contactin indicates a hydrophobic stretch of amino acids at the COOH-terminus, raising the possibility that human contactin is anchored to the membrane through a glycosyl phosphatidylinositol moiety (Ferguson and Williams, Annu. Rev. Biochem. ,
  • COS-7 cells were transfected with h-contactin 2 clone 157A, washed once with PBS and then treated with 0.4-0.75 U phosphatidylinositol specific phospholipase C (PI-PLC, Boehringer Mannheim Biochemicals, Indianapolis, IN) in buffer containing 50 mM TRIS, pH 7.6, 0.15 mM CaCl2, 1 mM PMSF, 50 ⁇ M leupeptin, 5 ⁇ M pepstatin, and 4 ⁇ g/ml aprotinin) at 37° C for 60 minutes.
  • PI-PLC phosphatidylinositol specific phospholipase C
  • the transfected cells expressed immuno- detectable h-contactin on the cell surface, while mock transfected cells were negative. After PI-PLC treatment, cell surface expression of h-contactin greatly diminished. Analysis of the PI-PLC-released proteins by Western blotting with polyclonal anti-human contactin antibodies revealed that contactin was released from the cell surface.
  • COS-7 cells were metabolically labeled with 3H-ethanolamine and cell-lysates immunoprecipitated with anti-human contactin mAb CF3. Equal numbers of contactin-transfected or control COS cells were grown in 5 cm dishes, washed twice with serum-free DMEM and metabolically labeled for 18 hours with 175 mCi [3H] -ethanolamine (1 mCi/ml, Amersham) containing DMEM with 10% dialyzed FCS.
  • the cells were washed in PBS and lysed on ice in 250 ml RIPA-buffer (0.15 M NaCl, 0.01 M phosphate buffer, pH 7.2, 1% Sodium deoxycholate, 1% NP40 and protease inhibitors as above) .
  • RIPA-buffer 0.15 M NaCl, 0.01 M phosphate buffer, pH 7.2, 1% Sodium deoxycholate, 1% NP40 and protease inhibitors as above
  • Monoclonal anti-human contactin antibody CF3 coupled to Protein G Sepharose (Pharmacia LKB, Uppsala, Sweden) (10 ⁇ g/ ⁇ l beads) was incubated with the lysate rotating overnight at 4° C.
  • the beads were washed 3 times, with RIPA buffer and once with PBS, and the bound proteins were dissociated from the beads by boiling in SDS-PAGE sample buffer.
  • Proteins were separated by SDS-PAGE on 7% reducing gels and detected with Coomassie Brilliant Blue. After destaining and incubation with Amplify (Amersham, Arlington Heights, IL) for 30 minutes, gels were dried and processed for fluorography. The fluorographs were exposed with Hyperfilm-MP (Amersham, Arlington Heights, IL) for 32 weeks.
  • the blots were probed with a probe representing h-contactin nucleotides 1302-1626 [SEQ ID NO:l] .
  • This probe was chosen because it hybridized under high stringency conditions to a single band of Taql digested human, but not hamster, genomic DNA.
  • the h-contactin probe was labeled by random priming and the filters were prehybridized and hybridized in 50% deionized formamide, x SSPE, 5% SDS, lx Denhardt solution and Salmon sperm DNA (100 ⁇ g/ml) at 46° C.
  • the filters were washed twice in 0.5x SSPE and 0.2% SDS at 62° C, for 10 minutes each, and then exposed for 170 hours to Hyper-MP film (Amersham) .
  • the hybridization signal was attributed to human chromosome 12 by discordance analysis according to the manufacturer' s instructions.
  • the chromosomes were first identified by G-banding with Giemsa (Seabright, M., Chromosoma, 36:204- 210 (1972) ) and photographed, then the same spreads were processed for in si tu hybridization with biotin-labeled human contactin DNA.
  • Giemsa-stained slides were destained in ethanol, methanol/acetic acid (3:1) and ethanol and then air-dried.
  • RNAse A Boehringer Mannheim Biochemicals, Indianapolis, IN
  • 2x SSC 3 M NaCl and 0.3 M Na-citrate, pH 7.0
  • probes representing either the complete 157A1 cDNA (5 kb) , the human contactin coding sequence (3.5 kb) or nt 1032-2112, were labeled with biotin-16-dUTP by Nick Translation (Boehringer Mannheim Biochemicals, Indianapolis, IN) .
  • the probes were mixed and diluted to a final concentration of 20 ng/ml (0.5 ⁇ g/slide) in hybridization buffer containing 10% dextran sulphate, 50% deionized formamide, 2x SSC, 0.05 M phosphate buffer, pH 7.0.
  • the samples were hybridized for 18 hours under sealed coverslips placed in a moist chamber at 37°C and washed at 40°C in 50% deionized formamide, 2x SSC, pH 7.0, followed by 2x SSC, pH 7.0 at RT.
  • Hybridized probes were detected with fluorescein-avidin (1/1000 in 5% BSA and 4x SSC, Vector Laboratories Inc, Burlingame, CA) .
  • fluorescein-avidin (1/1000 in 5% BSA and 4x SSC, Vector Laboratories Inc, Burlingame, CA) .
  • the slides were incubated with biotin-conjugated anti-avidin antibody (1/100 in 5% BSA, 4x SSC) , washed 4 times in 0.05% Tween-20/4x SSC, and reacted with fluorescein-avidin as above.
  • Chromosomes were counterstained with propidium-iodide (2 ⁇ g/ml) and examined under a Nikon Optiphot microscope (Nikon, Japan) with a lOOx plan objective or by confocal microscopy on a Zeiss LSM 10 instrument.
  • the specific hybridization signal for contactin was detected only on two chromosomes, on the q-arm close to the centromere.
  • the labeled chromosomes were identified as number 12, and by comparing 100 metaphases, the gene locus of h-contactin was attributed to position qll-ql2 (12qll-ql2) .
  • the human contactin gene locus maps close to the gene loci for several known human diseases.
  • the gene locus for progressive antnroophthalmopathy also called Stickler syndrome
  • Stickler syndrome an autosomal dominant hereditary disorder of connective tissue, can result from a mutation in the collagen type Ila gene located at 12ql3 or from another gene located in the same region.
  • the locus 12ql3 represents a breakpoint found in several malignant and benign prolierative disorders. Because recent studies have implicated cell adhesion molecules as tumor suppressors, the identification of the contactin gene locus close to this tumor breakpoint can indicate a potential role for contactin in controlling tumor formation or spreading.
  • nucleic acids encoding human contactin can be useful both as a probe in diagnostics and as well as useful in treating the various disorders in which contactin has been implicated to be involved.

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Abstract

Séquences nucléotidiques codant la contactine humaine, contactine humaine recombinée et procédés de production et d'utilisation de ces molécules pour stimuler la croissance de neurites et dans des thérapies en cas d'endommagement des neurones.
PCT/US1995/007408 1994-06-10 1995-06-09 Molecules d'acide nucleique codant la contactine humaine WO1995035373A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766922A (en) * 1995-05-26 1998-06-16 Sugen, Inc. Functional ligands for the axonal cell rcognition molecule contactin
WO2000070099A2 (fr) * 1999-05-19 2000-11-23 Mitokor Expression genique differente dans des regions specifiques du cerveau, dans des maladies neurodegeneratives
WO2001069261A2 (fr) * 2000-03-15 2001-09-20 Oxford Glycosciences (Uk) Ltd. Proteines, genes et leur utilisation dans le diagnostic et le traitement de la demence vasculaire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0618293A1 (fr) * 1993-03-26 1994-10-05 Becton, Dickinson and Company Contactine humaine, une molécule d'adhésion cellulaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0618293A1 (fr) * 1993-03-26 1994-10-05 Becton, Dickinson and Company Contactine humaine, une molécule d'adhésion cellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FASEB J., vol. 7, no. 7, 1993, page A1272, E.O. BERGLUND AND B. RANSCHT; 'Molecular cloning and chromosomal localization of human contactin' & joint meeting of the American Society for Biochemistry and Molecular Biology and the American Chemical Society, Division of Biological Chemistry, San Diego, Ca., USA. *
GENOMICS, vol. 21, 1994 pages 571-582, E.O. BERGLUND AND B. RANSCHT; 'Molecular cloning and in situ localization of the human contactin gene (CNTN1) on chromosome 12q11-q12' *
MOL. BRAIN RES., vol. 21, 1994 pages 1-8, R.A. REID ET AL.; 'Identification and characterization of the human cell adhesion molecule contactin' *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766922A (en) * 1995-05-26 1998-06-16 Sugen, Inc. Functional ligands for the axonal cell rcognition molecule contactin
WO2000070099A2 (fr) * 1999-05-19 2000-11-23 Mitokor Expression genique differente dans des regions specifiques du cerveau, dans des maladies neurodegeneratives
WO2000070099A3 (fr) * 1999-05-19 2002-04-04 Mitokor Expression genique differente dans des regions specifiques du cerveau, dans des maladies neurodegeneratives
WO2001069261A2 (fr) * 2000-03-15 2001-09-20 Oxford Glycosciences (Uk) Ltd. Proteines, genes et leur utilisation dans le diagnostic et le traitement de la demence vasculaire
WO2001069261A3 (fr) * 2000-03-15 2002-05-16 Oxford Glycosciences Uk Ltd Proteines, genes et leur utilisation dans le diagnostic et le traitement de la demence vasculaire

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AU2824195A (en) 1996-01-15

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