EP1060248A1 - Materiaux de lectomedines et procedes associes - Google Patents

Materiaux de lectomedines et procedes associes

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Publication number
EP1060248A1
EP1060248A1 EP99911080A EP99911080A EP1060248A1 EP 1060248 A1 EP1060248 A1 EP 1060248A1 EP 99911080 A EP99911080 A EP 99911080A EP 99911080 A EP99911080 A EP 99911080A EP 1060248 A1 EP1060248 A1 EP 1060248A1
Authority
EP
European Patent Office
Prior art keywords
lectomedin
polypeptide
polynucleotide
seq
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99911080A
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German (de)
English (en)
Inventor
Joel S. Hayflick
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Icos Corp
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Icos Corp
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Publication date
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Publication of EP1060248A1 publication Critical patent/EP1060248A1/fr
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Classifications

    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • GPCRs G-protein coupled receptors
  • GPCRs include receptors for opiates, adrenaline, histamine, polypeptide hormones, and photons, among other ligand s. These receptors are coupled to a wide variety of cellular second messenger pathways including, for example, pathways that alter intracellular calcium concentrations and cAMP levels.
  • CD97 appears to be representative of a sub-family of proteins which effect cellular adhesion [McKnight, et al., Immunol Today 77:283-287(1996)].
  • CD97 and related receptors are unique in that their structure includes a transmembrane domain that directly links a cytoplasmic domain that participates in GTP hydrolysis with extracellular protein binding domains that specifically participate in cell-cell adhesion.
  • the extracellular, amino terminal region of CD97 includes numerous cell-cell adhesive motifs, including multiple epidermal growth factor-like (EGF-like) repeats and an integrin binding site [Hamann J, et al., Immunol 155:1942-1950 (1996); Gray, et al., J.
  • CD97 include members of the integrin family of cell surface adhesion receptors.
  • Various integrins recognize and interact with their cognate ligands through a trimeric amino acid sequence of arginine-glycine-aspartic acid (denoted RGD in the single letter amino acid code) [D'Souza, etal, Trends Biochem. Sci., 16:246-250 (1991)] and this sequence has been identified in the extracellular region of CD97, between the EGF-like repeats and the transmembrane domain.
  • CD97 has been shown to undergo post-translational proteolytic processing which results in an extracellular (and potentially soluble) alpha subunit and a smaller, integral membrane beta subunit [Gray, et al, J. Immunol. 757:5438-5447 (1996)].
  • the two subunits are associated in a non-covalent manner and the alpha subunit is held at the cell surface through its interaction with the beta subunit.
  • the role of proteolysis is unclear, but it may be a mechanism for receptor down-regulation which is common among proteins, such as selectins and intercellular adhesion molecules (ICAMs), that participate in cell adhesion.
  • IAMs intercellular adhesion molecules
  • CD97 sub-family of GPCRs have been identified by amino acid sequence and structural homology and include human EMRl, HE6, BAI1, the calcium-independent receptor of latrotoxin (CIRL), latrophilin, and proteins encoded by the Caenorhabditis elegans open reading frames designated B0457.1 and B0286.2 [Baud N, et al, Genomics 2r5:334-344 (1995); McKnight, et al, J. Biol. Chem. 277:486-489 (1996); Krasnoperov, et al, Neuron 18:925-937 (1997); Lelianova, et al, J. Biol. Chem.
  • EMRl and its murine homolog F4/80, are macrophage-specific in expression and structurally related to CD97 in that they contain multiple extracellular EGF-like repeats, a rod-like stalk region, and the characteristic transmembrane domain of GPCRs [Baud V, et al, Genomics 25:334-344
  • CIRL has been shown to bind latrotoxin, a component of black widow spider venom, in the 0.5 to 1.0 nM range, and binding of the ligand to CIRL expressed in bovine chroma fin cells has been shown to result in exocytosis, a hallmark of toxin binding [Krasnoperov, et al, Neuron 75:925-937 (1997)].
  • Alpha latrotoxin binding has also been demonstrated at neuromuscular motor endplates, and this interaction elicits explosive secretory granule release of acetylcholine from presynaptic granules, resulting in muscle paralysis characteristic of the spider's bite [Petrenko, et al, F.E.B.S. Letts. 525:81-85 (1993)]. It is unclear, however, if the peripheral toxin effects result from binding to CIRL or some other related protein.
  • CD97-like receptors can permit identification and diagnosis of disease states which arise from aberrant signaling by the receptor, as well as disease states that arise from aberrant expression of the receptor itself.
  • the present invention provides purified and isolated human seven transmembrane receptor lectomedin polypeptides or fragments thereof, said polypeptides comprising extracellular lectin-binding, olfactomedin-like, and mucin-like domains.
  • Mature lectomedin polypeptides are also provided wherein signal or leader sequences are cleaved.
  • Preferred polypeptides of the invention comprise the amino acid sequence set out in SEQ ID NO: 2 or a fragment thereof, the amino acid sequence set out in SEQ ID NO: 4 or fragment thereof, the amino acid sequence set out in SEQ ID NO: 6 or fragment thereof, and the amino acid sequence set out in SEQ ID NO: 58 or fragment thereof.
  • the invention also provides polynucleotides encoding polypeptides of the invention. Preferred polynucleotides comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 57.
  • the invention also provide polynucleotides encoding a human lectomedin polypeptide selected from the group consisting of the polynucleotide set out in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
  • Preferred polynucleotides of the invention are DNA molecules.
  • Preferred DNA molecules are cDNA molecules and genomic DNA molecules.
  • the invention also provides DNA which is a wholly or partially chemically synthesized. Anti-sense polynucleotide which specifically hybridizes with a polynucleotide of the invention are also comprehended.
  • the invention also proved expression construct comprising the a polynucleotide of the invention, as well as host cells transformed or transfected with a polynucleotide or expression construct of the invention.
  • the invention also provides polynucleotide of the invention operably linked to a heterologous promoter, and host cells polynucleotides operably linked to a heterologous promoter.
  • the invention provides methods for producing a human lectomedin polypeptide comprising the steps of: a) growing the host cell of the invention under conditions appropriate for expression of the lectomedin polypeptide and b) isolating the lectomedin polypeptide from the host cell or the medium of its growth.
  • the invention also proved antibodies specifically immunoreactive with a polypeptide of the invention.
  • antibodies of the invention are monoclonal antibodies.
  • the invention also provides cells, e.g. hybridomas, that produce antibodies of the invention.
  • Anti-idiotype antibodies specifically immunoreactive with an antibody of the invention are also comprehended.
  • the invention also provides methods to identify a specific binding partner compound of a lectomedin polypeptide comprising the steps of: a) contacting the lectomedin polypeptide with a compound under conditions which permit binding between -5- the compound and the lectomedin polypeptide; b) detecting binding of the compound to the lectomedin polypeptide; and c) identifying the compound as a specific binding partner of the lectomedin polypeptide.
  • Methods of the invention embrace specific binding partner that modulate activity of the lectomedin polypeptide.
  • the compound inhibits activity of the lectomedin polypeptide, and in another aspect, the compound enhances activity of the lectomedin polypeptide.
  • the invention also provides methods to identify a specific binding partner compound of a lectomedin polynucleotide comprising the steps of: a) contacting the lectomedin polynucleotide with a compound under conditions which permit binding between the compound and the lectomedin polynucleotide; b) detecting binding of the compound to the lectomedin polynucleotide; and c) identifying the compound as a specific binding partner of the lectomedin polynucleotide.
  • Methods of the invention embrace specific binding partner that modulates expression of a lectomedin polypeptide encoded by the lectomedin polynucleotide.
  • the compound inhibits expression of the lectomedin polypeptide, and in another aspect, the compound enhances expression of the lectomedin polypeptide.
  • the invention also provides compounds identified by a method of the invention.
  • the invention comprehends composition comprising the compound identified by a method of the invention. and a pharmaceutically acceptable carrier.
  • the invention also provides use of a compound identified by a method of the invention for the preparation of a medicament to treat lectomedin related pathologies.
  • the invention also provides for use of a lectomedin polypeptide in the preparation of a medicament for the treatment of a lectomedin related disorder.
  • the present invention provides purified and isolated polypeptides and underlying polynucleotides for a novel family of transmembrane proteins designated lectomedins.
  • the invention includes both naturally occurring and non-naturally occurring lectomedin polynucleotides and polypeptide products thereof.
  • Naturally occurring lectomedin products include distinct gene and polypeptide species within the lectomedin family, including, for example, allelic variants, which are expressed within cells of the -6- same animal, as well as corresponding species homologs expressed in cells of other animals.
  • the invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts.
  • Non-naturally occurring lectomedin products include variants of the naturally occurring products such as analogs, fragments, fusion (or chimeric) proteins, and lectomedin products having covalent modifications.
  • the lectomedin family of proteins is distinguished from previously known seven transmembrane families of proteins in that the lectomedin proteins include at least one extracellular lectin binding-like domain and at least one extracellular olfactomedin domain. Unlike many other seven transmembrane proteins, the structure of proteins in the lectomedin family of proteins does not include EGF-like binding domains which effect cell/cell interactions.
  • the invention provides polynucleotides comprising the sequences set forth in SEQ ID NOs: 1, 3, 5 and 57.
  • the invention also embraces polynucleotides encoding the amino acid sequences set out in SEQ ID NOs: 2, 4, 6, and 58.
  • Presently preferred polypeptides of the invention comprises the amino acid sequences set out in SEQ ID NOs: 2, 4, 6, and 58.
  • the invention also provides expression constructs (or vectors) comprising polynucleotides of the invention, as well as host cells transformed, transfected, or electroporated to include a polynucleotide or expression construct of the invention. Methods to produce a polypeptide of the invention are also comprehended.
  • the invention further provides antibodies, preferably monoclonal antibodies, specifically immunoreactive with a polypeptide of the invention, as well as cell lines, e.g., hybridomas, that secrete the antibodies.
  • the present invention provides novel purified and isolated human polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the human lectomedins.
  • DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences.
  • Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and includes allelic variants of the preferred polynucleotides of the invention.
  • Genomic DNA of the invention is distinguishable from genomic DNAs encoding polypeptides other than lectomedin in that it includes the lectomedin coding region found in lectomedin cDNA of -7- the invention.
  • Genomic DNA of the invention can be transcribed into RNA, and the resulting RNA transcript may undergo one or more splicing events wherein one or more introns (i.e., non-coding regions) of the transcript are removed, or "spliced out.”
  • RNA transcripts that can be spliced by alternative mechanisms, and therefore be subjected to removal of different non-coding RNA sequences but still encode a lectomedin polypeptide are referred to in the art as splice variants, which are embraced by the invention.
  • Splice variants comprehended by the invention therefore, are encoded by the same DNA sequences but arise from distinct mRNA transcripts.
  • Allelic variants are known in the art to be modified forms of a wild type (predominant) gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are inherently naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).
  • the invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding lectomedin, followed by second strand synthesis of a complementary strand to provide a double stranded DNA.
  • DNA were produced by chemical means.
  • DNA sequences encoding human lectomedin polypeptides are set out in SEQ ID NOs: 1, 3, 5, and 57.
  • preferred DNAs of the invention comprise double stranded molecules, for example, the molecule having the sequence set forth in either SEQ ID NOs: 1, 3, 5, or 57, along with the complementary molecule (the "non-coding strand” or "complement") having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson- Crick base pairing rules for DNA.
  • the invention further embraces species, preferably mammalian, homologs of the human lectomedin DNA. Species homologs, in general, share at least 35%, at least -8-
  • Percent sequence "homology" with respect to polynucleotides of the invention is defined herein as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the lectomedin coding sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • polynucleotide sequence information makes possible large scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
  • Polynucleotides also permit identification and isolation of polynucleotides encoding related lectomedin polypeptides by well known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR), ligase chain reaction, as well as other amplification techniques.
  • PCR polymerase chain reaction
  • ligase chain reaction as well as other amplification techniques.
  • related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to lectomedins and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of lectomedin.
  • full length polynucleotides encoding lectomedin polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotides.
  • the invention therefore provides fragments of lectomedin coding polynucleotides. Such fragments comprise at least 10 to 20, and preferably at least 15, consecutive nucleotides of the polynucleotide.
  • the invention comprehends, however, fragments of various lengths.
  • fragment polynucleotides of the invention comprise sequences unique to the lectomedin coding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding lectomedin, or lectomedin fragments thereof containing the unique sequence.
  • Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full length sequence derived from introns, regulatory regions, and/or other non-translated sequences.
  • Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other -9- known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases.
  • the invention also provides fragment polynucleotides that are conserved in one or more polynucleotides encoding members of the lectomedin family of polypeptides.
  • Such fragments include sequences characteristic of the family of lectomedin polynucleotides, and are also referred to as "signature sequences.”
  • the conserved signature sequences are readily discernable following simple sequence comparison of polynucleotides encoding members of the lectomedin family. Fragments of the invention can be labeled in a manner that permits their detection, including radioactive and non- radioactive labeling.
  • Fragment polynucleotides are particularly useful as probes for detection of full length or other fragment lectomedin coding polynucleotides.
  • One or more fragment polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding lectomedin, or used to detect variations in a polynucleotide sequence encoding lectomedin.
  • the invention also embraces DNA sequences encoding lectomedin species which hybridize under moderately or highly stringent conditions to the non-coding strand, or complement, of the polynucleotide in SEQ ID NOs: 1, 3, 5, or 57.
  • DNA sequences encoding lectomedin polypeptides which would hybridize thereto but for the redundancy of the genetic code are further comprehended by the invention.
  • Exemplary highly stringent conditions are include hybridization at 45 °C in 5X SSPE and 45% formamide, and a final wash at 65 °C in 0. IX SSC.
  • Exemplary moderately stringent condition include a final wash at 55°C in IX SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology. John
  • hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook, et al, (Eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51. -10-
  • Expression constructs such as plasmid and viral DNA vectors incorporating lectomedin coding sequences are also provided.
  • Expression constructs wherein lectomedin-encoding polynucleotides are operably linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.
  • Expression control DNA sequences include promoters, enhancers, and operator, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
  • Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.
  • Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell.
  • Preferred constructs of the invention also include sequences necessary for replication in a host cell.
  • Expression constructs are preferably utilized for production of an encoded lectomedin protein, but may also be utilized to amplify the construct itself.
  • host cells including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention in a manner which permits expression of the encoded lectomedin polypeptide.
  • Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.
  • Methods for introducing DNA into the host cell well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
  • Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, and mammalian cells systems.
  • Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with lectomedin.
  • Host cells of the invention are also useful in methods for large scale production of lectomedin polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by purification methods known in the art, e.g., conventional chromatographic methods -11- including immunoafi_nity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC and the like.
  • HPLC high pressure liquid chromatography
  • Still other methods of purification include those wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.
  • the purified protein can be cleaved to yield the desired protein, or be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
  • Knowledge of lectomedin coding DNA sequences allows for modification of cells to permit, or increase, expression of endogenous lectomedin.
  • Cells can be modified (e.g., by homologous recombination) to provide increased lectomedin expression by replacing, in whole or in part, the naturally occurring lectomedin promoter with all or part of a heterologous promoter so that the cells express lectomedin at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operably linked to lectomedin- encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the lectomedin coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the lectomedin coding sequences in the cells.
  • the DNA sequence information provided by the present invention also makes possible the development through, e.g.
  • the invention also provides purified and isolated mammalian lectomedin polypeptides encoded by a polynucleotide of the invention. Presently preferred are -12- lectomedin polypeptides comprising the amino acid sequence set out in SEQ ID NO: 2, 4, 6, or 58.
  • the invention also embraces lectomedin polypeptides encoded by a DNA selected from the group consisting of : a) the DNA sequence set out in SEQ ID NO: 1, 3, 5 or 57; b) a DNA molecule which hybridizes under high stringent conditions to the noncoding strand of the protein coding portion of (a); and c) a DNA molecule that would hybridize to the DNA of (a) but for the degeneracy of the genetic code.
  • the invention also embraces variant (or analog) lectomedin polypeptides.
  • insertion variants are provided wherein one or more amino acid residues supplement a lectomedin amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the lectomedin amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels.
  • the invention provides deletion variants wherein one or more amino acid residues in a lectomedin polypeptide are removed.
  • Deletions can be effected at one or both termini of the lectomedin polypeptide, or with removal of one or more residues within the lectomedin amino acid sequence.
  • Deletion variants therefore, include all fragments of a lectomedin polypeptide.
  • the invention provides substitution variants of lectomedin polypeptides.
  • Substitution variants include those polypeptides wherein one or more amino acid residues of a lectomedin polypeptide are removed and replaced with alternative residues.
  • the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables A, B, or C below.
  • the invention also provides derivatives of lectomedin polypeptides.
  • Derivatives include lectomedin polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.
  • the modifications are covalent in nature, and include, for example, chemical bonding with polymers, lipids, non-naturally occurring amino acids, other organic, and inorganic moieties.
  • Derivatives of the invention may be prepared to increase circulating half-life of a lectomedin polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs. -13-
  • the invention also embraces polypeptides have at least 99%,at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention.
  • Percent amino acid sequence "identity" with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the lectomedin sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Percent sequence "homology" with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the lectomedin sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.
  • percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment [Dayhoff, in Altas of Protein Sequence and Structure. Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].
  • Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated form of lectomedin polypeptides are embraced.
  • Insertion variants include lectomedin polypeptides wherein one or more amino acid residues are added to a lectomedin acid sequence, or fragment thereof.
  • Variant products of the invention also include mature lectomedin products, i.e., lectomedin products wherein leader or signal sequences are removed, with additional -14- amino terminal residues.
  • the additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from a specific proteins.
  • Lectomedin products with an additional methionine residue at position -1 are contemplated, as are lectomedin products with additional methionine and lysine residues at positions -2 and -1
  • the invention also embraces lectomedin variants having additional amino acid residues which result from use of specific expression systems.
  • use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide.
  • GST glutathione-S-transferase
  • Insertional variants also include fusion proteins wherein the amino and/or carboxy termini of the lectomedin polypeptide is fused to another polypeptide.
  • fusion proteins are immunogenic polypeptides, proteins with long circulating half life, such as immunoglobulin constant regions, marker proteins (e.g., fluorescent) and proteins or polypeptide that facilitate purification of the desired lectomedin polypeptide,
  • ® e.g. FLAG tags or polyhistidine sequences.
  • Deletion variants include lectomedin polypeptides wherein one or more amino acid residues are deleted from the lectomedin amino acid sequence.
  • Deletion variants of the invention embrace polypeptide fragments of the sequence set out in SEQ ID NO: 2, 4, 6, or 58 wherein the fragments maintain biological or immunological properties of a lectomedin polypeptide. Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ED NO: 2, 4, 6, or 58 are comprehended by the invention.
  • Preferred polypeptide fragments display antigenic properties unique to or specific for the lectomedin family of polypeptides. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art. -15-
  • Substitution variants of the invention include lectomedin polypeptides, or fragments thereof, wherein one or more amino acid residues in the lectomedin amino acid sequence are deleted and replaced with another amino acid residue.
  • Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A (from WO 97/09433, page 10, published March 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below.
  • the invention further embraces lectomedin products, or fragments thereof, covalently modified or derivatized, e.g., to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • lectomedin products covalently modified with polyethylene glycol (PEG) subunits.
  • PEG polyethylene glycol
  • Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the lectomedin products, or randomly attached to one or more side chains of the polypeptide.
  • Additional derivatives include lectomedin species immobilized on a solid support, pin microparticle, or chromatographic resin, as well as lectomedin polypeptides modified to include one or more non-protein labels, tags, or chelating agents. -17-
  • antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and
  • CDR-grafted antibodies including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) and other binding proteins specific for lectomedin products or fragments thereof.
  • Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in
  • Antibody fragments including Fab, Fab', F(ab') 2 , and F w are also provided by the invention.
  • the term "specific for” indicates that the variable regions of the antibodies of the invention recognize and bind lectomedin polypeptides exclusively (i.e., able to distinguish single lectomedin polypeptides from the family of lectomedin polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S.
  • aureus protein A or other antibodies in ELISA techniques through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule.
  • Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies: A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 6.
  • Antibodies that recognize and bind fragments of the lectomedin polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, lectomedin polypeptides.
  • antibodies of the invention that recognize lectomedin fragments are those which can distinguish single and distinct lectomedin polypeptides from the family of lectomedin polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • Non-human antibodies may be humanized by any methods known in the art.
  • the non-human complementarity determining regions are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
  • Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of lectomedin), diagnostic purposes to detect or quantitate lectomedin, as well as purification of lectomedin. Antibodies are particularly useful for detecting and/or quantitating lectomedin expression in cells, tissues, organs and lysates and extracts thereof, as well as fluids, including serum, plasma, cerebrospinal fluind, urine, sputum, peritoneal fluid, pleural fluid, or pulmonary lavage. Kits comprising -19- an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.
  • Specific binding proteins can be identified or developed using isolated or recombinant lectomedin products, lectomedin variants, or cells expressing such products.
  • Binding proteins are useful for purifying lectomedin products and detection or quantification of lectomedin products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of lectomedin, especially those activities involved in signal transduction.
  • DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of lectomedins.
  • DNA and amino acid sequence information for lectomedin also permits identification of binding partner compounds with which a lectomedin polypeptide or polynucleotide will interact.
  • Agents that modulate (i.e., increase, decrease, or block) lectomedin activity or expression may be identified by incubating a putative modulator with a lectomedin polypeptide or polynucleotide and determining the effect of the putative modulator on lectomedin activity or expression.
  • the selectivity of a compound that modulates the activity of the lectomedin can be evaluated by comparing its binding activity to one particular lectomedin to its activity to other lectomedin polypeptides.
  • Cell based methods such as di-hybrid assays to identify DNAs encoding binding compounds and split hybrid assays to identify inhibitors of lectomedin polypeptide interaction with a known binding polypeptide, as well as in vitro methods, including assays wherein a lectomedin polypeptide, lectomedin polynucleotide, or a binding partner are immobilized, and solution assays are contemplated by the invention.
  • Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to a lectomedin polypeptide or a lectomedin-encoding nucleic acid, oligonucleotides which specifically bind to a lectomedin polypeptide or a lectomedin gene sequence, and other non-peptide compounds (e.g., isolated or synthetic organic and inorganic molecules) which specifically react with a lectomedin polypeptide or its underlying nucleic acid.
  • Mutant lectomedin polypeptides -20- which affect the enzymatic activity or cellular localization of the wild-type lectomedin polypeptides are also contemplated by the invention.
  • Presently preferred targets for the development of selective modulators include, for example: (1) regions of the lectomedin polypeptide which contact other proteins, (2) regions that localize the lectomedin polypeptide within a cell, (3) regions of the lectomedin polypeptide which bind substrate,
  • lectomedin polypeptide (4) allosteric regulatory binding site(s) of the lectomedin polypeptide, (5) site(s) of the lectomedin polypeptide wherein covalent modification regulates biological activity and (6) regions of the lectomedin polypeptide which are involved in multimerization of lectomedin subunits.
  • selective modulators include those that recognize specific lectomedin encoding and regulatory polynucleotide sequences. Modulators of lectomedin activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which lectomedin activity is known or suspected to be involved.
  • Lectomedin polypeptides of the invention are amenable to numerous cell based high throughput screening (HTS) assays known in the art, including melanophore assay to investigate receptor-ligand interaction, yeast based assay systems, and mammalian cell expression systems.
  • HTS high throughput screening
  • Chemical libraries consist of structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.
  • Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non- ribosomal peptides, and variants (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large -21- numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
  • DNA and amino acid sequences of the present invention are manifest.
  • knowledge of the sequence of a cDNA for lectomedin makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding lectomedin and lectomedin expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.
  • DNA/DNA hybridization procedures carried out with DNA sequences of the invention under moderately to highly stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of lectomedin; allelic variants are known in the art to include structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to lectomedin.
  • non-human species genes encoding proteins homologous to human lectomedin can also be identified by Southern and/or PCR analysis; species homologs of the invention are particularly useful in animal models for the study of lectomedin-related disorders.
  • complementation studies can be useful for identifying other human lectomedin products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of lectomedin.
  • Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express lectomedin. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a lectomedin locus that underlies a disease state or states. -22-
  • anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding lectomedin. Full length and fragment anti-sense polynucleotides are provided. The worker of ordinary skill will appreciate that fragment antisense molecules of the invention include (i) those which specifically recognize and hybridize to lectomedin RNA (as determined by sequence comparison of
  • Antisense polynucleotides that hybridize to RNA encoding other members of the lectomedin family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of lectomedin by those cells expressing lectomedin mRNA.
  • Antisense nucleic acids preferably 10 to 20 base pair oligonucleotides capable of specifically binding to lectomedin expression control sequences or lectomedin RNA are introduced into cells (e.g. , by a viral vector or colloidal dispersion system such as a liposome).
  • the antisense nucleic acid binds to the lectomedin target nucleotide sequence in the cell and prevents transcription or translation of the target sequence.
  • Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.
  • the antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.
  • the invention further contemplates methods to modulate lectomedin expression through use of ribozymes.
  • Ribozyme technology can be utilized to inhibit translation of lectomedin mRNA in a sequence specific manner through (i) the hybridization of a complementary RNA to a target mRNA and (ii) cleavage of the hybridized mRNA through nuclease activity inherent to the complementary strand.
  • Ribozymes can identified by empirical methods but more preferably are specifically designed based on accessible sites on the target mRNA (Bramlage, et al, Trends in Biotech 76:434-438 (1998).
  • ribozymes to target cells can be accomplished using either exogenous or endogenous delivery techniques well known and routinely practiced in the art.
  • Exogenous -23- delivery methods can include use of targeting liposomes or direct local injection.
  • Endogenous methods include use of viral vectors and non-viral plasmids.
  • Ribozymes can specifically modulate expression of lectomedin when designed to be complementary to regions unique to a polynucleotide encoding lectomedin. "Specifically modulate” therefore is intended to mean that ribozymes of the invention recognizes only a polynucleotide encoding lectomedin. Similarly, ribozymes can be designed to modulate expression of all or some of the lectomedin family of proteins. Ribozymes of this type are designed to recognize polynucleotide sequences conserved in all or some of the polynucleotides which encode the family of proteins. The invention further embraces methods to modulate transcription of lectomedin through use of oligonucleotide-directed triplet helix formation.
  • Triplet helix formation is accomplished using sequence specific oligonucleotides which hybridize to double stranded DNA in the major groove as defined in the Watson-Crick model. Hybridization of a sequence specific oligonucleotide can thereafter modulate activity of DNA-binding proteins, including, for example, transcription factors and polymerases.
  • Preferred target sequences for hybridization include promoter and enhancer regions to permit transcriptional regulation of lectomedin expression.
  • Oligonucleotides which are capable of triplet helix formation are also useful for site-specific covalent modification of target DNA sequences. Oligonucleotides useful for covalent modification are coupled to various DNA damaging agents as described in Lavrovsky, et al. [supra].
  • a functional lectomedin gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g. , adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g. , liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no.
  • the invention further embraces pharmaceutical compositions comprising a lectomedin polypeptide of the invention, generally in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions optionally may include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used.
  • Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
  • the pharmaceutical compositions can be packaged in forms convenient for delivery.
  • the compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the immunogenic composition into the recipient organism and, particularly, when the immunogenic composition is being delivered in unit dose form.
  • the dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
  • compositions may be introduced into the subject to be treated by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drug solutions) or subcutaneous injection (including depot administration for long term release) ; by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • lectomedin product compositions are generally injected in doses ranging from 1 ⁇ g/kg to 100 mg/kg per day, preferably at doses -25- ranging from 0.1 mg/kg to 50 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kg/day.
  • the lectomedin product composition may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product.
  • Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
  • the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area or organ size.
  • the pharmaceutical compositions and treatment methods of the invention may be useful in the fields of human medicine and veterinary medicine.
  • the subject to be treated may be a mammal, preferably human, or other -26- animals.
  • subjects include, for example, farm animals including cows, sheep, pigs, horses, and goats, companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks and geese.
  • Example 1 describes identification and characterization of cDNA encoding lectomedin polypeptides.
  • Example 2 relates to expression of recombinant lectomedin.
  • Example 3 described characterization of recombinant lectomedin. Ligand affinity chromatography with immobilized lectomedin is described in Example 4.
  • Example 5 describes Northern analysis of lectomedin expression.
  • Example 5 provides an assessment of tissue distribution of lectomedin in mammalian cell types, while Example 6 describes results from in situ hybridization. The chromosome localization of lectomedin is disclosed in Example 7.
  • Example 8 provides production of polyclonal and monoclonal antibodies specific for lectomedin.
  • Example 9 addresses lectomedin expression.
  • CD97-related sequences identified as corresponding to known proteins were ESTs representing CD97, human EMRl, and murine F4/80.
  • ESTs representing CD97, human EMRl, and murine F4/80.
  • several ESTs showed statistically significant values for relatedness in the transmembrane region of CD97, but they were not CD97, EMRl, or F4/80.
  • One of the identified ESTs One of the identified ESTs,
  • T47902 GenBank Accession No: 13
  • GenBank that the EST was derived from a human fetal spleen library.
  • a library probe was first generated by PCR based on the Genbank sequence of T47902. Primers used to amplify a T47902 sequence are set out in SEQ ID NOs: 14 and 15 below.
  • cDNA Mixed human adult spleen cDNA was prepared by standard methods and ligated into vector pcDNAl.amp (Invitrogen) [Van der Vieren, M. et al. Immunity 5:683- 690 (1995)]. The resulting plasmid mixture was transformed into E. coli and the bacteria were plated onto LB bacterial plates containing carbenicillin (100 ug/ml). Surviving colonies were collected by scraping and plasmid DNA was prepared by the alkaline lysis method. The cDNA mixture was used as a template for PCR in a reaction mixture including 350 ng cDNA, 100 ng each primer (SEQ ID NO. 14 and NO.
  • PCR was carried out with an initial denaturation step of seven minutes at 95 °C followed by 30 cycles of denaturation for one minute at
  • the resulting plasmid preparation was transformed into E. coli -28- and the cells plated as described above. Several colonies were selected for DNA minipreps and the cDNA inserts were sequenced.
  • the 306 bp insert was excised from the pCR2.1 TA Cloning vector by digestion with EcoRI. Following digestion, DNA fragments were fractionated on an agarose gel. A band of approximately 306 bp was eluted from the gel and labeled by random priming according to standard procedures. The labeled probe was used to screen the human spleen adult cDNA pcDNAl library (described above) immobilized on nylon membranes following colony lifts from cells spread on LB/carbenicillin plates.
  • cDNA clones Two cDNA clones, designated 3.3 and 15.3.1, were obtained and purified.
  • Clone 3.3 included an insert of approximately 4200 bp and clone 15.3.1 contained an insert of approximately 2750 bp. Both clones were sequenced by standard automated methods.
  • the nucleotide sequence for clone 3.3 is set out in S ⁇ Q ID NO: 9 and the predicted amino acid encoded is set out in S ⁇ Q ID NO: 10.
  • the nucleotide sequence for clone 15.3.1 is set out in S ⁇ Q ID NO: 16. It was initially presumed that the two clones represented a single cDNA sequence and, relying predominantly on the larger 3.3 insert, a contiguous cDNA was predicted.
  • PCR was carried out with an initial denaturation step for two minutes at 94 °C, followed by (i) five cycles of 0.05 minutes at 94 °C and seven minutes at 74 °C, (ii) five cycles of 0.05 minutes at 94°C and seven minutes at 72°C, and (iii) 25 cycles of 0.05 minutes at 94 °C and seven minutes at 70 °C.
  • reaction mixture was diluted 1 :50, and 5 ⁇ l was used in a second amplification reaction including 100 ng upstream internal gene specific primer and adapter primer AP2 (MarathonTM cDNA kit, Clontech) with the same cycling conditions as in the first amplification.
  • Amplification products from the second reaction were separated on a 0.9% agarose gel and a band of approximately 2 kb in the gel was eluted for subcloning into vector
  • the isolated fragment was designated RACE3.3.
  • the nucleotide and predicted amino acid sequences for the fragment are set out in SEQ ID NOs: 7 and 8, respectively.
  • the putative extracellular domain in the predicted amino acid sequence did not include EGF domains characteristic of the CD97-like protein family and the amino acid sequence of the transmembrane domain in the predicted protein was only about 45-60% identical to the transmembrane domains of CD97.
  • the predicted amino acid sequence deduced from the combined RACE3.3 and clone 3.3 open reading frame included potential lectin-binding, olfactomedin-like, and mucin-like extracellular domains not found in CD97. Based on the presence of the extracellular lectin binding-like and olfactomedin- like domains, the polypeptide encoded by the RACE3.3 and clone 3.3 sequences was designated lectomedin- l ⁇ .
  • Lectomedin- l ⁇ also includes a sequence at amino acid residues 809 to 814 (in SEQ ID NO: 2) which is similar to a proposed cleavage site conserved in both CD97 and latrophilin. It is possible that these three proteins are processed by an endoprotease (or related proteases) with similar primary sequence specificity.
  • lectomedin- l ⁇ may be related to a human homolog of the rat latrophilin and may participate in stimulation/secretion coupling in presynaptic termini and/or secretory granule release. Expression of lectomedin in cell and tissue types outside the central nervous system (discussed below), however, indicates that lectomedin is functionally distinct from latrophilin.
  • the overall amino acid sequence of lectomedin- l ⁇ was found to be approximately 80% identical to that of latrophilin, but the amino acid sequence of the latrophilin cytoplasmic domain was unrelated to the predicted cytoplasmic domain of lectomedin- l ⁇ .
  • the location of the initiating methionine in latrophilin differed from that in the predicted open reading frame of lectomedin- 1 ⁇ .
  • a methionine codon in a different reading frame was identified upstream from the originally predicted open reading frame.
  • the location of the upstream methionine codon (with respect to the transmembrane region) more closely corresponded to the position of the latrophilin initiating methionine and the first few amino acids in reading frame with the upstream methionine codon also corresponded to the sequence in latrophilin.
  • the polynucleotide sequence encoding the 1114 amino acid lectomedin- l ⁇ open reading frame was again compared to the raw data obtained during automated sequencing of the RACE3.3 cDNA. Further inspection showed that a guanosine nucleotide at position 454 had been entered in SEQ ID NO: 33, but was not present in the raw sequence data.
  • the corrected nucleotide sequence for RACE3.3 (i.e., having the extraneous guanosine nucleotide deleted) together with the sequence of clone 3.3 (SEQ ID NO: 9) showed an open reading -31- frame encoding 1177 amino acids.
  • the corrected open reading frame began with the newly identified initiating methionine and included the previously identified lectin bindinglike, olfactomedin-like, mucin-like, transmembrane and cytoplasmic domains of lectomedin- l ⁇ (SEQ ID NO: 1). Based on sequence homology with known proteins, domains in various regions of the lectomedin- l ⁇ protein were identified.
  • lectomedin- l ⁇ contained a region from amino acids 354 to 563 (SEQ ID NO: 2) with many serine and threonine residues (which may be O-glycosylated), as well as many proline residues (which break up alpha helices resulting in an extended structure with many beta turns), a mucin- like domain was identified.
  • the sequence for lectomedin- l ⁇ was based on the sequences determined for clone 3.3 and the fragment RACE3.3.
  • a second lectomedin cDNA could also be deduced based on the sequence of the second spleen clone 15.3.1.
  • the sequences for clones 3.3 and 15.3.1 it was first noted that the clones were substantially identical throughout both 5' regions, except that an adenosine required at position 1620 of clone 3.3 (SEQ ID NO : 7) was apparently not present in clone 15.3.1.
  • clone 15.3.1 did not encode a protein having the characteristic seven transmembrane domain found in lectomedin- 1 ⁇ .
  • the variant adenosine was inserted into the sequence for clone 15.3.1, the predicted protein sequence was consistent with the -32- lectomedin-l ⁇ amino acid sequence up to the first amino acid residue in the cytoplasmic domain.
  • This sequence suggested an alternative splice site in the cytoplasmic region of clone 15.3.1 that produced a shorter cDNA comprising a cytoplasmic domain of approximately forty-eight amino acids (as compared to 107 amino acids in the cytoplasmic domain of the lectomedin- l ⁇ cDNA derived solely from clone 3.3 sequences).
  • the lectomedin- l ⁇ cDNA deduced from clone 3.3 also terminated at an alternative poly(A + ) site 210 nucleotides upstream from the corresponding poly(A + ) site identified in clone 15.3.1.
  • the differences suggested that clone 15.3.1 represents a second member of the lectomedin family, which was designated lectomedin-l ⁇ .
  • a deduced polynucleotide sequence for lectomedin- 1 ⁇ was therefore generated using the overlapping sequence from clone 3.3 (which extended the 5' region of clone 15.3.1) and the RACE3.3 sequence (to provide an appropriate 5 ' end); the complete predicted cDNA and deduced amino acid sequences for lectomedin-l ⁇ are set out in SEQ ID NOs: 3 and 4, respectively.
  • Characterization of the predicted amino acid sequence for lectomedin-l ⁇ provides various domains similar (in both sequence and position) to those identified for lectomedin- l ⁇ .
  • An extracellular region of approximately 831 amino acids is predicted, including a D-galactoside-binding lectin-like domain (amino acids 36 to 131 of SEQ ID NO: 4), an olfactomedin-like domain (amino acids 135 to 327 of SEQ ID NO: 4), and a mucin-like domain (amino acids 354 to 563 of SEQ ID NO: 4).
  • a seven transmembrane domain (amino acids 832 to 1075 of SEQ ID NO: 4) was located adjacent the extracellular domain, and a cytoplasmic region of 48 amino acids (residues 1076 to 1123 of SEQ ID NO: 4) was located adjacent the transmembrane region.
  • lectomedin-l ⁇ is predicted to include (i) an extracellular region of approximately 831 amino acids with a D-galactoside binding lectin-like domain (amino acids 36 to 131 of SEQ ID NO: 6), an olfactomedin-like domain (amino acids 135 to 327 of SEQ ID NO: 6) and a mucin-like domain (amino acids
  • Lectomedin l and rat latrophilin were used as query sequences in a subsequent BLAST search in an attempt to identify any additional ESTs with sequence homology.
  • Two human ESTs designated AA683020 (SEQ ID NO: 22) and M79057 (SEQ ID NO: 23) were identified as being closely related to both lectomedin-l ⁇ and rat latrophilin.
  • the sequence for EST AA683020 corresponds to the region from nucleotide 3275 to 3643 in the lectomedin-l ⁇ sequence (SEQ ID NO: 22) and the sequence for EST M79057 corresponds to nucleotides 2561 to 2842 in lectomedin-l ⁇ .
  • BLAST search results indicated that both ESTs were more closely related to the sequence encoding rat latrophilin than to the nucleotide sequence encoding lectomedin- l ⁇ , further distinguishing lectomedin-l ⁇ from the rat protein and suggesting that the human ESTs may be more closely related to a putative human homolog of rat latrophilin.
  • the AA683020 and M79057 ESTs were determined to represent unique lectomedin-2 and lectomedin-3 species.
  • primers were designed based on the EST sequences for both lectomedin-2 and lectomedin-3 to amplify probes for library screening.
  • Primers for amplifying a lectomedin-2 sequence were NHlect2.5 (SEQ ID NO: 35) and NHlect2.3 (SEQ ID NO: 36), and primers for the lectomedin-3 sequence were Nhlct.5 (SEQ ID NO: 37) and NHlct.3 (SEQ ID NO: 38).
  • PCR was carried out using a Clontech human brain Marathon-ReadyTM cDNA library as template. Reaction conditions included an initial incubation at 94 °C for five minutes, followed by 25 cycles at 94 °C for 30 seconds, 55 °C for 30 seconds, and
  • ® ® amplification products were separately cloned into vector pCRII with a TA Cloning kit (Invitrogen), and sequencing was carried out to identify errors associated with PCR.
  • Probes for cDNA library screening were prepared by purifying EcoRI
  • the lectomedin-2 digestion products provided two -35- fragments, 274 and 158 bp, and the 274 bp fragment was purified.
  • the lectomedin-3 digestion resulted in a 297 bp EcoRI fragment.
  • a human fetal brain cDNA library in the LAMBDA ZAP II vector was purchased from Stratagene (La Jolla, CA). Approximately 50,000 pfu were plated on twenty 150 mm LBM agar plates with L ⁇ 392 E. coli. Plates were inverted and incubated at 37 °C overnight. The next day, the plates were chilled at 4°C for two hours before
  • the plates were screened a second time as described above using 82 mm
  • Positive plaques from the second screening were screened for a third time to ensure that only positive plaques were picked for the phage rescue.
  • ® Positive plaques were prepared using an ⁇ xassist /SOLR phage rescue system (Stratagene, La Jolla, CA) according to the manufacturer's suggested protocol.
  • the rescue procedure produced E. coli colonies containing the DNA of interest cloned
  • Plasmid DNA was
  • the resulting purified clones were analyzed by DNA sequencing at both the 5 ' and 3 ' ends. Of the positive clones identified by the probe derived from EST M79057, several of the longest isolates were chosen for complete DNA sequence analysis. Two clones (designated 2.1 and 2.4) were found to comprise overlapping DNA sequences totaling 5611 bp including a complete open reading fame encoding 1470 amino acids.
  • the organization of various domains in the predicted polypeptide sequence oflectomedin-2 was related to that in lectomedin- 1.
  • the approximately 851 amino acid extracellular domain of lectomedin-2 included a region with homology to the D- galactoside binding lectin-like domain (amino acids 36 to 131 of SEQ ID NO.: 57), an olfactomedin-like domain (amino acids 135 to 325 of SEQ ID NO: 57), three extracellular and three intracellular domains separated by seven transmembrane domains (approximately amino acids 852 to 1095 of SEQ ID NO: 57) and a cytoplasmic region (approximately amino acids 1096 to 1470 of SEQ ID NO: 57).
  • the cytoplasmic region of lectomedin-2 was most similar in length and sequence identity to lectomedin-l ⁇ .
  • Primer JD#1 (SEQ ID NO: 61) incorporated a H dIII site to facilitate cloning.
  • the resulting 843 bp amplified product was digested withHr ⁇ dlll and BamHL and a 535 bp DNA fragment was isolated.
  • the 535 bp fragment from clone 2-1 was ligated with a 1912 bp BamHUSaA fragment from clone 2-1, a 2904 bp SafiJEcoKV DNA fragment from clone 2-4, and pcDNA3 (Invitrogen) previously digested with Hwdlll and EcoRV.
  • a BLAST search of the GenBank ⁇ ST database with lectomedin-l ⁇ or lectomedin-2 as query sequences identified ⁇ ST sequences identical to lectomedin- 1, ⁇ ST sequences identical to lectomedin-2 and ⁇ STs that were significantly related to but distinct from both known lectomedins. See Table 1.
  • One of these unique lectomedin ⁇ STs (GenBank Acc# R50822) was derived from clone #37438. Clone #37438 was purchased and its DNA sequence completely determined. The polynucleotide sequence for clone #37438 is set out in S ⁇ Q ID NO: 59, and the encoded amino acid sequence is set out in S ⁇ Q ID NO: 60.
  • the 3' sequence of clone #37438 is comprised of an untranslated region preceded by a predicted coding sequence for a protein with significant amino acid homology to the cytoplasmic domains of lectomedin-l ⁇ and lectomedin-2.
  • the 5' end of the sequence of clone #37438 was unrelated to the lectomedins, but was identical to the nucleotide sequence for a tRNA synthetase.
  • This clone may represent a partially spliced mRNA or it might be a cloning artifact.
  • the cDNA clone #37438 was used to generate a labeled probe to screen approximately one million clones from a human fetal brain cDNA library by techniques standard in the art. Hybridization was carried out at 43 °C in the presence of 45% formamide and filters were washed in 0. IX SSC at 68°C for 90 minutes. Positive clones identified were isolated to homogeneity and partial sequence analysis was carried out with eleven of the clones. The DNA sequence of one clone (#11) was determined to have 3' sequences identical to clone #37438 and 5 'sequences within the upstream coding sequences similar to, but distinct from, lectomedin- 1 and lectomedin-2. -38-
  • Expression vectors encoding lectomedin isoforms were constructed by combining DNA fragments from clone 3.3 and RACE3.3 described above.
  • both clone 3.3 and RACE3.3 polynucleotides are first modified in the overlapping regions by insertion of a silent mutation to introduce a S ⁇ cl restriction site.
  • PCR is employed using primers (SEQ ID NOs: 25 and 26) to amplify a 5' sequence from the RACE3.3 cDNA template that changes G to C at position 1455 to create the desired restriction site.
  • the 5' primer (SEQ ID NO: 26) is designed based on sequences at the ATG start codon; the primer introduces a Bam ⁇ l restriction site to facilitate cloning and a Kozak consensus start sequence.
  • the 3 ' primer is based on sequences at the stop site of translation and is designed to introduce a NotI restriction site.
  • BamHL site and a 3' S ⁇ cl site, and a clone 3.3 D ⁇ A with a 5 'S ⁇ cl site and a 3' NotI site, are digested with appropriate enzymes, ligated together, and cloned into the mammalian expression vector pcD ⁇ A+3, (Invitrogen, Carlsbad, CA) previously digested with Bam ⁇ l and NotI.
  • a lectomedin- 1 ⁇ -encoding D ⁇ A is generated using PCR with the 5' primer used to amplify RACE3.3 described above and the 3 ' primer used to amplify the clone 3.3 D ⁇ A also described above.
  • both RACE3.3 and clone 3.3 D ⁇ A are combined with the two primers.
  • the RACE3.3 and clone 3.3 D ⁇ A will anneal across the overlapping regions and the double stranded region will serve as primers in the first extension that produces a complete double stranded lectomedin-l ⁇ D ⁇ A.
  • Subsequent amplifications will result from extension from the 5' and 3' primers.
  • the amplification product is then purified, digested with Bam ⁇ l and NotI, and inserted into the pcD ⁇ A vector previously digested with the same enzymes.
  • an expression vector encoding lectomedin-l ⁇ was constructed in a two step procedure. First, PCR was carried out using a Xbal fragments of clone 3.3 and primers 3.3.24 (SEQ ID NO: 52) and Lecto 3' express (SEQ ID NO: 27) along with Taq polymerase.
  • a second PCR was carried out using an EcoRI fragment of RACE 3.3 as template DNA and primers Lecto 5' express (SEQ ID NO: 26) and Lecto ⁇ (SEQ ID NO: 25) with Vent R polymerase (New England Biolabs, Beverly, MA).
  • the two amplification products were purified, denatured, and annealed. Because the two fragments overlap in a region of approximately 100 nucleotides, annealing results in a partially double stranded molecule spanning the entire lectomedin-l ⁇ coding region.
  • Extension with Taq polymerase first produces a double stranded lectomedin-l ⁇ coding region. The double stranded molecule was then amplified using primers in SEQ ID NOs: 26 and 27.
  • the SEQ ID NO: 26 primer was designed to introduce a Bam ⁇ l restriction site, followed by a Kozak consensus start site.
  • the resulting amplification product was digested with NotI and Bam ⁇ l, and the lectomedin-l ⁇ fragment was gel purified.
  • the fragment was ligated into pcD ⁇ A3 (Invitrogen, Carlsbad, CA) previously digested with Bam ⁇ l and NotI.
  • Sequence analysis of the resulting plasmid designated pcD ⁇ A3 Lectomedin- l ⁇ #2, indicated that several errors were introduced in the amplification process.
  • the correct lectomedin-l ⁇ coding sequence was constructed from regions of pcDNA3 Lectomedin- l ⁇ #2 without errors ligated to fragments of RACE3.3 and clone 3.3 as follows.
  • pBSKS pBluescript KS+
  • BsiXl BsiXl
  • Apal BsiXl
  • the resulting plasmid was designated pBSKSlectoHindIII/ApaI#14.
  • a 306 bp ApaVEcoKl fragment from clone 3.3 and a 2486 bp EcoRI/EcoRI fragment from clone 3.3 were ligated in the presence of pBSKS previously digested with Apal and EcoRI.
  • the resulting plasmid was designated pBSKSlectolalpha ⁇ coRI/ApaI#6.
  • Plasmids pNEF6 and pDEF2 encode promoter regions and a 5' intron from the gene encoding Chinese hamster ovary elongation factor 1, in addition to neomycin
  • Plasmid pEFl/XN was generated by ligation of an 11 kb Notl/Xbal fragment from ⁇ SK/EF1.12 (WO 98/49289, published November 5, 1998, incorporated -41- herein by reference), having the Xbal site blunt ended with Klenow polymerase, with a 2.22 Notl/Smal fragment from pDC31 (WO 98/49289).
  • Plasmid pNEF3 was generated by ligation of a 4.19 kb SaWNs ⁇ l fragment (theNsz ' I site blunt ended with Klenow polymerase) from pSKEFl.7 (WO 98/49289) with a 7.96 kb Sali/Pmel fragment from p ⁇ EFl (WO 98/49289).
  • Plasmid p ⁇ EF5 was constructed with a 9.2 kb Ascl/Not ⁇ fragment from pNEF3 and an 11 kb Ascl/Notl fragment from pEXl/XN.
  • Plasmid pNEF6 was constructed by ligation of a 19.7 kb NZ> ⁇ I/-4.s/>718 fragment fom pNEF5 with a 0.844 kb Xbal/Asp718 fragment from pRc/CMv (Invitrogen).
  • a 736 bp Notl/Hindl ⁇ fragment (including the intron sequence) was isolated from pDEF2 and combined with a 1571 HindUVApal fragment (including the Kozak sequence, translation start site, and coding region for amino acids 1 to 515) from pBSKSlectoHindIII/ApaI#14, a 3714 bp ApallXbal fragment (encoding amino acids 516 to 1177 of lectomedin-l ⁇ and including a stop codon and untranslated sequences) from pBSKSlectolalphaEcoRI/Apa#6, and pNEF6 previously digested with Xbal and NotI.
  • the resulting plasmid was designated pNEF6LectomedinlA#3.1.
  • Clone 15.3.1 and clone 71509 were separately amplified with primers lecto3.3.10 and 3.3.19.
  • the resulting amplification products were digested with St ⁇ l and Ncol, and a 113 bp fragment from 15.3.1 (lectomedin-l ⁇ ) and a 210 bp fragment from 71509 (lectomedin-l ⁇ ) were purified. Each fragment was separately ligated into pBSKSlectolalphaEcoRI/ApaI#6 previously digested with Stul and Ncol. The resulting plasmids were designated pBSKSlectolbetaEcoRI/ApaI#7 and pBSKSlectolgammaEcoRI/ApaI#6. -42-
  • Plasmid pDC37 encoding human VCAM-1 with human IgGl hinge-CH2 coding sequences, is a derivative of pDC31 generated by digestion with Sail, filled in with Klenow polymerase, and blunt end ligated to eliminate the Sail site.
  • Plasmid pDC37/VCAMl.Igl.2c was digested withH/wdlll and Sail and a fragment lacking the NACM-1 coding region was isolated.
  • the isolated Hind ⁇ l/Sall fragment was ligated with a 1571 bp HindllllApal fragment from pcD ⁇ A3Lectomedin- la#2 and an amplification product from clone 3.3 prepared using primers lecto Sal Ig (SEQ ID NO: 51) and 3.3.24 (SEQ ID NO: 52).
  • Primer lecto Sal Ig generated a unique Sail site in the amplification product (after codon 811 of lectomedin- 1) to permit in-frame ligation to IgGl coding sequences.
  • the resulting plasmid was designated pDC37Lecto.Ig#7. -43-
  • a 736 bp Notl/HindUl fragment from pDEF2 was ligated with a 1571 bp Htnd ⁇ i/-4 7 ⁇ III fragment (encoding the Kozak sequence, start site, and amino acids 1 to 515 from lectomedin- l ⁇ ) from pcDNA3 Lectomedin- la#2, a 1788 bp Apal/Xbal fragment (encoding amino acids 516 to 811 from lectomedin- 1 fused to IgG sequences) from pDC37Lecto.Ig#7, and pDEF14 previously digested with NotI and Xbal.
  • the resulting plasmid was designated pDEF14Lecto.Ig#2.
  • Plasmid pDEF14Lecto.Ig#2 was transfected into D ⁇ FR " DG44 CHO cells and stably transfected cells were selected.
  • Characterization of the protein expression level in recombinant cells is carried out using polyclonal antisera (produced as described in Example 8), and functional analysis, with respect to latrotoxin binding (discussed below) and/or release of secretory granule contents, is performed as previously described.
  • CHO cells Chinese hamster ovary (CHO) cells were transfected by standard methods (i.e., calcium phosphate or cationic lipids) with lectomedin-l ⁇ expression construct. After 48 hours incubation, the cells were lysed in
  • ⁇ A constructs For functional characterization, secretory cells of the endocrine system are employed which readily accept D ⁇ A constructs by transfection.
  • Cell lines useful in functional characterization include, for example, mouse anterior pituitary corticotroph continuous cells (AtT20; ATCC CCL 89), rat pancreatic islet insulinoma continuous cells
  • RenM5F human pituitary somatotroph continuous cells
  • GH3 human pituitary somatotroph continuous cells
  • incubation of the -44- transfected cells with alpha-latrotoxin, or another ligand is followed by detection of stimulated secretion of proteins using enzyme-linked immunosorbant assay or radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • lectomedin- 1 is a G-protein coupled receptor
  • ligand binding would be expected to trigger intracellular second messenger effector pathway activity changes such as, for example, increased production of cyclic AMP (cAMP) or changes in intracellular calcium concentration. Changes of these types are measured by standard techniques, for example, RIA detection of cAMP or fluorescence detection of calcium binding indicators (i.e., Fura 2).
  • Example 2 was purified using protein A Sepharose (Amersham Pharmacia Biotech,
  • Amino terminal sequencing indicated that the mature amino terminus of the protein was identified as a phenylalanine residue at position 26. This observation indicated that the recombinant protein was recognized and cleaved by a signal peptidase in the CHO cells and that the amino terminus was not blocked.
  • Size exclusion chromatography suggested a protein with a molecular weight of approximately 650 kDal as compared to the molecular weight determined on SDS PAGE of approximately 170 kDal.
  • the gel filtration result suggested that four monomers combined to produce the 650 kDal protein.
  • N-glycosidase F Treatment of the protein with N-glycosidase F, O-glycosidase, and/or neurominidase (Boehringer Mannheim) in 10 mM N-I 2 HPO 4 (pH 6.8), 5 mM EDTA, 0.25% Triton® X-100, 0.5% SDS, and 0.5% ⁇ -mercaptoethanol (BME), at 37°C, resulted in reduction of protein molecular weight. After treatment with N-glycosidase F alone, monomeric protein molecular weight was approximately 130 kDal.
  • a detergent extract was prepared from human spleen (3.48 g wet weight).
  • Tissue was homogenized in 15 ml buffer containing 1% Triton X-100, 25 mM Tris, pH
  • Mac-2 (galectin-3) is synthesized by numerous immune cell populations and epithelia, and is a major non-integrin laminin binding protein [Perillo, et al, J. Mol. Med. 76:402-412 (1998)]. Recent observations indicated that Mac-2 was expressed in vessels in early atherosclerotic lesions in association with infiltrating monocytes. Expression was not detected in normal vessels. Expression was also detected in aortic smooth muscle cells in culture, as well as in animals following a hypercholesterolemic feeding regimen and post balloon angioplasty [Arar, et al, FEBS Letts.430:30 -311 (1998), Nachtigal, etal, AmJ.Pathol 752:1199-1208 (1998)].
  • Mac-2 stimulates normal fibroblast proliferation, neural cell adhesion, and neurite outgrowth [Inohjara, et la., Exp. Cell. Res. 245:294-302 (1998); Pesheva, et al, J. Neurosci. Res. 5 ⁇ :639-654 (1998)].
  • Example 5 Northern Analysis In an attempt to assess human lectomedin-l ⁇ expression, Northern blot analysis was performed using a commercial multi-tissue blot (Clontech, Palo Alto, CA) with RNA derived from various human tissue sources. The probe used was a 531 bp 7?stXI fragment derived from the extracellular region of clone 3.3 (bases 1860 to 2350 in SEQ ID NO: 7). Hybridization was carried out in Express-Hyb TM Solution (Clontech) at 68°C for two hours; the final wash was carried out at 68°C in 0.1X SSC for one hour.
  • Probes for in situ hybridization analysis were prepared as follows. Clone 3.3 was engineered by PCR to include a S ⁇ cl site near the 5' end of the cDNA by changing a G nucleotide to C at position 1459 of the composite sequence. A 626 bp
  • S ⁇ cI/EcoRI fragment was prepared and subcloned into pBSSK (Stratagene, La Jolla, CA).
  • the resulting plasmid was linearized with either EcoRI or S ⁇ cl.
  • the ends of the S ⁇ cl linearized DNA were made blunt by standard procedures using T4 DNA polymerase.
  • Linear DNAs were used to generate 35 S-labeled sense or antisense strand probes for in situ hybridization with tissue sections from spleen, lung, prostate, heart, thymus, duodenum.
  • BamHl/Xbal fragment of lectomedin-l (S ⁇ Q ID NO: 1) was subcloned and a representative clone was isolated and designated as probe BX.
  • 35 S-labeled sense and antisense probes from both BS and BX were prepared by methods standard in the art and hybridized with tissue sections from human brain occipital cortex, cerebellum and thalamus; interventricular septum, sino-atrial node and atrium of the heart; small intestine, spleen, lung, prostate, adrenal gland and pancreas.
  • the antisense BX probe produced similar patterns except for the presence of specific signals in a subset of secretory cells of the prostate.
  • the contiguous lectomedin-l ⁇ cDNA deduced from combining clone 3.3 and RACE3.3 sequences was used as a query to search the NCBI Sequence-Tagged Sites (STS) database in an attempt to map the chromosomal location of a gene encoding lectomedin-l ⁇ .
  • STS NCBI Sequence-Tagged Sites
  • An E. coli expression vector was constructed encoding the extracellular region of lectomedin-l (residues 432 to 852 of SEQ ID NO: 1) as a fusion protein with a biotinylated tag at the amino terminus.
  • the plasmid, designed "Biolectol st ECD" was constructed as follows.
  • PCR primers "lecto-1” and “lecto-2” were used to amplify a 1283 bp fragment of clone 3.3 (nucleotides 1508-
  • Primer lecto- 1 introduced an Ncol restriction site (underlined above) in the amplification product and primer lecto-2 introduced a BgRl restriction site (underlined above) and a translational termination site.
  • the amplified fragment was subcloned into plasmid arabio lb previously digested with Ncol and BglE to form plasmid Bio 1 ecto 1 stECD .
  • the plasmid arabio lb contains the Salmonella typhimurium arabinose promoter and araC gene, as well as the biotin transferase gene [Kashishian, et al, J. Bio. Chem. 275:27412- 27419 (1998)].
  • the expression product of the final construct provides a fusion protein of approximately 55 kDa with the biotin tag at the ⁇ -terminus, and the lectomedin- 1 amino acids 432 to 852 at the C-terminus.
  • the plasmid Biolectol stECD was transformed into E. coli using standard procedures and single colonies were isolated and grown for plasmid preparation.
  • a -51- culture including the desired plasmid was grown at 30°C in LB/carbenicillin supplemented with biotin (4 ⁇ M) and induced in the presence of arabinose (0.5%) for 16 hours.
  • Bacteria were collected by centrifugation and lysed with hen egg lysozyme (10 ⁇ g/ml) in TEN buffer (50 mM Tris-HCl, pH 7.5 at 25 °C, 0.5 mM EDTA, 0.3 M NaCl) on ice for 15 minutes.
  • NP-40 detergent was added to 0.2% final concentration and the resulting mixture sonicated briefly on ice. Insoluble material was removed by centrifugation at 15,000 x g for ten minutes, after which the pellet washed five times with the additional of TEN buffer followed by sonication and centrifugation. The final pellet was solubilized in 2X sample loading buffer for preparative SDS-PAGE separation.
  • a major band of 55 kDa was detected after treating the gel for 30 minutes in 0.4 M KCl.
  • the 55 kDa band was excised from the gel and the fusion protein eluted in dialysis tubing using 0.5% SDS-PAGE running buffer. The collected protein was
  • the purified protein was used to immunize two rabbits to generate antisera according to well known procedures. Briefly, two New Zealand white rabbits (designated #7234 and #7278) were prebled to obtain preimmune serum and then immunized with 250 ⁇ g of purified Biolectol stECD fusion protein emulsified with complete Freund's adjuvant. The rabbits were boosted repeatedly with 250 ⁇ g of purified fusion protein in incomplete
  • Fruend's adjuvant The first three boosts were given at one month intervals, the third and fourth boosts following a three month interval, and the fourth and fifth boost following an additional one month interval.
  • Blood was drawn by ear vein puncture two weeks after the second, third, fourth, and fifth boosts to determine antibody titers. Immunoprecipitation was carried out with the resulting polyclonal sera using extracts from tissues/cell lines, including brain cortex, lung, spleen, liver, skeletal muscle, hippocampus, and prostate carcinoma cell line PC-3 (ATCC, CRL 1435).
  • Protein species having molecular weights of 200, 180, 170, and 70 kDa were detected which may have represented full length proteins, proteolytic fragments, or isoforms of lectomedin including the ⁇ , ⁇ , and ⁇ proteins . -52-
  • Serum obtained from rabbit #7234 after the fifth boost was subjected to antigen-specific affinity chromatography by methods standard in the art. Briefly, 10 ml of 0.45 or 0.8 microfiltered serum (100 x g supernatant) adjusted to 10 mM Tris, pH 7.5, was incubated with sLecto-Hg agarose beads for 16 hours at 4°C with rotation. The beads were drained and washed with 20 bed volumes of 0.5 M NaCl, 10 mM Tris, pH
  • Peptides specific to the carboxy terminal cytoplasmic regions of ⁇ , ⁇ , and ⁇ isoforms of lectomedin- 1 were synthesized as immunogens for producing polyclonal antisera in New Zealand White rabbits.
  • the peptides were designed from the DNA sequence in the cytoplasmic region of lectomedin l (SEQ ID NO: 53), lectomedin l ⁇
  • Peptides comprising the carboxy terminal regions of the related lectomedin-2 (SEQ ID NO: 63) and lectomedin-3 (SEQ ID NO: 64) proteins were also synthesized.
  • Immulon 4 (Dynax Technologies, Chantilly, VA) plates were coated with unconjugated peptide at 2 ⁇ g/ml. Plates were blocked with 0.5% fish skin gelatin and washed. Serial dilutions of the pre-immune serum and test bleeds from each rabbit were incubated on the peptide-coated plates. After washing, goat anti-rabbit- horseradish peroxidase (HRP) conjugated secondary antibody was added. The plates were washed and signal detected by tetramethyl benzidine (TMB) (Sigma) reagent.
  • TMB tetramethyl benzidine
  • Serum from rabbits #6484 and #6453 immunized with the lectomedin-l ⁇ peptide showed reactivity three-fold greater than pre-immune serum at a 3000-fold dilution.
  • Serum from rabbits #6868 and #6307 immunized with lectomedin-l ⁇ showed three-fold greater reactivity over pre-immune serum at a 3000-fold dilution.
  • Serum from rabbits #7347 and #6490 immunized with lectomedin-l ⁇ showed three-fold greater reactivity at a 200-fold dilution.
  • mice Five 6 to 12 week old BALB/c mice were prebled on day 0 and immunized by subcutaneous injection with 20 ⁇ g of the lectomedin- l ⁇ , lectomedin-l ⁇ , or lectomedin- l ⁇ peptides (SEQ ID NOs: 53, 54, and 55) described above (60 ⁇ g total) in complete Freund's adjuvant. On Days 21, 41, and 62, each mouse was boosted with 10 ⁇ g of each peptide (30 ⁇ g total) in incomplete Freund's adjuvant.
  • Test bleeds were drawn on day 72 and reactivity determined by ELISA against individual peptides as described in generation of polyclonal antisera, with the exception that specific mouse antibody was detected with a goat anti-mouse-HRP.
  • Immune serum from all five mice showed reactivity to lectomedin- 1 ⁇ and lectomedin-l ⁇ peptides greater than pre-immune serum at a 12800-fold dilution. Serum from all of the mice showed modest reactivity to lectomedin-l ⁇ peptide.
  • Additional peptides comprising the carboxyl termini of lectomedin-2 and lectomedin-3 (SEQ ID NOs: 58 and ) were synthesized to screen for cross reactive antibodies recognizing similar epitopes found in termini of lectomedin-l ⁇ , lectomedin-2 and lectomedin-3.
  • mice were immunized with the second lectomedin-l ⁇ peptide (SEQ ID NO: 56) described above. Immune serum from each of the mice is tested for lectomedin- l ⁇ reactivity by ELISA (described above) prior to fusion and hybridoma cloning.
  • the spleen of the immunized animal is removed aseptically and a single-cell suspension is formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640 (Gibco, Canada) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • the cell suspension is filtered with a sterile 70-mesh Nitex cell strainer
  • Thymocytes taken from naive Balb/c mice are prepared in the same manner.
  • NS-1 cells Approximately 2 x 10 8 spleen cells are combined with 4 x 10 7 NS-1 cells (kept in log phase in RPMI with 11% fetal bovine serum [FBS] for three days prior to fusion). The cells are collectedy centrifugation and the supernatant is aspirated. The cell pellet is dislodged and 2 ml of 37°C PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer Mannheim) is added while stirring over the course of one minute, followed by the addition of 14 ml of serum free RPMI over seven minutes. Additional RPMI can be added and the cells are centrifuged at 200 x g for 10 minutes.
  • 37°C PEG 1500 50% in 75 mM HEPES, pH 8.0
  • the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim), and 1.5 x 10 6 thymocytes/ml.
  • the suspension is dispensed into ten 96-well flat bottom tissue culture plates (Corning, United Kingdom) at 200 ⁇ l/well. Cells are fed on days 2, 4, and 6 days post-fiision by aspirating 100 ⁇ l from each well and adding 100 ⁇ l/well plating medium containing 10 U/ml IL-6 and lacking thymocytes.
  • ® ELISAs are performed as follows. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) are coated at 4°C with 50 ⁇ l/well with 100 ng/well of immunogen in 50
  • NDRI Philadelphia, PA
  • human brain cortex and cerebellum
  • heart septum and atrium
  • prostate septum and atrium
  • lung liver
  • spleen small intestines
  • adrenal gland artery
  • artery renal, pulmonary and subclavian
  • Endogenous peroxidase activity was quenched by incubating the fixed cryosections in buffer containing 0.1% sodium azide and 0.33% hydrogen peroxide in phosphate buffered saline (PBS) during a 15 minute incubation. All incubations were carried out at room temperature
  • Biotinylated goat anti-rabbit antibody conjugated to peroxidase (Vector Labs) was diluted 1:200 in blocking solution and applied to the slides for 30 minutes. Slides were washed for five minutes and incubated for 30 minutes with ABC Elite reagent (avidin-biotin-peroxidase kit PK-6100, Vector Labs). Slides were washed -57- twice for five minutes per wash in TSBT. Substrate solution (DAB substrate kit for peroxidase, Vector Labs) was applied to the slides and the desired color intensity was allowed to develop over approximately five minutes.
  • ABC Elite reagent avidin-biotin-peroxidase kit PK-6100, Vector Labs
  • human brain cortex labeling with 7234 sera was detected in a subset of neurons (including large and small pyrimidal neurons), granule cells, and smooth muscle cells of the vasculature.
  • Human cerebellum staining with 7234 sera was localized to purkinje neurons and neurons of the granular cell layer.
  • Human heart (septal and atrial sections) showed cardiomyocyte immunoreactivity, most prominently at cardial myocyte cell junctions transverse to the plane of the contractile apparatus called intercalated dicks.
  • Sections of human prostate showed weak stromal cell labeling and cytoplasmic skeletal muscle staining. Lung staining was found in cartilage and some bronchial smooth muscle cells, with certain cells staining more strongly than others. The medulla of the adrenal gland showed strong positive staining.
  • Human liver, spleen, and small intestines exhibited a non-specific pattern of immunoreactivity.
  • Human aorta showed immunoreactivity with 7234 sera in the vessel wall that was primarily located in the tunica intima (Iumenal muscle layer) and tunica media (intermediate muscle layer).
  • Thoracic aorta, pulmonary artery, and renal antery each showed similar staining patterns.
  • lectomedin- 1 immunoreactivity was found primarily in the same cells (i.e., smooth muscle cells).

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Abstract

L'invention porte sur de nouveaux polypeptides récepteurs heptatransmembranaires de structure extracellulaire caractéristique comportant des domaines: de fixation de la lectine, du genre olfactomédine, et du genre mucine.
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US6479256B1 (en) * 1998-03-04 2002-11-12 Icos Corporation Lectomedin materials and methods
US20020055626A1 (en) * 2000-06-09 2002-05-09 Turner C. Alexander Novel human seven transmembrane proteins and polynucleotides encoding the same
WO2000058462A1 (fr) * 1999-03-25 2000-10-05 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines de recepteur couplees aux proteines de liaison a la guanosine triphosphate (gtp), bg3
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