EP0731836A1 - Recepteur p95 de l'ovocyte du poussin (recepteur de lipomicrons/vitellogenine) - Google Patents

Recepteur p95 de l'ovocyte du poussin (recepteur de lipomicrons/vitellogenine)

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Publication number
EP0731836A1
EP0731836A1 EP95903784A EP95903784A EP0731836A1 EP 0731836 A1 EP0731836 A1 EP 0731836A1 EP 95903784 A EP95903784 A EP 95903784A EP 95903784 A EP95903784 A EP 95903784A EP 0731836 A1 EP0731836 A1 EP 0731836A1
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Prior art keywords
asp
ser
cys
gly
leu
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EP95903784A
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German (de)
English (en)
Inventor
Wolfgang Johann Schneider
Hideaki Bujo
Johannes Nimpf
Seitaro Sugawara
Tokuo Yamamoto
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Progen Biotechnik GmbH
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Progen Biotechnik GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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 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.
  • 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.
  • ligands a select set of molecules present in the extracellular milieu
  • VLDL very low density lipoprotein
  • VVTG vitellogenin
  • 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.
  • LDLR low density lipoprotein receptor
  • 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) .
  • 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.
  • 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) .
  • the nucleic acid molecule codes for a polypeptide comprising at least one immunological and/or biological property of the major chicken oocyte receptor P95.
  • 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.
  • LDLR low density lipoprotein receptor
  • the nucleic acid molecule of the present invention is prefer ⁇ ably a DNA-molecule and more preferably a cDNA-molecule.
  • 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.
  • 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..
  • LDL receptor family polypeptide e.g. the human LDL receptor, the rabbit VLDL receptor etc.
  • 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.
  • the nucleic acid molecule codes for a polypeptide which does not have any O-glycosylation sites.
  • 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.
  • detectable label e.g. a radioactive label, a fluorescent label, a chemiluminescent labels, an enzymatic label, or an affinity label, such as biotin.
  • 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.
  • origin of replication from the well-known plasmid pBR-322 which is suitable for most gram-negative bacteria, the 2 ⁇ -plasmid origin for yeast and various viral origins (SV40, polyoma, adeno virus etc.) which are useful for cloning vectors in eukaryotic cells.
  • the present invention also encompasses vectors which integrate into the genome of the host cell, e.g. the E.coli bacteriophage ⁇ .
  • Selection genes 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.
  • 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.
  • promoters for use with prokaryotic hosts include the ⁇ -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.
  • 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.
  • 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.
  • 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
  • the present invention refers to a process for the production of a recombinant polypeptide comprising the steps of:
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is a further advantage in attempts to obtain biologically active protein in heterologous systems.
  • well defined recombinant P95 or fragments thereof allow reproducible preparations for standardized application methods.
  • a process 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.
  • a liquid sample such as a body fluid, e.g. serum, or a tissue extract
  • the present invention refers to a process for the quantitative and/or qualitative determination of receptor ligands in a liquid sample, comprising
  • 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
  • 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.
  • 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.
  • 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 ⁇ -galactosidase is especially preferred.
  • 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, ⁇ . 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 are known to a person skilled in the art.
  • 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.
  • 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.
  • the process according to the present invention comprises:
  • the label measured on the solid phase is proportional to the functionality and/or activity of the receptor ligand to be measured.
  • the label measured on the solid phase is proportional to the functionality and/or activity of the receptor ligand to be measured.
  • Fig. 1 Northern blot - Distribution of P95 mRNA in chicken tissues.
  • Poly- (A) -RNA (2.5 ⁇ g 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.
  • 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.
  • VLDLR rabbit VLDL receptor
  • LDLR rabbit LDL receptor
  • COS-7 cells were transiently transfected with the P95 expression plasmid pCDMCVR-1 (lanes 2,5 and 8; 70 ⁇ g protein/lane) or vector alone (lanes 3, 6 and 9; 70 ⁇ g cell protein/lane) , and processed for immunoblotting following SDS-PAGE under nonreducing conditions as described in Example 3.
  • Lanes 1 and 7 contained 5 ⁇ g
  • lane 4 contained 1 ⁇ g of oocyte membrane protein.
  • Immunoblotting incubations were performed with 2 ⁇ g/ml anti-P95 IgG (lanes 1-3) , 20 ⁇ g/ml anti-carboxyterminal IgG (lanes 4-6) , and 20 ⁇ g/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.
  • panel C an aliquot of the cells (60 ⁇ g protein/lane) used in A and B were subjected to ligand blotting with the same 125 I-labelled ligands at 4 ⁇ g/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 ⁇ g 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 ⁇ g oocyte membrane protein; lanes 2 and 3, 60 ⁇ g protein of pCDMCVR-1 transfected or control COS-7 cells, respectively.
  • 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.
  • 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) .
  • the laying hen does not display detectable P95 message in any other tissue than ovary, heart, and muscle under these conditions.
  • 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:
  • COS-7 cells (American Type Culture Collection) were seeded at a density of 1.5 x 10 6 per 80 cm 2 dish and incubated overnight in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM glutamine and 0.05 mM ⁇ - 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 ⁇ g 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 10 5 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.
  • 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)
  • IgG fractions were purified from sera on columns of protein A-Sepharose CL-4B (Beisiegel et al.
  • 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 ⁇ M leupeptin.
  • Cells were pelleted by centrifugation and detergent extracts were prepared as follows. The cell pellet derived from one dish (80 cm 2 ) was resuspended in 75 ⁇ l of ice-cold solubilization buffer containing 200 mM Tris-maleate (pH 6.5), 2 mM CaCl 2 , 0.5 mM PMSF, 2.5 ⁇ M 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.
  • SDS-polyacrylamide gel electrophoresis was performed using a minigel system (Bio-Rad, Mini-ProteanTM 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 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 HyperfilmTM-ECL (Amersham) .
  • 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.
  • VLDL and VTG were radiolabelled with 125 I 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.
  • 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 ⁇ g/ml.
  • 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.
  • truncated receptor sP95
  • kb 1.2 kilobasepair
  • pBKV/sP95 was inserted into the Sail and BamHI sites of the expression vector pBK-CMV (Stratagene) to yield pBKV/sP95.
  • pBKV/sP95 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.
  • COS-7 cells were transiently transfected with plasmids pBK-CMV, pBKV/sP95, or pBKCVLR (20 ⁇ g 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.
  • 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

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Abstract

La présente invention se rapporte à une molécule d'acide nucléique purifiée et isolée codant (a) un polypeptide comprenant la séquence d'acide nucléique d'au moins un des éléments structuraux du récepteur P95 de l'ovocyte du poussin mature tel que défini dans la figure 2, ou (b) un polypeptide comprenant une séquence d'acides aminés qui a une forte homologie avec au moins un des éléments structuraux de (a).
EP95903784A 1993-11-30 1994-11-30 Recepteur p95 de l'ovocyte du poussin (recepteur de lipomicrons/vitellogenine) Withdrawn EP0731836A1 (fr)

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EP93119293 1993-11-30
EP93119293 1993-11-30
PCT/EP1994/003983 WO1995015379A1 (fr) 1993-11-30 1994-11-30 Recepteur p95 de l'ovocyte du poussin (recepteur de lipomicrons/vitellogenine)

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EP0731836A1 true EP0731836A1 (fr) 1996-09-18

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