EP0731836A1 - Chicken oocyte receptor p95 (vldl/vtg receptor) - Google Patents

Abstract

The present invention refers to a purified and isolated nucleic acid molecule encoding (a) a polypeptide comprising the amino acid sequence of at least one of the structural elements of the mature chicken oocyte receptor P95 as defined in Fig. 2, or (b) a polypeptide comprising an amino acid sequence which has high homologyto at least one of the structural elements of (a).

Description

CHICKEN OOCYT-E RECEPTOR P95 (VLDL/VTG RECEPTOR)

The invention provides, for the first time, recombinant DNA molecules which code for polypeptides, and the polypeptides per se, that have at least one of the structural elements (i.e. continuous sequence of amino acid residues) of a protein (P95) localized in oocytes of the chicken (Gallus gallus domesticus) , or the entire protein, and exhibit one or more of the immunological or biological properties (e.g. recognizing complementary ligands with high affinity and specificity) of the naturally occurring P95, including allelic variants thereof. Furthermore, the invention provides a new process for the determination of receptor ligands, e.g. ligands of the LDL receptor family.

The storage compartment of the cytoplasm of the oocytes of oviparous (egg-laying) species, termed yolk, contains a significant portion of the nutrients that the developing embryo must sustain on. In birds, yolk is accumulated during the third phase of oocyte growth, which spans the last 5 to 9 days before ovulation. Accumulation of yolk occurs, apparently exlusively, through the uptake from the bloodstream of plasma components across the plasma membrane of the oocyte. The plasma components include lipoproteins, vitamines bound to proteinaceous carriers, trace metals carried by proteins, albumin, immunoglobulins, carbohydrates, and other hitherto unidentified molecules. Most, if not all, of these components are transported across the oocyte plasma membrane by receptor-mediated endocytosis. In this process, receptors in the surface membrane bind a select set of molecules (termed ligands) present in the extracellular milieu, and carry them into the cell (i.e. endocytose them) via a ligand-receptor complex present in a membrane-enclosed vesicle that enters the cell.

Receptor-mediated endocytosis of chicken yolk precursor molecules has been characterized for the major lipoproteins, very low density lipoprotein (VLDL) and vitellogenin (VTG) . See Barber, D.L. et al, J. Biol. Chem. 266: 18761-18770 (1991) . P95 binds and internalizes these two ligands by interaction with certain structural elements within them; these elements consist of clusters of stretches of positively charged and hydrophobic residues. Such recognition epitopes are, based on their primary structure in combination with their interaction with receptors of the low density lipoprotein receptor (LDLR) family, present in apolipoprotein (apo)B-lOO of all species tested so far, vitellogenin, apo E (variations affecting the affinity for P95 occur in isoforms thereof) , lipoprotein lipase, alpha-2-macroglobulin, pregnancy zone protein, complexes between urokinase- and/or tissue-type plasminogen activator inhibitor-1 (PA/PAI-1 complexes) , Pseudomonas aeruginosa exotoxin A, the 39-kDa receptor-associated protein (RAP) , chicken riboflavin binding protein, a region of the Rous sarcoma virus (subgroup A) envelope protein (Bates P. et al., Cell 74 (1993), 1043- 1051) , a surface epitope of vesicular stomatitis virus (Fischer D.G. et al. , Science 262 (1993), 250-253), and pos¬ sibly other yet unidentified macromolecules.

P95 is a small LDLR with demonstrated affinity for VLDL, VTG, alpha-2-macroglobulin, human apo E isoforms, and riboflavin binding protein. It can reasonably be expected that P95 is capable of similar high affinity interaction with the other ligands of LDLRs and homologues identified so far and possibly others, as .noted above. The chicken oocyte plasma membrane is a source for P95, which can be isolated therefrom in pure form. See Barber, D.L. et al. , J. Biol. Chem. 266: 18761-18770 (1991) .

This invention concerns P95, a major surface protein of the chicken oocyte, or fragments thereof. P95 is the key receptor on the surface of growing chicken oocytes in that it mediates the uptake from the bloodstream of the two major yolk compo¬ nents, VLDL and VTG, which make up more than 2/3 of the dry mass of the fully grown oocyte (i.e. egg yolk) . P95, or certain structural elements thereof (as specified below) , interact in specific fashion and with high affinity with macromolecules that contain structural elements sufficiently similar to that mediating the interaction of human apo E with LDLRs. Binding of P95 to VLDL, VTG, and human apo E is known (see Steyrer, E. et al., J. Biol. Chem. 265: 19575-19581, 1990) .

The present invention provides recombinant DNA molecules which contain a nucleotide sequence that codes for a polypeptide which exhibits the same or similar immunological and/or biological properties as natural P95. The invention also relates to nucleotide sequences coding for polypeptides from species other than chicken which comprise at least one structural element with an amino acid sequence identical to that portion of P95 or at least having a high amino acid homology to that portion of P95. The invention provides the complete cDNA sequence of P95 and hence the complete deduced amino acid sequence (see Fig. 2) .

Subject-matter of the present invention is a purified and isolated nucleic acid molecule encoding (a) a polypeptide comprising the amino acid sequence of at least one of the structural elements of the major chicken oocyte receptor P95 as defined in Fig. 2, or (b) a polypeptide comprising an amino acid sequence which has high homology to at least one of the structural elements of (a) . The nucleotide and amino acid sequence of the P95 receptor is shown in SEQ. ID. NO. 1 and SEQ. ID. NO. 2.

The structural elements of the mature chicken oocyte receptor P95 as defined in Fig. 2 are contiguous stretches of amino acid residues and comprise 8 ligand-binding repeats 1-8 of approximately 120 nucleotides, 3 EGF-precursor homology repeats A-C, a structural element comprising 5 repeats with consensus tetrapeptide, a membrane spanning domain and a cytoplasmic tail as defined below.

Ligand-binding repeat 1 is located from nucleotide 1 to 117 of the nucleic acid sequence which is depicted in Fig. 2. Repeat 2 is located from nucleotide 118 to nucleotide 240. Repeat 3 is located from nucleotide 241 to nucleotide 363. Repeat 4 is located from nucleotide 364 to nucleotide 480. Repeat 5 is located from nucleotide 481 to nucleotide 624. Repeat 6 is located from nucleotide 625 to nucleotide 735. Repeat 7 is located from nucleotide 736 to nucleotide 852. Repeat 8 is located from nucleotide 853 to nucleotide 981.

The EGF-precursor homology repeat A is located from nucleotide 994 to nucleotide 1,101. EGF-precursor homology repeat B is located from nucleotide 1,114 to nucleotide 1,221. EGF-precursor homology repeat C is located from nucleotide 2,038 to nucleotide 2,169.

A further structural element containing 5 repeats with the consensus tetrapeptide Tyr/Phe-Trp-Xxx-Asp is located from nucleotide 1,222 to nucleotide 2,037.

A membrane spanning domain is located from nucleotide 2,227 to nucleotide 2,292.

The cytoplasmic tail which contains the internalization signal Asn-Phe-Asp-Asn-Pro-Val-Tyr is located from nucleotide 2,293 to 2,451 (the carboxy terminus of the molecule).

The present invention encompasses a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of at least one of the above mentioned structural elements, or a polypeptide comprising an amino acid sequence which has a high homology to at least one of the above-defined structural elements. The term "high homology" preferably means an amino acid homology of at least 90% in the area of the ligand- binding repeats 1-8 and the EGF-precursor homology repeats A- C, a homology of at least 80% in the area of the element containing the 5 tetrapeptide repeats and the transmembrane structural element and a homology of at least 95% in the cytoplasmic tail. More preferably "high homology" means an amino acid homology of at least 95% in one of the above- mentioned structural elements.

A further subject matter of the present invention is a puri¬ fied and isolated nucleic acid molecule encoding (a) a polypeptide comprising the entire amino acid sequence of the mature chicken oocyte receptor P95 as defined in Fig. 2 or (b) a polypeptide comprising an amino acid sequence which has a homology of at least 75%, preferably at least 80%, more preferably 85% and most preferably 90% to the polypeptide of (a) .

In a preferred embodiment of the present invention the nucleic acid molecule codes for a polypeptide comprising at least one immunological and/or biological property of the major chicken oocyte receptor P95. The term "immunological property" refers to the reaction with specific antibodies and the term "biological property" refers to the binding of specific macromolecules defined by having certain structural elements within them, wherein these elements consist of clusters of stretches of positively charged and hydrophobic residues. Such recognition epitopes are based on the primary structure in combination with their interaction with receptors of the low density lipoprotein receptor (LDLR) family present in apo lipoprotein (apo) B-100 of all species tested so far, vitellogenins, apo E (variations affecting the affinity for P95 occur in isoforms thereof) , lipoprotein lipase, alpha-2-macroglobulin, pregnancy zone protein, complexes between urokinase- and/or tissue-type plasminogen activator and plasminogen activator inhibitor 1 (PA/PAI-1 complexes) , Pseudomonas aeruginosa exotoxin A, the 39-kDa receptor-associated protein (RAP) , a riboflavin binding protein, a region of the Rous sarcoma virus (subgroup A) envelope protein (Bates P. et al. , Cell 74 (1993), 1043- 1051) , a surface epitope of vesicular stomatitis virus (Fischer D.G. et al. , Science 262 (1993), 250-253), and other macromolecules having similar receptor-binding abilities.

The nucleic acid molecule of the present invention is prefer¬ ably a DNA-molecule and more preferably a cDNA-molecule. In an especially preferred aspect the present invention refers to a nucleic acid molecule comprising the nucleic acid sequence as defined in Fig. 2, optionally without the non- coding regions and/or the signal peptide-coding region.

An especially preferred embodiment of the present invention are nucleic acid molecules as defined above, which encode a soluble polypeptide, i.e. a polypeptide wherein the membrane spanning domain is no longer functionally active, e.g. by means of complete or partial deletion. This soluble polypeptide is especially suitable for the determination of receptor ligands in a liquid sample.

Still another especially preferred embodiment of the present invention are chimeric nucleic acids comprising operably linked (a) a nucleic acid sequence encoding at least one of the ligand-binding repeats 1-8 as defined above and (b) a nucleic acid sequence encoding at least one ligand-binding repeat of another LDL receptor family polypeptide, e.g. the human LDL receptor, the rabbit VLDL receptor etc.. By combining the ligand binding repeats of two or more LDL receptor family polypeptides it is possible to provide new chimeric receptor polypeptides which have new ligand specificities. The nucleotide and amino acid sequence of a P95/human LDL receptor chimaera is shown in SEQ. ID. NO. 3 and SEQ. ID. NO. 4. In a further preferred aspect of the present invention the nucleic acid molecule codes for a polypeptide which does not have any O-glycosylation sites.

In a further aspect the present invention refers to a nucleic acid which is a single-stranded DNA. The nucleic acid molecule can also be covalently associated with a detectable label, e.g. a radioactive label, a fluorescent label, a chemiluminescent labels, an enzymatic label, or an affinity label, such as biotin. Methods of covalently labeling nucleic acid molecules are common knowledge in the field of molecular biology and need not be discussed in detail.

A further subject-matter of the invention is a vector com¬ prising at least one copy of the nucleic acid molecule according to the present invention. The vector is preferably an expression and/or cloning vector that enables the vector to replicate in one or more selected host cells. The vector usually comprises a nucleic acid sequence which enables the vector to replicate independently of the host chromosomes, such as origins of replications or autonomously replicating sequences. Such sequences are well known for a variety of organisms, including bacteria, yeast and viruses. Specific examples are the origin of replication from the well-known plasmid pBR-322 which is suitable for most gram-negative bacteria, the 2μ-plasmid origin for yeast and various viral origins (SV40, polyoma, adeno virus etc.) which are useful for cloning vectors in eukaryotic cells.

It should be however noted that the present invention also encompasses vectors which integrate into the genome of the host cell, e.g. the E.coli bacteriophage λ.

Expression and/or cloning vectors should also contain a selection gene, also termed a selectable marker. This is a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate or tetracyclin, or that complement auxothrophic deficiencies.

Expression vectors, unlike cloning vectors, should contain an expression signal which is recognized by the host organism as operably linked to the nucleic acid molecule according to the present invention. Expression signals are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100-1,000 bp) that control the transcription and translation of nucleic acid under their control.

Expression signals or promoters typically fall into two clas¬ ses, inducible and constitutive. Inducible expression signals are promoters that initiate increased levels of transcription under their control in response to some change in the environment, e.g. the presence or absence of a particular chemical compound or a change in temperature.

The nucleic acid molecule of the present invention is operably linked with a promoter in an expression vector. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and in the case of a leader sequence contiguous and in a reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Examples for promoters for use with prokaryotic hosts include the β-lactamase and lactose promoter systems, a tryptophan promoter system, and hybrid promoters, such as the tac- promoter. Suitable promoters for use with yeast host cells include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes. Suitable vectors and promoters for use in yeast expression are in more detail described in EP-A-0 073 657.

Promoters which are suitable for eukaryotic host cells include promoters obtained from the genoms of viruses, such as SV40, CMV and baculo virus. Especially preferred eukaryotic expression systems are the commercially available expression vectors pBK-CMV (Stratagene) and pCDM-8 (Invitrogen) which are suitable for recombinant expression of foreign DNA in monkey (COS) cells.

A further subject matter of the present invention is a cell which is transformed with a nucleic acid molecule or a vector according to the present invention. Suitable host cells for cloning or expressing the vectors are prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells include gram-negative or gram-positive organisms, for example E.coli or bacilli. Preferred cloning hosts are gram-negative prokaryotic organisms, especially E.coli.

In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast are suitable host cells. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains, are commonly available and useful.

Suitable host cells for the expression can also be derived from multi-cellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. The propagation of such cells in culture is well-known. Examples for useful eukaryotic host cell lines are Chinese hamster ovary (CHO) -cell lines, COS- cells and HeLa cells. Host cells are transformed with the above described expression and/or cloning vectors encultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting transformants containing amplified genes. The culture conditions, such as temperature, pH and the like, suitably are those previously used with the host cell selected for cloning or expression, as the case the may be, and will be apparent to a person skilled in the art.

Furthermore, the present invention refers to a process for the production of a recombinant polypeptide comprising the steps of:

(a) inserting a nucleic acid molecule as defined above into a suitable host cell,

(b) cultivating said host cell under conditions which are suitable for the expression of said nucleic acid molecule and

(c) isolating the expression product.

A preferable host cell is a prokaryotic cell, particularly an E.coli cell. A further preferable host cell is a eukaryotic cell such as a mammalian cell, e.g. a COS-cell, or an insect cell, e.g. a Sf9 cell.

A detailed discussion of cloning and/or expression vectors, the transformation of host cells with recombinant DNA and the expression of recombinant DNA in host cells is described in Molecular Cloning. A Laboratory Manual, 2nd edition, Sambrook et al. (1989) , Cold Spring Harbor, Laboratory Press, USA, particularly chapters 1, 2, 3, 4, 5, 10, 15, 16, 17 and 18, which are hereby incorporated by reference.

Still a further subject-matter of the present invention is a polypeptide produced by a process as defined above, which differs from naturally occurring chicken oocyte receptor P95 by means of glycosylation pattern and/or amino acid sequence. Preferably the polypeptide is non-glycosylated. The polypeptide can also be a fragment of the P95 protein, which preferably contains at least one complete structural element as defined above.

In an especially preferred embodiment the polypeptide contains at least one of the ligand-binding repeats 1-8 as defined in Fig. 2. If the protein contains more than one of the ligand-binding repeats also a shuffled arrangement of these repeats is possible, i.e. the order of the individual repeats may differ from the naturally occurring order.

The polypeptide can also be a chimeric receptor polypeptide comprising operably linked (a) at least one of the ligand- binding repeats 1-8 as defined in Fig. 2 and (b) at least one ligand-binding repeat of another LDL receptor family polypeptide.

In a further embodiment the polypeptide does not contain the amino acids of the membrane spanning domain, which are encoded by nucleotides 2,227 to 2,292 of Fig. 2.

The above mentioned fragments of the P95 protein are available by recombinant DNA technology. More specifically a nucleic acid comprising the desired portions of the sequence of Fig. 2 is constructed (e.g. by site directed mutagenesis) from the complete P95 cDNA sequence and expressed in an appropriate host.

This invention also provides compositions containing polypeptides which exhibit the same or similar immunological and/or biological properties of parts or the whole of P95. It is possible to use P95 and polypeptide fragments thereof as binding components in qualitative and quantitative diagnostic tests (such as ELISA, Ligand Blots, ligand/receptor coprecipitation procedures, and others known in the art) . Through site-specific mutagenesis of the native P95, it is possible to alter it so as to recognize other ligands than the ones already known. Since apo E concentrations are reported to be inversely related to the risk for atherogenesis, and the apo E4 allele has been reported to be associated with increased incidence of late onset Alzheimer's disease (see Corder, E.H. et al., Science 261, 921-923, 1993) , the P95 or apo E-binding fragments thereof could be used for qualitative and quantitative determination of this serum component.

The present invention further refers to a composition for qualitative and/or quantitative diagnostic tests comprising a polypeptide which exhibits at least one immunological and/or biological property of at least one of the structural elements of the mature chicken oocyte receptor as defined in Fig. 2. The composition is preferably in a liquid or lyophilized form.

The smaller size of P95 in comparison to all other LDLRs cha¬ racterized so far, is a distinct advantage over LDLRs from other sources and makes P95 advantageous to (i) determine ligand concentrations quantitatively, and to (ii) determine the isoform of certain ligands, e.g. of apo E, qualitatively.

Diagnostic kits for these purposes require larger amounts of P95 or fragments thereof than would easily be obtainable by purification from chicken oocytes. Therefore, the recombinant DNA production of P95 or fragments thereof is cheaper and more efficient than protein purification methodology. The carbohydrate-poor nature of P95 (see below, Fig. 4) is a further advantage in attempts to obtain biologically active protein in heterologous systems. Furthermore, well defined recombinant P95 or fragments thereof allow reproducible preparations for standardized application methods.

Thus, a process is provided for the quantitative and/or qualitative determination of P95 ligands in a liquid sample such as a body fluid, e.g. serum, or a tissue extract, comprising contacting said sample with a composition as defined above.

Further, the present invention refers to a process for the quantitative and/or qualitative determination of receptor ligands in a liquid sample, comprising

contacting said liquid sample with (i) an immobilised antibody or antibody-fragment, preferably a monoclonal antibody or a fragment thereof (e.g. a Fab, F(ab') or F(ab')₂ fragment) specific for a predetermined receptor ligand and (ii) a soluble receptor polypeptide specific for said predetermined receptor ligand and

measuring the binding of said soluble receptor polypeptide to said predetermined receptor ligand.

The soluble receptor polypeptide is preferably selected from receptors of the low-density lipoprotein receptor family, e.g. the P95 receptor, the LDL receptor, the VLDL receptor or chimaeras of said receptors. More preferably the soluble receptor polypeptide comprises at least one of the ligand- binding repeats 1-8 of P95 as defined in Fig. 2. The solubility of the receptor polypeptide is provided by complete or partial deletion of the respective membrane- spanning domain. Moreover, other domains, such as the cytoplasmic tail and the EGF-precursor-homologous-domains, which are not necessary for ligand-binding can be partially or completely deleted.

To provide an effective measurement of the binding of the soluble receptor to the receptor ligand, the soluble receptor is generally labelled. The label can be a direct label which is covalently attached to the receptor polypeptide or an indirect label, e.g. a receptor specific antibody, which in turn carries a label. The label can be anyone of the labels used in immunoassays of the prior art, e.g. a radioactive label, a fluorescent label, a chemiluminescent label, an enzymatic label, an NMR-active label or an affinity label. An enzymatic label, such as alkaline phosphatase, peroxidase or β-galactosidase is especially preferred. Methods of covalently labelling polypeptide molecules are common knowledge in the field of molecular biology and immunology and need not be discussed in detail.

The receptor ligand, which is to be determined in the process of the present invention, is preferably a ligand of a receptor of the LDL receptor family and more preferably selected from the group comprising apo lipoprotein B, apo lipoprotein E, lipoprotein lipase, α.₂-macroglobulin, pregnancy zone protein, vitellogenin, plas inogen activator - plasminogen activator inhibitor 1 complex, Pseudomonas aeruginosa exotbxin A, 39 kDa receptor-associated protein, riboflavin binding protein, Rous sarcoma virus envelope protein, and vesicular stomatitis virus surface epitope. It should however be noted that any other receptor ligand which can bind to a soluble receptor can be determined in the process according to the present invention.

The immobilisation of antibodies or antibody fragments to a solid phase, such as a test vessel, are known to a person skilled in the art. For example, the antibody can be immobilised to the solid carrier via a divalent spacer molecule or by means of a specific binding pair, e.g. a streptavidin-coated carrier and a biotinylated antibody.

Antibodies specific for predetermined receptor ligands are known in the prior art. Examples for such antibodies are monoclonal antibodies for apo lipoproteins B and E or Pseudomonas exotoxin A. These antibodies or fragments thereof are suitable for the process of the present invention.

Specific examples for antibodies which react with apo lipoproteins B and E are described by Fievet, C. et al. , J. Lipid Res. 30, 1015-1024, 1987; Koffigan, M. et al. , Clin. Chim. Acta 163, 245-256, 1987; Krul, E.S. et al. , J. Lipid Res. 29, 937-947, 1988; Tikkanen, M.J. et al. , Arterio¬ sclerosis 4, 138-146, 1984; Salmon, S. et al. , Biochem. Biophys. Res. Commun. 125, 704-711, 1984; Tikkanen et al., J. Lipid Res. 23, 1032-1038, 1982; Wilkinson, J. et al. , J. Lipid Res., 34, 815-825, 1993; Gherardi, E.A. et al. , Biochem. J. , 252, 237-245, 1988.

A great advantage of the process according to the present invention over the prior art is based on the fact that the determination of the receptor ligand is carried out via a combination of (i) an immunological binding with an antibody and (ii) a receptor-ligand activity binding. The binding of the receptor ligand to an antibody is highly specific for a predetermined ligand, and enables a quantitative measurement of the ligand concentration. It can however generally not be used to determine the functionality and/or activity of the bound ligand. The receptor-ligand binding is not as specific as the binding of the antibody to the ligand, however the functionality and activity of the bound ligand can be determined very accurately by its ability to bind to the receptor. By this combination of binding methods not only the concentration of a predetermined ligand but also its functionality and activity can be determined.

Preferably the process according to the present invention comprises:

contacting the liquid sample containing the predetermined receptor ligand to be measured with a receptor ligand- specific antibody or antibody fragment, which is immobilised on a solid phase,

separating the liquid sample from the solid phase, on which the receptor ligand in the liquid sample has been fixed by binding to the antibody,

contacting the solid phase with a receptor ligand- specific, soluble receptor polypeptide which is directly or indirectly labelled, and

measuring the label on said solid phase.

By means of the above process the label measured on the solid phase is proportional to the functionality and/or activity of the receptor ligand to be measured. Of course it is possible to carry out several parallel determinations of several predetermined receptor ligands using different ligand- specific antibodies and/or different soluble receptor polypeptides.

The following figures and sequence listings aid in under¬ standing the field and scope of the invention.

Fig. 1: Northern blot - Distribution of P95 mRNA in chicken tissues.

Poly- (A) -RNA (2.5 μg per lane) from the indicated chicken tissues was electrophoresed on a 1.5 % agarose gel and subjected to Northern Hybridization. Prehybridization was for 5 hr in 50% formamide, 5 X SSC, 5 X Denhardt's solution and 0.1% SDS at 42°C, and hybridization for 20 hr under identical conditions with the addition of 32-P-labeled random-primed probe. The probe was a mixture of 1.1 kb Xho I (vector) -Bgl II (nt 1.035) fragment and 0.7 kb Bgl II (nt 1.036) -Bgl II (nt 1.746) fragment of the Bluescript IIKS + (Stratagene Cloning Systems, La Jolla, CA, USA) plasmid which contained full-length P95 cDNA. The final wash was for 1 hr in 0.1 x SSC, 0.1% SDS at 50°C. The membrane was exposed with an intensifying screen for 48 hr. The Hindlll digest of phage lambda DNA was used for size markers (in kb) . Fig. 2: cDNA sequence and deduced amino acid sequence of P95.

Following the signal sequence (boxed) , there are eight repeats of approximately 40 residues each, each containing 6 cysteinyl residues (Repeat I to VIII) , two repeats with homology to EGF-precursor repeats (labeled A and B, respectively) , a region containing 5 repeated elements with the consensus tetrapeptide Tyr/Phe-Trp-Xxx-Asp (asterisks) , another EGF-precursor-homologous region (C) , the membrane spanning domain (boxed) , and the cytoplasmic tail that contains the internalization signal AsnPhe-Asp-Asn-Pro-Val- Tyr (doubly underlined) .

Fig. 3: Comparison of the derived amino acid sequence of P95 with the amino acid sequences of VLDL receptor from rabbit (see Takahashi, S. et al. , Proc. Natl. Acad. Sci. U.S.A. 89, 9252-9256, 1992) , and LDL receptors from rabbit, man, and Xenopus (see Mehta, K.D. et al. , J. Biol. Chem. 266, 10406- 10414, 1991) . Note the absence in P95 (Chicken V/VR) of a region thought to contain carbohydrate in O-glycosidic linkage in LDL receptors (dashed line) .

Fig. 4: Extent of identity between P95 and similar receptors.

Structural elements of P95 (Chicken VTG/VLDLR) as defined in the legend to Fig. 2 are aligned with homologous elements in the rabbit VLDL receptor (VLDLR) (see Takahashi, S. et al. , Proc. Natl. Acad. Sci. U.S.A., 89, 9252-9256, 1992) and the rabbit LDL receptor (LDLR) (see Yamamoto, T. et al. Science 232, 1230-1237, 1986) . The percentage of identical amino acid residues for each domain or subset of domains is indicated.

Fig. 5:

Immunoblotting analysis of expressed P95 in COS-7 cells

COS-7 cells were transiently transfected with the P95 expression plasmid pCDMCVR-1 (lanes 2,5 and 8; 70 μg protein/lane) or vector alone (lanes 3, 6 and 9; 70 μg cell protein/lane) , and processed for immunoblotting following SDS-PAGE under nonreducing conditions as described in Example 3. Lanes 1 and 7 contained 5 μg, and lane 4 contained 1 μg of oocyte membrane protein. Immunoblotting incubations were performed with 2 μg/ml anti-P95 IgG (lanes 1-3) , 20 μg/ml anti-carboxyterminal IgG (lanes 4-6) , and 20 μg/ml nonimmune IgG (lanes 7-9) . Exposure times were 5 min. (lanes 1-3 and 7- 9) and 2 min. (lanes 4-6) , respectively. The position of migration of the 95-kDa receptor is indicated by a closed circle.

Fig. 6:

Functional analysis of P95 expressed in COS-7 cells

Surface binding of (A)¹²⁵I-VLDL (482 cpm/ng) and (B)¹²⁵I-VTG (613 cpm/ng) to monolayers of pCDMCVR-1-transfected (closed circles) and vector-only-transfected (open circles) . COS-7 cells were determined as described in Example 4. The data are the average of duplicate determinations and represent high- affinity binding, which is the difference between binding in the absence and presence of excess unlabelled ligand (1 mg/ml VLDL, panel A; and 750 μg/ml VTG, panel B) . In panel C, an aliquot of the cells (60 μg protein/lane) used in A and B were subjected to ligand blotting with the same ¹²⁵I-labelled ligands at 4 μg/ml. Lanes 1 and 4, 2 dishes each of pCDMCVR-1 transfected cells; lanes 2 and 5, 2 dishes each of control cells; lanes 3 and 6, 0.15 μg of oocyte membrane protein. Autoradiography was for 30 h. The arrow indicates the position of the 95-kDa VLDL/VTGR. The insert in panel A shows the results of immunoblotting with the anti-carboxyterminal IgG performed as in Fig. 5; lane 1, 1 μg oocyte membrane protein; lanes 2 and 3, 60 μg protein of pCDMCVR-1 transfected or control COS-7 cells, respectively.

Fig. 7:

Functional analysis of the soluble P95 in conditioned medium of transfected COS-cells.

48 hr after the transfection with pBKV/ps95 (lanes 2 and 3) or vector only, pBKCMV (lane 1) , the conditioned medium was collected from each dish and subjected to ligand blotting (one tenth of a dish/lane) with ¹²⁵I-VLDL (4 μg/ml, 482 cpm/ng) following SDS-PAGE under nonreducing conditions as described in Example 4. In lane 3, 50 fold excess of unlabelled VLDL was added to the incubation. Exposure time was 30 hr. The position of migration of the truncated soluble receptor is indicated by an arrowhead. Molecular masses in kDa are indicated.

Fig. 8:

Functional analysis of the P95/human LDL receptor chimaera in transfected COS cells.

48 hr after transfection with pBKCVLR (lanes 3 and 4) or vector only (lanes 1 and 2) , the cells were collected from each dish and subjected to ligand blotting (80 μg/lane) with ¹²⁵I-VTG (4 μg/ml, 613 cpm/ng, lanes 1 and 3) or ^12SI-VLDL (4 μg/ml, 482 cpm/ng, lanes 2 and 4) following SDS-PAGE under nonreducing conditions as described in Example 4. Exposure time was 30 hr. The position of migration of the chimaeric receptor is indicated by an arrowhead. Molecular masses in kDa are indicated.

SEQ ID NO. 1: P95 cDNA sequence

Nucleotide 1 in SEQ ID NO. 1 corresponds to nucleotide -150 in Fig. 2. The signal sequence starts at nucleotide 13 and the sequence coding for the mature protein starts at nucleotide 151. The coding sequence ends with nucleotide 2661.

SEQ ID NO. 2: P95 amino acid sequence The deduced amino acid sequence of P95 is shown in the three- letter-code. The signal peptide sequence ranges from position -46 to -1. The sequence coding for the mature protein starts at position 1 with the sequence Ala-Lys-Ala-Lys-Lys.

SEQ. ID. NO. 3: cDNA sequence of a P95/LDL receptor chimaera

The signal sequence starts at nucleotide 13 and the sequence coding for the mature protein starts at nucleotide 150. The P95 part ends at nucleotide 267. The LDL receptor part starts with nucleotide 268 and ends with nucleotide 2784.

SEQ. ID. NO. 4: Amino acid sequence of a P95/LDL receptor chimaera

The signal peptide sequence ranges from -46 to -1. The sequence coding for the mature polypeptide starts with position 1. The sequence coding for the P95 part ends with position 39. The sequence coding for the LDL receptor part starts at position 40 with the sequence Ala-Val-Gly-Asp-Arg.

Examples

The invention can be understood by reference to the following examples:

1. Tissue distribution of P95 mRNA.

As expected of a receptor that is the key molecule for the import of yolk components into growing oocytes, mRNA levels as detected by Northern blotting are by far the highest in the ovary (Fig. 1) . P95 mRNA (approx. 3.5 kb) can also be detected, but at low levels compared to the ovary, in heart and skeletal muscle, which in contrast are by far the major sites of expression of the VLDL receptor in rabbits (see Takahashi, S. et al. , Proc. Natl. Acad. Sci. U.S.A. 89, 9252- 9256, 1992) . Importantly, also in contrast to the rabbit VLDL receptor, the laying hen does not display detectable P95 message in any other tissue than ovary, heart, and muscle under these conditions.

2. cDNA cloning of P95

Chicken ovarian follicles of laying hens contain high levels of transcripts coding for P95 (Fig. 1) . A cDNA library was constructed in an Okayama-Berg vector (Okayama, H. and Berg, P. Mol. Cell. Biol. 2, 161-170, 1983) with poly(A)⁺RNA isolated from chicken embryo. Screening of the cDNA library under low-stringency conditions with a probe derived from the rabbit VLDL receptor cDNA (Takahashi, S., Kawarabayashi, Y., Nakai, T., Sakai, J. and Yamamoto, T. Proc. Natl. Acad. Sci. U.S.A. 89, 9252-9256, 1992) yielded several hybridizing cDNA clones. Partial nucleotide sequence analysis identified one of the clones as encoding the full-length of chicken counterpart of the rabbit VLDL receptor. The cDNA insert of the recombinant phage was subcloned into Bluescript II vector (Stratagene Cloning Systems, La Jolla, CA) , and sets of nested deletions were prepared from the cDNA insert by digestion with Exonuclease III and SI nuclease (Pharmacia) . The complete nucleotide sequence of the cDNA clone was determined on both strands by the dideoxy chain determination method with T7 DNA polymerase (United States Biochemical Corp.) or the large fragment of DNA polymerase I (Gibco-BRL) . All molecular biological techniques were performed essentially as described (Sambrook, J. , Fritsch, E.F., and Maniatis, T. Molecular Clonig: A laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY, 2nd Ed., 1989) .

Fig. 2 shows the sequence of the cDNA that codes for P95, and the deduced amino acid of the coding region. Nucleotide residues are numbered in the 5' to 3' direction, beginning with the codon specifying the N-terminal residue of the proposed mature protein. Analysis of nucleotide and amino acid sequences was performed using GeneWorks software (IntelliGenetics, Inc.) . The complete coding region spans 2589 nucleotides, coding for 863 amino acid residues. Fig. 3 provides a comparison of P95 protein sequence with homologous proteins from man, frog, and rabbit. Figs. 3 and 4 also illustrate the modular (domain) structure of P95. Its 863 amino acid residues are arranged, from the aminoterminal end, into:

1) a signal sequence,

2) 8 successive similar stretches, each consisting of approximately 40 amino acid residues of which 6 are cysteinyl residues (the 8 repeats span a total of 327 amino acid residues) ,

3) 2 different successive repeats with 6 cysteinyl residues each,

4) 5 repeats that contain the signature tetrapeptide Y/FWXD,

5) 1 repeat as described in 3) ,

6) a membrane spanning domain, and

7) the intracellular domain of 50 amino acid residues which contains the sequence NFDNPVY, required for receptor in- ternalization and identical in all LDL and VLDL receptors characterized so far.

These contiguous sequences of amino acids define distinct structural elements of P95 that likely function distinctly to assure the biological action of the protein.

3. Recombinant expression of P95

3.1 Transformation of COS-7 cells

COS-7 cells (American Type Culture Collection) were seeded at a density of 1.5 x 10⁶ per 80 cm² dish and incubated overnight in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 0.05 mM β- mercaptoethanol (standard medium) . The P95 cDNA in Bluescript was excised by Xho I and Not I, and subcloned into the cytomegalovirus promoter-driven expression vector pCDM8 (Invitrogen) . The resulting recombinant plasmid, pCDMCVR-1, or the starting plasmid pCDM8 (20 μg per dish) , was transiently transfected into COS-7 cells by electroporation using a Bio-Rad Gene Pulser. Dishes (60 mm diameter) were seeded with 4 x 10⁵ cells each in standard medium; after 48 h, the cells were either prepared for immunoblotting, ligand blotting, or cell surface binding assays as described below.

3.2 Antibody production and immunoblotting

Antiserum against the carboxytermmus of the chicken P95 was prepared as follows. A synthetic peptide corresponding to the last 14 amino acids of the deduced amino acid sequence of the cloned cDNA for the chicken P95 was coupled to keyhole limpet hemocyanin (KLH)

(Schneider et al. (1983), J. Cell. Biol. 97, 1635-1640) and used for immunization of New Zealand White rabbits as described (Nimpf et al. (1988), J. Lipid Res. 29, 657-667) . IgG fractions were purified from sera on columns of protein A-Sepharose CL-4B (Beisiegel et al.

(1981), J. Biol. Chem. 256, 11923-11932). Rabbit anti- VLDL/VTGR IgG was obtained by immunization with 95 kDa protein and purified as described previously (Barber et al. (1991), J. Biol. Chem. 266, 18761-18770).

Transiently transfected COS-7 cells were washed 3 times with PBS and harvested in PBS containing 0.5 mM phenylmethanesulfonyl fluoride (PMSF) and 2.5 μM leupeptin. Cells were pelleted by centrifugation and detergent extracts were prepared as follows. The cell pellet derived from one dish (80 cm²) was resuspended in 75 μl of ice-cold solubilization buffer containing 200 mM Tris-maleate (pH 6.5), 2 mM CaCl₂, 0.5 mM PMSF, 2.5 μM leupeptin, and 1% Triton X-100 and kept on ice for 10 min. The extraction mixture was centrifuged at 300,000 g for 40 min. at 4 °C and the resulting supernatant was used for immunoblotting. Protein concentrations were determined by the method of Lowry et al. (1951), J. Biol. Chem. 193, 265-275.

One-dimensional gradient (4.5-18%) SDS-polyacrylamide gel electrophoresis (PAGE) was performed using a minigel system (Bio-Rad, Mini-Protean™ II Slab Cell) . Samples were prepared in the absence of dithiothreitol (DTT) and without heating (nonreducing conditions) . Electrophoresis was performed at 180 V for 60 min. Broad range M_r standards (Bio-Rad) were used. Electrophoretic transfer of the proteins to nitrocellulose membrane (Bio-Rad, pore size 0.45 μm) was performed in transfer buffer (26 mM Tris, 192 mM glycin) for 2 h at 200 mA, on ice, using the Bio-Rad Mini Transblot system. The transferred proteins were stained with 0.2% Ponceau S in 3% (w/v) TCA and destained with water. Western blotting was performed using specific rabbit antibodies at the concentrations indicated in the Figure legends, followed by protein-A-horseradish peroxidase (HRP-Sigma) and the chemiluminescence detection method (ECL system, Amersham) . Membranes were exposed for 0.1-5 min. on Hyperfilm™-ECL (Amersham) .

3.3 Expression and immunological identification of recombinant P95

In order to test whether pCDMCVR-1 indeed leads to the expression of the same receptor that is present in chicken oocytes, we used two polyclonal antibodies in immunoblots of detergent extracts from oocyte membranes and transfected COS-7 cells (Figure 5) . One of the newly-raised antibodies, raised against purified P95, reacts with the 95-kDa protein as well as another protein in oocyte extracts which represents an oocyte- specific lipo- protein receptor polypeptide (approximate size, 380 kDa, Fig. 5, lane 1) . COS-7 cells transfected with the P95 cDNA containing plasmid, but not with the vector alone, expressed a single crossreactive 95-kDa protein comigrating with the native oocyte protein (Fig. 5, lanes 1-3) . The other antibody, raised against an oligopeptide corresponding to the carboxyterminal 14 residues of the cloned receptor, reacted with the product of pCDMCVR-1 expression in COS-7 cells (lane 5) , and importantly, also with the bona-fide receptor of oocytes (lane 4) . This antibody shows no crossreactivity with the oocytic LRP, nor with any other protein in chicken oocytes (lane 4) ; in COS-7 cells transfected with pCDMCVR-1 (lane 5) or vector alone (lane 6) , there is a weakly crossreactive large protein, possibly a simian member of the LDLR gene family. These results demonstrate unambiguously that the cloned chicken cDNA encodes the previously described oocyte-specific VLDL/VTG receptor P95.

4. Ligand binding function of recombinant P95

4.1 Surface binding of VLDL and VTG to transfected COS-7 cells and ligand blotting

VLDL and VTG were radiolabelled with ¹²⁵I as described previously (Barber et al. (1991) Supra) to a specific radioactivity of 482 cpm/ng and 613 cpm/ng, respectively. All assays were performed on ice. Monolayers of COS-7 cells transiently transfected with pCDMCVR-1, and control cells (transfected with pCDM8) , were incubated for 3 h in standard medium containing 2 mg/ml bovine serum albumin and the concentrations of radioiodinated and unlabelled ligands indicated in the legend to Fig. 6. The medium was then removed and the monolayers carefully washed to remove unbound ligand as described previously (Hayashi et al. (1989), J. Biol. Chem. 264, 3131-3139) . Cell-associated radioactivity was determined by liquid scintillation counting following solubilization of the cells in 1 ml of 0.1 N NaOH. Ligand blotting following SDS-PAGE and electrophoretic transfer were performed as described above.

.2 Ligand binding function of the receptor expressed in COS-7 cells

As demonstrated in Fig. 6, A and B, the cells expressing P95 showed saturable, high affinity binding of the receptor ligands VLDL and VTG, with maximal amounts of binding 2- to 3-fold higher than that of control- transfected cells. An exact determination of binding parameters for the expressed heterologous receptor is not possible due to saturable ligand binding to endogenous sites (open circles) ; however, maximum binding of VLDL and VTG to transfected cells were comparable, and the K_d values for both ligands were in the range of 3-5 μg/ml.

The combination of results of ligand- and immunoblotting demonstrates that the expression of chicken cDNA in a heterologous cell system leads to a functionally active P95 receptor.

5. Construction of truncated soluble P95 and a chimaera of P95 and the human LDL receptor

cDNAs encoding a truncated, soluble P95 (all 8 ligand binding repeats and part of EGF precursor region A) , and a chimeric receptor consisting of part of P95 (signal sequence and binding repeat 1) and LDL receptor (mature form of the receptor, i.e., without signal sequence) were constructed in expression plasmids for transfection into cultured mammalian cells as follows. For the truncated receptor (sP95) , the 1.2 kilobasepair (kb) SalI/BglII fragment from P95 cDNA (Fig. 2) was inserted into the Sail and BamHI sites of the expression vector pBK-CMV (Stratagene) to yield pBKV/sP95. For the construction of a chimeric receptor (chP95) , two synthetic oligonucleotides were prepared: LV-F, 5'- GATGAAAGTGCTTGTGCAGTGGGCGACAGA-3'and VL-R, TCTGTCGCCCACTGCACAAGCACTTTCATC-3' . The nucleotide sequences of primers LV-F and VL-R were corresponding to the sense and antisense sequences of the annealing region in the chimaera of P95 and human LDL receptor, respectively (the end of binding repeat 1 in P95 and the beginning of binding domain 1 of the human LDL receptor) . pBKV/sP95 was amplified by polymerase chain reaction (PCR) with T3 primer (Stratagene) and VL-R. Human LDL receptor cDNA was inserted into the HindiII site of pBK-CMV, and the resultant plasmid pBKLDLR was amplified by PCR with LV-F and T7 primer (Stratagene) . The mixed aliquots of the resultant PCR products were re-amplified with T3 and T7 primers. PCR parameters were 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, for 30 cycles. Finally, the amplified " 3kb fragment was cleaved with Sail and Notl, and inserted into the same sites of pBK-CMV to yield pBKCVLR. The nucleotide sequence of chimeric receptor in the expression vector was confirmed using Sequenase (US Biochemical) . The nucleotide and amino acid sequence is shown in SEQ. ID. NO. 3 and SEQ. ID. NO. 4.

6. Ligand blot analysis of the transformed cultured COS cells

COS-7 cells were transiently transfected with plasmids pBK-CMV, pBKV/sP95, or pBKCVLR (20 μg per dish), as described in Example 3. The ligand blot analysis was carried out as described in Example 4.

Fig. 7 and Fig. 8 demonstrate that the recombinant soluble P95 and the recombinant P95 / human LDL receptor chimaera are functionally active.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: PROGEN Biotechnik GmbH

(B) STREET: Im Neuenheimer Feld 519

(C) CITY: Heidelberg

(E) COUNTRY: Deutschland

(F) POSTAL CODE (ZIP) : 69120

(ii) TITLE OF INVENTION: DNA-sequences encoding chicken oocyte receptor P95 (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2781 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Gallus gallus (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (ix) FEATURE:

(A) NAME/KEY: mat_j>eptide

(B) LOCATION: 151..2601 (ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 13..150 ( ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 13..2601

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CTCGGCGCGG CG ATG CGG TCG AGC CGG CAG CGC GGA GAC CGG AGC GCG 48

Met Arg Ser Ser Arg Gin Arg Gly Asp Arg Ser Ala -46 -45 -40 -35

GCG ACC GGC GGC GGG TGT GGG GCG CGG CGG TGG GCG CTC CCG CGC TGC 96 Ala Thr Gly Gly Gly Cys Gly Ala Arg Arg Trp Ala Leu Pro Arg Cys -30 -25 -20

GGG GCG CTC TGC CTG CTG CTC GCC CTC GGC TGC CTG CGT ACT GCC ACC 144 Gly Ala Leu Cys Leu Leu Leu Ala Leu Gly Cys Leu Arg Thr Ala Thr -15 -10 -5

GAC GGT GCA AAA GCA AAA TGT GAG GAG TCC CAG TTC CAG TGT AGT AAT 192 Asp Gly Ala Lys Ala Lys Cys Glu Glu Ser Gin Phe Gin Cys Ser Asn 1 5 10

GGA CGC TGT ATT CCT TTA CTC TGG AAA TGT GAT GGT GAT GAA GAC TGT 240 Gly Arg Cys lie Pro Leu Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys 15 20 25 30

TCA GAC GGC AGT GAT GAA AGT GCT TGT GTC AAG AAG ACA TGT GCT GAA 288 Ser Asp Gly Ser Asp Glu Ser Ala Cys Val Lys Lys Thr Cys Ala Glu 35 40 45

TCT GAC TTT GTG TGT AAC AGT GGT CAG TGT GTG CCG AAC AGA TGG CAG 336 Ser Asp Phe Val Cys Asn Ser Gly Gin Cys Val Pro Asn Arg Trp Gin 50 55 60

TGT GAT GGG GAT CCG GAC TGT GAG GAT GGG TCT GAC GAG AGT GCT GAA 384 Cys Asp Gly Asp Pro Asp Cys Glu Asp Gly Ser Asp Glu Ser Ala Glu 65 70 75

CTG TGC CAT ATG AGA ACA TGC CGG GTA AAT GAG ATC AGC TGT GGT CCT 432 Leu Cys His Met Arg Thr Cys Arg Val Asn Glu lie Ser Cys Gly Pro 80 85 90

CAG TCA ACC CAG TGT ATC CCA GTG TCC TGG AAA TGT GAT GGT GAA AAA 480 Gin Ser Thr Gin Cys lie Pro Val Ser Trp Lys Cys Asp Gly Glu Lys 95 100 105 110 GAC TGT GAC AGT GGA GAA GAT GAA GAG AAT TGT GGC AAT GTG ACT TGT 528 Asp Cys Asp Ser Gly Glu Asp Glu Glu Asn Cys Gly Asn Val Thr Cys 115 120 125

AGT GCA GCA GAG TTC ACA TGC AGT AGT GGG CAG TGT ATT TCC AAG AGC 576 Ser Ala Ala Glu Phe Thr Cys Ser Ser Gly Gin Cys lie Ser Lys Ser 130 135 140

TTT GTC TGC AAT GGT CAA GAT GAC TGC AGT GAT GGT AGT GAT GAG TTG 624 Phe Val Cys Asn Gly Gin Asp Asp Cys Ser Asp Gly Ser Asp Glu Leu 145 150 155

GAG TGT GCA CCT CCA ACA TGT GGT GTT CAT GAG TTC CAG TGC AAG AGC 672 Glu Cys Ala Pro Pro Thr Cys Gly Val His Glu Phe Gin Cys Lys Ser 160 165 170

TCC ACT TGC ATC CCT ATC AGC TGG GTG TGT GAT GAT GAT GCT GAC TGC 720 Ser Thr Cys lie Pro lie Ser Trp Val Cys Asp Asp Asp Ala Asp Cys 175 180 185 190

TCT GAC CAC TCT GAT GAA TCT TTG GAG CAG TGT GGC CGA CAG CCT GCA 768 Ser Asp His Ser Asp Glu Ser Leu Glu Gin Cys Gly Arg Gin Pro Ala 195 200 205

CCT CCT GTG AAG TGC TCT ACC AGT GAG GTG CAG TGC GGC TCA GGT GAA 816 Pro Pro Val Lys Cys Ser Thr Ser Glu Val Gin Cys Gly Ser Gly Glu 210 215 220

TGT ATC CAC AAG AAG TGG CGC TGT GAT GGA GAT CCT GAC TGC AAA GAT 864 Cys lie His Lys Lys Trp Arg Cys Asp Gly Asp Pro Asp Cys Lys Asp 225 230 235

GGA AGT GAT GAA ATC AAC TGC CCT TCT CGG ACC TGC AGA CCA GAC CAG 912 Gly Ser Asp Glu lie Asn Cys Pro Ser Arg Thr Cys Arg Pro Asp Gin 240 245 250

TTT AGG TGT GAA GAT GGG AAC TGC ATC CAT GGG AGC AGG CAG TGC AAT 960 Phe Arg Cys Glu Asp Gly Asn Cys lie His Gly Ser Arg Gin Cys Asn 255 260 265 270

GGT GTG AGA GAC TGT CTA GAT GGC ACT GAT GAA GCA AAC TGT AAC AAT 1008 Gly Val Arg Asp Cys Leu Asp Gly Thr Asp Glu Ala Asn Cys Asn Asn 275 280 285

GTT ATT CAG TGT TCT GGA CCT GGC AAA TTC AAG TGC AGA AGT GGA GAA 1056 Val lie Gin Cys Ser Gly Pro Gly Lys Phe Lys Cys Arg Ser Gly Glu 290 295 300 TGC ATA GAT ATT AAT AAA GTG TGT AAC CAT CAC GGA GAC TGC AAG GAC 1104 Cys He Asp He Asn Lys Val Cys Asn His His Gly Asp Cys Lys Asp 305 310 315

TGG AGT GAT GAG CCT CTC AAG GAA TGT AAC ATA AAT GAG TGT TTG GTC 1152 Trp Ser Asp Glu Pro Leu Lys Glu Cys Asn He Asn Glu Cys Leu Val 320 325 330

AAC AAT GGT GGA TGC TCG CAC ATC TGC AGA GAT CTT GTT ATT GGC TAT 1200 Asn Asn Gly Gly Cys Ser His He Cys Arg Asp Leu Val He Gly Tyr 335 340 345 350

GAA TGT GAC TGT CCA GCT GGG TTT GAG CTT GTA GAC AGG AGA ACC TGT 1248 Glu Cys Asp Cys Pro Ala Gly Phe Glu Leu Val Asp Arg Arg Thr Cys 355 360 365

GGA GAT ATT GAT GAA TGC CAG AAT CCT GGT ATC TGT AGC CAA ATC TGT 1296 Gly Asp He Asp Glu Cys Gin Asn Pro Gly He Cys Ser Gin He Cys 370 375 380

ATC AAC CTG AAA GGG GGA TAC AAG TGT GAA TGT AGC CGT GGC TAT CAG 1344 He Asn Leu Lys Gly Gly Tyr Lys Cys Glu Cys Ser Arg Gly Tyr Gin 385 390 395

ATG GAT CTT GCT ACA GGA GTG TGC AAG GCT GTG GGG AAA GAA CCA TGT 1392 Met Asp Leu Ala Thr Gly Val Cys Lys Ala Val Gly Lys Glu Pro Cys 400 405 410

CTG ATT TTC ACC AAC CGA CGG GAT ATC AGG AAG ATT GGC CTT GAG AGA 1440 Leu He Phe Thr Asn Arg Arg Asp He Arg Lys He Gly Leu Glu Arg 415 420 425 430

AAA GAA TAC ATT CAG CTA GTA GAG CAG CTA AGA AAC ACA GTT GCT CTA 1488 Lys Glu Tyr He Gin Leu Val Glu Gin Leu Arg Asn Thr Val Ala Leu 435 440 445

GAT GCT GAT ATT GCT GAG CAA AAG CTT TAT TGG GCT GAC TTC AGC CAA 1536 Asp Ala Asp He Ala Glu Gin Lys Leu Tyr Trp Ala Asp Phe Ser Gin 450 455 460

AAA GCA ATT TTC AGT GCC TCT ATT GAT ACC CGT GAT AAA GTT GGA ACA 1584 Lys Ala He Phe Ser Ala Ser He Asp Thr Arg Asp Lys Val Gly Thr 465 470 475

CAC ACT AGA ATC CTA GAC AAC ATA CAC AGC CCT GCA GGA ATT GCT GTT 1632 His Thr Arg He Leu Asp Asn He His Ser Pro Ala Gly He Ala Val 480 485 490 GAC TGG ATT TAT AAG AAC ATC TAC TGG ACT GAC TCA TCT GCA AAG ACC 1680 Asp Trp He Tyr Lys Asn He Tyr Trp Thr Asp Ser Ser Ala Lys Thr 495 500 505 510

ATT TCA GTG GCC AGC CTG AAT GGC AAG AAA AGA AAG GTT TTA TTT CTT 1728 He Ser Val Ala Ser Leu Asn Gly Lys Lys Arg Lys Val Leu Phe Leu 515 520 525

TCT GAG CTG AGA GAG CCA GCT TCT ATT GCT GTA GAT CCT CTC TCT GGC 1776 Ser Glu Leu Arg Glu Pro Ala Ser He Ala Val Asp Pro Leu Ser Gly 530 535 540

TTT ATG TAC TGG TCA GAC TGG GGT GAG CCA GCA AAA ATT GAA AAA GCA 1824 Phe Met Tyr Trp Ser Asp Trp Gly Glu Pro Ala Lys He Glu Lys Ala 545 550 555

GGA ATG AAT GGA TTT GAC AGA CAG CAG CTT GTG ACA ACA GAA ATC CAA 1872 Gly Met Asn Gly Phe Asp Arg Gin Gin Leu Val Thr Thr Glu He Gin 560 565 570

TGG CCT AAT GGC ATT GCT TTA GAT CTT GTA AAA AGC CGT TTG TAT TGG 1920 Trp Pro Asn Gly He Ala Leu Asp Leu Val Lys Ser Arg Leu Tyr Trp 575 580 585 590

CTT GAT TCT AAA CTA CAT ATG CTC TCA AGT GTG GAT CTG AAT GGC CAG 1968 Leu Asp Ser Lys Leu His Met Leu Ser Ser Val Asp Leu Asn Gly Gin 595 600 605

GAT CGT AGA CTT GTG CTC AAG TCT CAT ATG TTC CTT CCT CAT CCT CTT 2016 Asp Arg Arg Leu Val Leu Lys Ser His Met Phe Leu Pro His Pro Leu 610 615 620

GCT CTA ACA ATA TTT GAG GAT CGT GTA TTC TGG ATT GAC GGA GAG AAC 2064 Ala Leu Thr He Phe Glu Asp Arg Val Phe Trp He Asp Gly Glu Asn 625 630 635

GAG GCA GTC TAT GGT GCC AAC AAA TTT ACT GGA GCT GAA TTG GTC ACC 2112 Glu Ala Val Tyr Gly Ala Asn Lys Phe Thr Gly Ala Glu Leu Val Thr 640 645 650

CTA GTA AAC AAC CTC AAT GAT GCG CAG GAC ATC ATT GTT TAT CAT GAA 2160 Leu Val Asn Asn Leu Asn Asp Ala Gin Asp He He Val Tyr His Glu 655 660 665 670

CTT GTT CAA CCT TCA GGC AGG AAC TGG TGT GAA GAG AAC ATG GTA AAT 2208 Leu Val Gin Pro Ser Gly Arg Asn Trp Cys Glu Glu Asn Met Val Asn 675 680 685 GGA GGC TGT AGC TAC CTG TGC CTG CCT GCT CCT CAG ATA AAT GAA CAC 2256 Gly Gly Cys Ser Tyr Leu Cys Leu Pro Ala Pro Gin He Asn Glu His 690 695 700

TCT CCG AAG TAT ACT TGC ACA TGT CCT GCT GGA TAT TTC TTG CAG GAG 2304 Ser Pro Lys Tyr Thr Cys Thr Cys Pro Ala Gly Tyr Phe Leu Gin Glu 705 710 715

GAC GGT CTG AGA TGT GGA GGA TTC AAC ATC AGT AGT GTG GTG TCT GAA 2352 Asp Gly Leu Arg Cys Gly Gly Phe Asn He Ser Ser Val Val Ser Glu 720 725 730

GTA GCT GCA AGA GGA GCA GCA GGA GCT TGG GCT GTT CTT CCT ATC TTA 2400 Val Ala Ala Arg Gly Ala Ala Gly Ala Trp Ala Val Leu Pro He Leu 735 740 745 750

CTG CTG GTG ACG GCT GCA TTG GCT GGC TAC TTC ATG TGG CGT AAT TGG 2448 Leu Leu Val Thr Ala Ala Leu Ala Gly Tyr Phe Met Trp Arg Asn Trp 755 760 765

CAG CAC AAG AAC ATG AAA AGC ATG AAT TTT GAT AAT CCC GTC TAT CTG 2496 Gin His Lys Asn Met Lys Ser Met Asn Phe Asp Asn Pro Val Tyr Leu 770 775 780

AAA ACT ACA GAA GAG GAC CTC ACA ATT GAT ATT GGC AGA CAC AGT GGT 2544 Lys Thr Thr Glu Glu Asp Leu Thr He Asp He Gly Arg His Ser Gly 785 790 795

TCA GTG GGA CAC ACC TAC CCT GCA ATA TCT GTT GTA AGC ACA GAT GAT 2592 Ser Val Gly His Thr Tyr Pro Ala He Ser Val Val Ser Thr Asp Asp 800 805 810

GAT ATG CTG TGAGTGCTGG ATCAGCAATC ACTTTCAGTT TACTTTGTGT 2641

Asp Met Leu

815

TTTACACTTA CGGGGATGAT AAACATGCTT GTGGCTGAAA GACTTCCTCC ATTCTTGGAA 2701

GAATGAAGAA ACTTTCTCTG TGTATGGAAC ACTTACATAA TTAGCTGTTT TATACAGCTT 2761

AACAACCAAC TCTGTAAATA 2781 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 863 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Arg Ser Ser Arg Gin Arg Gly Asp Arg Ser Ala Ala Thr Gly Gly -46 -45 -40 -35

Gly Cys Gly Ala Arg Arg Trp Ala Leu Pro Arg Cys Gly Ala Leu Cys -30 -25 -20 -15

Leu Leu Leu Ala Leu Gly Cys Leu Arg Thr Ala Thr Asp Gly Ala Lys -10 -5 1

Ala Lys Cys Glu Glu Ser Gin Phe Gin Cys Ser Asn Gly Arg Cys He 5 10 15

Pro Leu Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys Ser Asp Gly Ser 20 25 30

Asp Glu Ser Ala Cys Val Lys Lys Thr Cys Ala Glu Ser Asp Phe Val 35 40 45 50

Cys Asn Ser Gly Gin Cys Val Pro Asn Arg Trp Gin Cys Asp Gly Asp 55 60 65

Pro Asp Cys Glu Asp Gly Ser Asp Glu Ser Ala Glu Leu Cys His Met 70 75 80

Arg Thr Cys Arg Val Asn Glu He Ser Cys Gly Pro Gin Ser Thr Gin 85 90 95

Cys He Pro Val Ser Trp Lys Cys Asp Gly Glu Lys Asp Cys Asp Ser 100 105 110

Gly Glu Asp Glu Glu Asn Cys Gly Asn Val Thr Cys Ser Ala Ala Glu 115 120 125 130

Phe Thr Cys Ser Ser Gly Gin Cys He Ser Lys Ser Phe Val Cys Asn 135 140 145

Gly Gin Asp Asp Cys Ser Asp Gly Ser Asp Glu Leu Glu Cys Ala Pro 150 155 160 Pro Thr Cys Gly Val His Glu Phe Gin Cys Lys Ser Ser Thr Cys He 165 170 175

Pro He Ser Trp Val Cys Asp Asp Asp Ala Asp Cys Ser Asp His Ser 180 185 190

Asp Glu Ser Leu Glu Gin Cys Gly Arg Gin Pro Ala Pro Pro Val Lys 195 200 205 210

Cys Ser Thr Ser Glu Val Gin Cys Gly Ser Gly Glu Cys He His Lys 215 220 225

Lys Trp Arg Cys Asp Gly Asp Pro Asp Cys Lys Asp Gly Ser Asp Glu 230 235 240

He Asn Cys Pro Ser Arg Thr Cys Arg Pro Asp Gin Phe Arg Cys Glu 245 250 255

Asp Gly Asn Cys He His Gly Ser Arg Gin Cys Asn Gly Val Arg Asp 260 265 270

Cys Leu Asp Gly Thr Asp Glu Ala Asn Cys Asn Asn Val He Gin Cys 275 280 285 290

Ser Gly Pro Gly Lys Phe Lys Cys Arg Ser Gly Glu Cys He Asp He 295 300 305

Asn Lys Val Cys Asn His His Gly Asp Cys Lys Asp Trp Ser Asp Glu 310 315 320

Pro Leu Lys Glu Cys Asn He Asn Glu Cys Leu Val Asn Asn Gly Gly 325 330 335

Cys Ser His He Cys Arg Asp Leu Val He Gly Tyr Glu Cys Asp Cys 340 345 350

Pro Ala Gly Phe Glu Leu Val Asp Arg Arg Thr Cys Gly Asp He Asp 355 360 365 370

Glu Cys Gin Asn Pro Gly He Cys Ser Gin He Cys He Asn Leu Lys 375 380 385

Gly Gly Tyr Lys Cys Glu Cys Ser Arg Gly Tyr Gin Met Asp Leu Ala 390 395 400

Thr Gly Val Cys Lys Ala Val Gly Lys Glu Pro Cys Leu He Phe Thr 405 410 415 Asn Arg Arg Asp He Arg Lys He Gly Leu Glu Arg Lys Glu Tyr He 420 425 430

Gin Leu Val Glu Gin Leu Arg Asn Thr Val Ala Leu Asp Ala Asp He 435 440 445 450

Ala Glu Gin Lys Leu Tyr Trp Ala Asp Phe Ser Gin Lys Ala He Phe 455 460 465

Ser Ala Ser He Asp Thr Arg Asp Lys Val Gly Thr His Thr Arg He 470 475 480

Leu Asp Asn He His Ser Pro Ala Gly He Ala Val Asp Trp He Tyr 485 490 495

Lys Asn He Tyr Trp Thr Asp Ser Ser Ala Lys Thr He Ser Val Ala 500 505 510

Ser Leu Asn Gly Lys Lys Arg Lys Val Leu Phe Leu Ser Glu Leu Arg 515 520 525 530

Glu Pro Ala Ser He Ala Val Asp Pro Leu Ser Gly Phe Met Tyr Trp 535 540 545

Ser Asp Trp Gly Glu Pro Ala Lys He Glu Lys Ala Gly Met Asn Gly 550 555 560

Phe Asp Arg Gin Gin Leu Val Thr Thr Glu He Gin Trp Pro Asn Gly 565 570 575

He Ala Leu Asp Leu Val Lys Ser Arg Leu Tyr Trp Leu Asp Ser Lys 580 585 590

Leu His Met Leu Ser Ser Val Asp Leu Asn Gly Gin Asp Arg Arg Leu 595 600 605 610

Val Leu Lys Ser His Met Phe Leu Pro His Pro Leu Ala Leu Thr He 615 620 625

Phe Glu Asp Arg Val Phe Trp He Asp Gly Glu Asn Glu Ala Val Tyr 630 635 640

Gly Ala Asn Lys Phe Thr Gly Ala Glu Leu Val Thr Leu Val Asn Asn 645 650 655

Leu Asn Asp Ala Gin Asp He He Val Tyr His Glu Leu Val Gin Pro 660 665 670 Ser Gly Arg Asn Trp Cys Glu Glu Asn Met Val Asn Gly Gly Cys Ser 675 680 685 690

Tyr Leu Cys Leu Pro Ala Pro Gin He Asn Glu His Ser Pro Lys Tyr 695 700 705

Thr Cys Thr Cys Pro Ala Gly Tyr Phe Leu Gin Glu Asp Gly Leu Arg 710 715 720

Cys Gly Gly Phe Asn He Ser Ser Val Val Ser Glu Val Ala Ala Arg 725 730 735

Gly Ala Ala Gly Ala Trp Ala Val Leu Pro He Leu Leu Leu Val Thr 740 745 750

Ala Ala Leu Ala Gly Tyr Phe Met Trp Arg Asn Trp Gin His Lys Asn 755 760 765 770

Met Lys Ser Met Asn Phe Asp Asn Pro Val Tyr Leu Lys Thr Thr Glu 775 780 785

Glu Asp Leu Thr He Asp He Gly Arg His Ser Gly Ser Val Gly His 790 795 800

Thr Tyr Pro Ala He Ser Val Val Ser Thr Asp Asp Asp Met Leu 805 810 815

( 2 ) INFORMATION FOR SEQ ID NO : 3 : ( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 2878 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : both

(D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : cDNA

( iii) HYPOTHETICAL : NO ( iii ) ANTI- SENSE : NO

( ix) FEATURE :

(A) NAME/KEY: sig_peptide

(B) LOCATION: 13..150 (ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 151..2784 (ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 13..2784

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

CTCGGCGCGG CG ATG CGG TCG AGC CGG CAG CGC GGA GAC CGG AGC GCG 48

Met Arg Ser Ser Arg Gin Arg Gly Asp Arg Ser Ala -46 -45 -40 -35

GCG ACC GGC GGC GGG TGT GGG GCG CGG CGG TGG GCG CTC CCG CGC TGC 96 Ala Thr Gly Gly Gly Cys Gly Ala Arg Arg Trp Ala Leu Pro Arg Cys -30 -25 -20

GGG GCG CTC TGC CTG CTG CTC GCC CTC GGC TGC CTG CGT ACT GCC ACC 144 Gly Ala Leu Cys Leu Leu Leu Ala Leu Gly Cys Leu Arg Thr Ala Thr -15 -10 -5

GAC GGT GCA AAA GCA AAA TGT GAG GAG TCC CAG TTC CAG TGT AGT AAT 192 Asp Gly Ala Lys Ala Lys Cys Glu Glu Ser Gin Phe Gin Cys Ser Asn 1 5 10

GGA CGC TGT ATT CCT TTA CTC TGG AAA TGT GAT GGT GAT GAA GAC TGT 240 Gly Arg Cys He Pro Leu Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys 15 20 25 30

TCA GAC GGC AGT GAT GAA AGT GCT TGT GCA GTG GGC GAC AGA TGT GAA 288 Ser Asp Gly Ser Asp Glu Ser Ala Cys Ala Val Gly Asp Arg Cys Glu 35 40 45

AGA AAC GAG TTC CAG TGC CAA GAC GGG AAA TGC ATC TCC TAC AAG TGG 336 Arg Asn Glu Phe Gin Cys Gin Asp Gly Lys Cys He Ser Tyr Lys Trp 50 55 60

GTC TGC GAT GGC AGC GCT GAG TGC CAG GAT GGC TCT GAT GAG TCC CAG 384 Val Cys Asp Gly Ser Ala Glu Cys Gin Asp Gly Ser Asp Glu Ser Gin 65 70 75

GAG ACG TGC TTG TCT GTC ACC TGC AAA TCC GGG GAC TTC AGC TGT GGG 432 Glu Thr Cys Leu Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly 80 85 90

GGC CGT GTC AAC CGC TGC ATT CCT CAG TTC TGG AGG TGC GAT GGC CAA 480 Gly Arg Val Asn Arg Cys He Pro Gin Phe Trp Arg Cys Asp Gly Gin 95 100 105 110 GTG GAC TGC GAC AAC GGC TCA GAC GAG CAA GGC TGT CCC CCC AAG ACG 528 Val Asp Cys Asp Asn Gly Ser Asp Glu Gin Gly Cys Pro Pro Lys Thr 115 120 125

TGC TCC CAG GAC GAG TTT CGC TGC CAC GAT GGG AAG TGC ATC TCT CGG 576 Cys Ser Gin Asp Glu Phe Arg Cys His Asp Gly Lys Cys He Ser Arg 130 135 140

CAG TTC GTC TGT GAC TCA GAC CGG GAC TGC TTG GAC GGC TCA GAC GAG 624 Gin Phe Val Cys Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu 145 150 155

GCC TCC TGC CCG GTG CTC ACC TGT GGT CCC GCC AGC TTC CAG TGC AAC 672 Ala Ser Cys Pro Val Leu Thr Cys Gly Pro Ala Ser Phe Gin Cys Asn 160 165 170

AGC TCC ACC TGC ATC CCC CAG CTG TGG GCC TGC GAC AAC GAC CCC GAC 720 Ser Ser Thr Cys He Pro Gin Leu Trp Ala Cys Asp Asn Asp Pro Asp 175 180 185 190

TGC GAA GAT GGC TCG GAT GAG TGG CCG CAG CGC TGT AGG GGT CTT TAC 768 Cys Glu Asp Gly Ser Asp Glu Trp Pro Gin Arg Cys Arg Gly Leu Tyr 195 200 205

GTG TTC CAA GGG GAC AGT AGC CCC TGC TCG GCC TTC GAG TTC CAC TGC 816 Val Phe Gin Gly Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys 210 215 220

CTA AGT GGC GAG TGC ATC CAC TCC AGC TGG CGC TGT GAT GGT GGC CCC 864 Leu Ser Gly Glu Cys He His Ser Ser Trp Arg Cys Asp Gly Gly Pro 225 230 235

GAC TGC AAG GAC AAA TCT GAC GAG GAA AAC TGC GCT GTG GCC ACC TGT 912 Asp Cys Lys Asp Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys 240 245 250

CGC CCT GAC GAA TTC CAG TGC TCT GAT GGA AAC TGC ATC CAT GGC AGC 960 Arg Pro Asp Glu Phe Gin Cys Ser Asp Gly Asn Cys He His Gly Ser 255 260 265 270

CGG CAG TGT GAC CGG GAA TAT GAC TGC AAG GAC ATG AGC GAT GAA GTT 1008 Arg Gin Cys Asp Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val 275 280 285

GGC TGC GTT AAT GTG ACA CTC TGC GAG GGA CCC AAC AAG TTC AAG TGT 1056 Gly Cys Val Asn Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys 290 295 300 CAC AGC GGC GAA TGC ATC ACC CTG GAC AAA GTC TGC AAC ATG GCT AGA 1104 His Ser Gly Glu Cys He Thr Leu Asp Lys Val Cys Asn Met Ala Arg 305 310 315

GAC TGC CGG GAC TGG TCA GAT GAA CCC ATC AAA GAG TGC GGG ACC AAC 1152 Asp Cys Arg Asp Trp Ser Asp Glu Pro He Lys Glu Cys Gly Thr Asn 320 325 330

GAA TGC TTG GAC AAC AAC GGC GGC TGT TCC CAC GTC TGC AAT GAC CTT 1200 Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys Asn Asp Leu 335 340 345 350

AAG ATC GGC TAC GAG TGC CTG TGC CCC GAC GGC TTC CAG CTG GTG GCC 1248 Lys He Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gin Leu Val Ala 355 360 365

CAG CGA AGA TGC GAA GAT ATC GAT GAG TGT CAG GAT CCC GAC ACC TGC 1296 Gin Arg Arg Cys Glu Asp He Asp Glu Cys Gin Asp Pro Asp Thr Cys 370 375 380

AGC CAG CTC TGC GTG AAC CTG GAG GGT GGC TAC AAG TGC CAG TGT GAG 1344 Ser Gin Leu Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys Gin Cys Glu 385 390 395

GAA GGC TTC CAG CTG GAC CCC CAC ACG AAG GCC TGC AAG GCT GTG GGC 1392 Glu Gly Phe Gin Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly 400 405 410

TCC ATC GCC TAC CTC TTC TTC ACC AAC CGG CAC GAG GTC AGG AAG ATG 1440 Ser He Ala Tyr Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met 415 420 425 430

ACG CTG GAC CGG AGC GAG TAC ACC AGC CTC ATC CCC AAC CTG AGG AAC 1488 Thr Leu Asp Arg Ser Glu Tyr Thr Ser Leu He Pro Asn Leu Arg Asn 435 440 445

GTG GTC GCT CTG GAC ACG GAG GTG GCC AGC AAT AGA ATC TAC TGG TCT 1536 Val Val Ala Leu Asp Thr Glu Val Ala Ser Asn Arg He Tyr Trp Ser 450 455 460

GAC CTG TCC CAG AGA ATG ATC TGC AGC ACC CAG CTT GAC AGA GCC CAC 1584 Asp Leu Ser Gin Arg Met He Cys Ser Thr Gin Leu Asp Arg Ala His 465 470 475

GGC GTC TCT TCC TAT GAC ACC GTC ATC AGC AGG GAC ATC CAG GCC CCC 1632 Gly Val Ser Ser Tyr Asp Thr Val He Ser Arg Asp He Gin Ala Pro 480 485 490 GAC GGG CTG GCT GTG GAC TGG ATC CAC AGC AAC ATC TAC TGG ACC GAC 1680 Asp Gly Leu Ala Val Asp Trp He His Ser Asn He Tyr Trp Thr Asp 495 500 505 510

TCT GTC CTG GGC ACT GTC TCT GTT GCG GAT ACC AAG GGC GTG AAG AGG 1728 Ser Val Leu Gly Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg 515 520 525

AAA ACG TTA TTC AGG GAG AAC GGC TCC AAG CCA AGG GCC ATC GTG GTG 1776 Lys Thr Leu Phe Arg Glu Asn Gly Ser Lys Pro Arg Ala He Val Val 530 535 540

GAT CCT GTT CAT GGC TTC ATG TAC TGG ACT GAC TGG GGA ACT CCC GCC 1824 Asp Pro Val His Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala 545 550 555

AAG ATC AAG AAA GGG GGC CTG AAT GGT GTG GAC ATC TAC TCG CTG GTG 1872 Lys He Lys Lys Gly Gly Leu Asn Gly Val Asp He Tyr Ser Leu Val 560 565 570

ACT GAA AAC ATT CAG TGG CCC AAT GGC ATC ACC CTA GAT CTC CTC AGT 1920 Thr Glu Asn He Gin Trp Pro Asn Gly He Thr Leu Asp Leu Leu Ser 575 580 585 590

GGC CGC CTC TAC TGG GTT GAC TCC AAA CTT CAC TCC ATC TCA AGC ATC 1968 Gly Arg Leu Tyr Trp Val Asp Ser Lys Leu His Ser He Ser Ser He 595 600 605

GAT GTC AAT GGG GGC AAC CGG AAG ACC ATC TTG GAG GAT GAA AAG AGG 2016 Asp Val Asn Gly Gly Asn Arg Lys Thr He Leu Glu Asp Glu Lys Arg 610 615 620

CTG GCC CAC CCC TTC TCC TTG GCC GTC TTT GAG GAC AAA GTA TTT TGG 2064 Leu Ala His Pro Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp 625 630 635

ACA GAT ATC ATC AAC GAA GCC ATT TTC AGT GCC AAC CGC CTC ACA GGT 2112 Thr Asp He He Asn Glu Ala He Phe Ser Ala Asn Arg Leu Thr Gly 640 645 650

TCC GAT GTC AAC TTG TTG GCT GAA AAC CTA CTG TCC CCA GAG GAT ATG 2160 Ser Asp Val Asn Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met 655 660 665 670

GTC CTC TTC CAC AAC CTC ACC CAG CCA AGA GGA GTG AAC TGG TGT GAG 2208 Val Leu Phe His Asn Leu Thr Gin Pro Arg Gly Val Asn Trp Cys Glu 675 680 685 AGG ACC ACC CTG AGC AAT GGC GGC TGC CAG TAT CTG TGC CTC CCT GCC 2256 Arg Thr Thr Leu Ser Asn Gly Gly Cys Gin Tyr Leu Cys Leu Pro Ala 690 695 700

CCG CAG ATC AAC CCC CAC TCG CCC AAG TTT ACC TGC GCC TGC CCG GAC 2304 Pro Gin He Asn Pro His Ser Pro Lys Phe Thr Cys Ala Cys Pro Asp 705 710 715

GGC ATG CTG CTG GCC AGG GAC ATG AGG AGC TGC CTC ACA GAG GCT GAG 2352 Gly Met Leu Leu Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu 720 725 730

GCT GCA GTG GCC ACC CAG GAG ACA TCC ACC GTC AGG CTA AAG GTC AGC 2400 Ala Ala Val Ala Thr Gin Glu Thr Ser Thr Val Arg Leu Lys Val Ser 735 740 745 750

TCC ACA GCC GTA AGG ACA CAG CAC ACA ACC ACC CGG CCT GTT CCC GAC 2448 Ser Thr Ala Val Arg Thr Gin His Thr Thr Thr Arg Pro Val Pro Asp 755 760 765

ACC TCC CGG CTG CCT GGG GCC ACC CCT GGG CTC ACC ACG GTG GAG ATA 2496 Thr Ser Arg Leu Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu He 770 775 780

GTG ACA ATG TCT CAC CAA GCT CTG GGC GAC GTT GCT GGC AGA GGA AAT 2544 Val Thr Met Ser His Gin Ala Leu Gly Asp Val Ala Gly Arg Gly Asn 785 790 795

GAG AAG AAG CCC AGT AGC GTG AGG GCT CTG TCC ATT GTC CTC CCC ATC 2592 Glu Lys Lys Pro Ser Ser Val Arg Ala Leu Ser He Val Leu Pro He 800 805 810

GTG CTC CTC GTC TTC CTT TGC CTG GGG GTC TTC CTT CTA TGG AAG AAC 2640 Val Leu Leu Val Phe Leu Cys Leu Gly Val Phe Leu Leu Trp Lys Asn 815 820 825 830

TGG CGG CTT AAG AAC ATC AAC AGC ATC AAC TTT GAC AAC CCC GTC TAT 2688 Trp Arg Leu Lys Asn He Asn Ser He Asn Phe Asp Asn Pro Val Tyr 835 840 845

CAG AAG ACC ACA GAG GAT GAG GTC CAC ATT TGC CAC AAC CAG GAC GGC 2736 Gin Lys Thr Thr Glu Asp Glu Val His He Cys His Asn Gin Asp Gly 850 855 860

TAC AGC TAC CCC TCG AGA CAG ATG GTC AGT CTG GAG GAT GAC GTG GCG 2784 Tyr Ser Tyr Pro Ser Arg Gin Met Val Ser Leu Glu Asp Asp Val Ala 865 870 875 TGAAACAACT CCTCGGCGCG GCGATGCGGT CGAGCCGGCA GCGCGGAGAC CGGAGCGCGG 2844

CGACCGGCGG CGGGTGTGGG GCGCGGCGGT GTCG 2878

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 924 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Arg Ser Ser Arg Gin Arg Gly Asp Arg Ser Ala Ala Thr Gly Gly -46 -45 -40 -35

Gly Cys Gly Ala Arg Arg Trp Ala Leu Pro Arg Cys Gly Ala Leu Cys -30 -25 -20 -15

Leu Leu Leu Ala Leu Gly Cys Leu Arg Thr Ala Thr Asp Gly Ala Lys -10 -5 1

Ala Lys Cys Glu Glu Ser Gin Phe Gin Cys Ser Asn Gly Arg Cys He 5 10 15

Pro Leu Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys Ser Asp Gly Ser 20 25 30

Asp Glu Ser Ala Cys Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe 35 40 45 50

Gin Cys Gin Asp Gly Lys Cys He Ser Tyr Lys Trp Val Cys Asp Gly 55 60 65

Ser Ala Glu Cys Gin Asp Gly Ser Asp Glu Ser Gin Glu Thr Cys Leu 70 75 80

Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn 85 90 95

Arg Cys He Pro Gin Phe Trp Arg Cys Asp Gly Gin Val Asp Cys Asp 100 105 110

Asn Gly Ser Asp Glu Gin Gly Cys Pro Pro Lys Thr Cys Ser Gin Asp 115 120 125 130 Glu Phe Arg Cys His Asp Gly Lys Cys He Ser Arg Gin Phe Val Cys 135 140 145

Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro 150 155 160

Val Leu Thr Cys Gly Pro Ala Ser Phe Gin Cys Asn Ser Ser Thr Cys 165 170 175

He Pro Gin Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly 180 185 190

Ser Asp Glu Trp Pro Gin Arg Cys Arg Gly Leu Tyr Val Phe Gin Gly 195 200 205 210

Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu 215 220 225

Cys He His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp 230 235 240

Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu 245 250 255

Phe Gin Cys Ser Asp Gly Asn Cys He His Gly Ser Arg Gin Cys Asp 260 265 270

Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn 275 280 285 290

Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu 295 300 305

Cys He Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp 310 315 320

Trp Ser Asp Glu Pro He Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp 325 330 335

Asn Asn Gly Gly Cys Ser His Val Cys Asn Asp Leu Lys He Gly Tyr 340 345 350

Glu Cys Leu Cys Pro Asp Gly Phe Gin Leu Val Ala Gin Arg Arg Cys 355 360 365 370

Glu Asp He Asp Glu Cys Gin Asp Pro Asp Thr Cys Ser Gin Leu Cys 375 380 385 Val Asn Leu Glu Gly Gly Tyr Lys Cys Gin Cys Glu Glu Gly Phe Gin 390 395 400

Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser He Ala Tyr 405 410 415

Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met Thr Leu Asp Arg 420 425 430

Ser Glu Tyr Thr Ser Leu He Pro Asn Leu Arg Asn Val Val Ala Leu 435 440 445 450

Asp Thr Glu Val Ala Ser Asn Arg He Tyr Trp Ser Asp Leu Ser Gin 455 460 465

Arg Met He Cys Ser Thr Gin Leu Asp Arg Ala His Gly Val Ser Ser 470 475 480

Tyr Asp Thr Val He Ser Arg Asp He Gin Ala Pro Asp Gly Leu Ala 485 490 495

Val Asp Trp He His Ser Asn He Tyr Trp Thr Asp Ser Val Leu Gly 500 505 510

Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe 515 520 525 530

Arg Glu Asn Gly Ser Lys Pro Arg Ala He Val Val Asp Pro Val His 535 540 545

Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys He Lys Lys 550 555 560

Gly Gly Leu Asn Gly Val Asp He Tyr Ser Leu Val Thr Glu Asn He 565 570 575

Gin Trp Pro Asn Gly He Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr 580 585 590

Trp Val Asp Ser Lys Leu His Ser He Ser Ser He Asp Val Asn Gly 595 600 605 610

Gly Asn Arg Lys Thr He Leu Glu Asp Glu Lys Arg Leu Ala His Pro 615 620 625

Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp Thr Asp He He 630 635 640 Asn Glu Ala He Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn 645 650 655

Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met Val Leu Phe His 660 665 670

Asn Leu Thr Gin Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu 675 680 685 690

Ser Asn Gly Gly Cys Gin Tyr Leu Cys Leu Pro Ala Pro Gin He Asn 695 700 705

Pro His Ser Pro Lys Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu 710 715 • 720

Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala 725 730 735

Thr Gin Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val 740 745 750

Arg Thr Gin His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu 755 760 765 770

Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu He Val Thr Met Ser 775 780 785

His Gin Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro 790 795 800

Ser Ser Val Arg Ala Leu Ser He Val Leu Pro He Val Leu Leu Val 805 810 815

Phe Leu Cys Leu Gly Val Phe Leu Leu Trp Lys Asn Trp Arg Leu Lys 820 825 830

Asn He Asn Ser He Asn Phe Asp Asn Pro Val Tyr Gin Lys Thr Thr 835 840 845 850

Glu Asp Glu Val His He Cys His Asn Gin Asp Gly Tyr Ser Tyr Pro 855 860 865

Ser Arg Gin Met Val Ser Leu Glu Asp Asp Val Ala 870 875

Claims

47

Patent Claims

A purified and isolated nucleic acid molecule encoding

(a) a polypeptide comprising the amino acid sequence of at least one of the structural elements of the mature chicken oocyte receptor P95 as defined in Fig. 2, or

(b) a polypeptide comprising an amino acid sequence which has high homology to at least one of the structural elements of (a) .

A purified and isolated nucleic acid molecule encoding

(a) a polypeptide comprising the entire amino acid se¬ quence of the mature chicken oocyte receptor P95 as defined in Fig. 2, or

(b) a polypeptide comprising an amino acid sequence which has a homology of at least 75% to the polypeptide of (a) .

The nucleic acid molecule according to claim 1 or 2 encoding a polypeptide comprising at least one immunological and/or biological property of P95.

The nucleic acid molecule according to any one of the claims 1-3, which is a DNA.

The nucleic acid molecule according to claim 4, which is a cDNA.

The nucleic acid molecule according to claim 4 or 5 com¬ prising the nucleic acid sequence as defined in Fig. 2 optionally without the non-coding regions and/or the signal peptide-coding region.

The nucleic acid molecule according to any one of the claims 1-6, which encodes a soluble polypeptide. 8. The nucleic acid molecule according to any one of the claims 1-7, comprising operably linked

(a) a nucleic acid sequence encoding at least one of the ligand-binding repeats 1-8 as defined in Fig. 2 and

(b) a nucleic acid sequence encoding at least one ligand-binding repeat of another polypeptide of the LDL receptor family.

9. The nucleic acid molecule according to any one of the claims 1-8 encoding a polypeptide which does not have any 0-glycosylation sites.

10. The nucleic acid molecule according to claim 4 which is a single stranded DNA.

11. The nucleic acid molecule according to any one of the claims 1-10 which is covalently associated with a detectable label.

12. The nucleic acid molecule according to claim 11 wherein said label is a radioactive label, a fluorescent label, a chemiluminescent label, an enzymatic label or an affinity label.

13. A vector comprising at least one copy of the nucleic acid molecule according to any one of the claims 1-12.

14. A cell which is transformed with a nucleic acid molecule according to any one of the claims 1-12, or a vector according to claim 13.

15. The cell according to claim 14 which is a prokaryotic cell.

16. The cell according to claim 15 which is an E.coli cell. 17. The cell according to claim 14 which is a eukaryotic cell .

18. A process for the production of a recombinant polypeptide comprising the steps of

(a) inserting a nucleic acid molecule according to any one of the claims 1-12 into a suitable host cell,

(b) cultivating said host cell under conditions which are suitable for the expression of said nucleic acid molecule and

(c) isolating the expression product.

19. The process according to claim 18, wherein said host cell is a prokaryotic cell.

20. The process according to claim 19, wherein said host cell is an E.coli cell.

21. The process according to claim 18, wherein said host cell is a eukaryotic cell.

22. A polypeptide produced by a process according to any one of the claims 18-21.

23. The polypeptide according to claim 22, which is non- glycosylated.

24. The polypeptide according to claim 22 or 23, which is a fragment of the chicken oocyte receptor P95.

25. The polypeptide according to claim 24, which contains at least one of the ligand-binding repeats 1-8 as defined in Fig. 2.

26. The polypeptide according to any one of the claims 22- 25, which is a soluble polypeptide. 27. The polypeptide according to claims 22-26, which is a chimaeric polypeptide comprising operably linked

(a) at least one of the ligand-binding repeats 1-8 as defined in Fig. 2 and

(b) at least one ligand-binding repeat of another polypeptide of the LDL receptor family.

28. A composition for qualitative and/or quantitative diagnostic tests comprising a polypeptide which exhibits at least one immunological and/or biological property of at least one of the structural elements of the mature chicken oocyte receptor as defined in Fig. 2.

29. The composition according to claim 28 comprising a poly¬ peptide according to any one of the claims 22-27.

30. The composition according to claim 28 or 29 for the quantitative determination of P95 ligand concentrations.

31. The composition according to claim 28 or 29 for the qualitative determination of P95 ligand isoforms.

32. A process for the quantitative and/or qualitative determination of P95 ligands in a liquid sample, comprising contacting said sample with a composition according to claim 28.

33. A process for the quantitative and/or qualitative determination of receptor ligands in a liquid sample, comprising contacting said liquid sample with

(i) and immobilized antibody or antibody fragment specific for a predetermined receptor ligand and (ii) a soluble receptor polypeptide specific for said predetermined receptor ligand and measuring the binding of said soluble receptor polypeptide to said predetermined receptor ligand.

34. The process according to claim 33, wherein said soluble receptor polypeptide is labelled.

35. The process according to claim 33 or 34, wherein said soluble receptor polypeptide carries an enzyme label.

36. The process according to any one of the claims 33-35, wherein said receptor ligand is selected from the group comprising apo lipoprotein B, apo lipoprotein E, lipoprotein lipase, α.₂-macroglobulin, pregnancy zone protein, vitellogenin, plasminogen activator - plasminogen activator inhibitor 1 complex, Pseudomonas aeruginosa exotoxin A, 39 kDa receptor-associated protein, riboflavin binding protein, Rous sarcoma virus envelope protein, and vesicular stomatitis virus surface epitope.

37. The process according to any one of the claims 33-36, wherein said soluble receptor polypeptide is selected from receptors of the LDL receptor family.

38. The process according to any one of the claims 33-37, wherein said soluble receptor polypeptide comprises at least one of the ligand-binding repeats 1-8 as defined in Fig. 2.

39. The process according to any one of the claims 33-38, comprising contacting, said liquid sample with a receptor ligand-specific antibody or antibody fragment, which is immobilized on a solid phase,

separating the liquid sample from said solid phase, contacting said solid phase with receptor ligand- specific soluble receptor polypeptide, which is labelled, and

measuring the label on said solid phase.

40. The process according to any one of the claims 33-39, wherein several receptor ligands are measured.