WO1996015801A1 - Proteine humaine detectrice du calcium, fragments de cette proteine et adn codant pour elle - Google Patents

Proteine humaine detectrice du calcium, fragments de cette proteine et adn codant pour elle Download PDF

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WO1996015801A1
WO1996015801A1 PCT/US1995/015203 US9515203W WO9615801A1 WO 1996015801 A1 WO1996015801 A1 WO 1996015801A1 US 9515203 W US9515203 W US 9515203W WO 9615801 A1 WO9615801 A1 WO 9615801A1
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gly
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PCT/US1995/015203
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WO1996015801A9 (fr
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Goran Amerstrom
Claes Juhlin
Lars Rask
Goran Hjalm
Clarence C. Morse
Edward M. Murray
Gregg R. Crumley
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Rhone-Poulenc Rorer Pharmaceuticals Inc.
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Priority to JP51705696A priority Critical patent/JP2002500501A/ja
Priority to AU42867/96A priority patent/AU726886B2/en
Priority to EP95941448A priority patent/EP0792159A4/fr
Priority to US08/652,877 priority patent/US6187548B1/en
Publication of WO1996015801A1 publication Critical patent/WO1996015801A1/fr
Publication of WO1996015801A9 publication Critical patent/WO1996015801A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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  • the present invention relates to a cDNA clone encoding a human calcium sensor protein of parathyroid, placental, and kidney tubule cells.
  • WO 88/03271 there is described monoclonal antiparathyroid antibodies identifying a parathyroid cell membrane-bound calcium receptor or sensor, crucially involved in calcium regulation of the parathyroid hormone (PTH) release
  • the receptor function is essential for maintenance of normal plasma calcium concentrations, and reduced receptor expression within proliferating parathyroid cells of patients with hyperparathyroidism (HPT) results in calcium insensitivity of the PTH secretion and variably severe hypercalcemia (3-6) .
  • Reactivity with the antiparathyroid antibodies was also demonstrated for proximal kidney tubule cells and cytotrophoblast cells of the human placenta, and the cytotrophoblasts were demonstrated to exhibit an almost parathyroid-identical regulation of cytoplasmic calcium
  • [Ca2 + i] (7,8) The antibody-reactive structure was found to exert calcium sensing function also in the cytotrophoblasts, and as these cells constitute part of the syncytium, the calcium sensor was suggested to be actively involved in the calcium homeostasis of the fetus (7,8). It was proposed that the antibody-reactive structure of the proximal kidney tubule cells exerts a similar calcium sensing function, and that the calcium sensor, thus, plays a more universal role in calcium regulation via different organ systems (1,7,9,10).
  • the calcium sensor/receptor has been revealed as a 500 kDa single chain glycoprotein (7) .
  • the amino acid sequence as well as the corresponding DNA sequences thereof are hitherto unknown.
  • an object of the present invention was to provide sufficient structural data of the calcium sensor/receptor to enable complete characterization thereof.
  • the present invention provides complete amino acid sequence of the human calcium sensor protein of parathyroid, placental and kidney tubule cells.
  • the invention provides nucleic acid sequence encoding the human calcium sensor and nucleic acid probes for identifying other novel calcium sensor proteins.
  • Another object is to use said structural data to design novel treatment methods as well as compounds and compositions for treating calcium related disorders.
  • the present invention provides identification of peptide regions within the calcium sensor protein cytoplasmic domain which are homologous to SH2 and SH3 binding motifs involved in signal transduction pathways.
  • cells expressing the calcium sensor protein or a fragment thereof or comprising the cDNA encoding the calcium sensor protein of the present invention may be utilized in an assay to identify molecules which block or enhance the activity of the calcium sensor protein, including signal transduction pathways associated with the activity of the sensor. These molecules will be useful in the treatment of mammalian pathological conditions associated with perturbations in the levels of PTH, vitamins D3 production, estrogen, osteoclast acti.vi.ty so?r osteoblast activity (therefore, bone resorption and/or formation) , calcium secretion and calcium ion homeostasis.
  • the present invention describes the isolation and characterization of cDNA clones encoding the calcium sensor/- receptor in human placenta and Northern blots verifying the presence of the corresponding mRNA within the parathyroid and kidney.
  • Close sequence similarity between the calcium sensor and a rat Heymann nephritis antigen, gp330 (11, 67) suggests that the common calcium sensor of the placenta, the parathyroid and kidney tubule is related to this antigen, represents the human homologue of gp330, and belongs to a family of large glycoproteins with receptor function and calcium binding ability. Therefore, a further object of this invention is to provide diagnostic assays and therapeutic methods based on human gp330.
  • Pig 1 Isolation by HPLC of peptides obtained after digestion of the calcium sensor protein with Lys-C endoprotease (solid line) . Dashed line represents the chromatography of an identical reaction where the calcium-sensor was omitted. The flow rate was kept at 100 ⁇ l/min. Two peptide fractions which gave easily interpretable sequences are denoted by arrows.
  • Pig 3 Partial nucleotide sequence (SEQ ID No. 3) and deduced amino acid sequence (SEQ ID No. 4) of the-cDNA clone, pCAS-2, encoding part of the calcium-sensor protein. Portions of the deduced amino acid sequence identical to the peptides 292 and 293 are underlined.
  • Fig 4. Alignment of the amino acid sequence of the calcium-sensor protein (SEQ ID No. 4) to corresponding portions of the Heymann antigen (HEYMANN, SEQ ID No. 5), low density lipoprotein receptor (LDL-RC, SEQ ID No. 6), and LDL related receptor protein (LDLRRP, SEQ ID No. 7) .
  • Stars denote residues identical between the calcium sensor protein and any of the H other sequences.
  • X denotes a position in the Heymann antigen sequence where identity has not been published.
  • Pig 5 Northern blot analysis of total RNA from parathyroid adenoma (1), kidney (2), liver (3), placenta (4), pancreas (5), adrenal gland (6), small gut (7). Filters were hybridized with the 2.8 kb pCAS-2 insert probe, and reactions visualized by a phosphorimager. Locations of 28S and 18S ribosomal RNA are indicated.
  • Fig. 6 Complete nucleotide (SEQ ID No. 11) and amino acid (SEQ ID No. 12) sequence of the human calcium sensor 2.8 kb cDNA clone.
  • the transmembrane domain of the sensor is shown in bold type.
  • the three SH3 binding regions are underlined or overlined and the SH2 binding region is shown in strikethru.
  • Fig 7. Amino acid sequence of the calcium sensor cytoplasmic domain (SEQ ID No. 13) and comparison of the three calcium sensor SH3 binding regions (SEQ ID Nos. 14-16) to known SH3 binding motifs (SEQ ID Nos. 20-37).
  • Pig. 8 Comparison of relative binding strengths between a calcium sensor SH3 binding region and various GST fusion proteins comprising an SH3 domain.
  • Fig. 9 Comparison of the calcium sensor SH2 binding region (SEQ ID No. 19) with amino acid sequence requirements necessary for interaction with the SH2 region of the p85 regulatory subunit of PI3K (SEQ ID Nos. 38-78).
  • Pig. 10 Structure of human gp330, including the EGF repeat, growth factor repeats and YWTD spacer regions. N depicts the amino terminus of the protein and C the carboxyl-terminus. The arrow indicates the location of the transmembrane region.
  • Pig. 11 Strategy for extending CAS sequence from pCAS-2.
  • polypeptide means a linear array of amino acids connected one to the other by peptide bonds between the ⁇ -amino and carboxy groups of adjacent amino acids. "Substantially purified” is used herein to mean
  • substantially homogeneous which is defined as a material which is substantially free of compounds normally associated with it in its natural state (e.g., other proteins or peptides, carbohydrates, lipids) .
  • substantially purified is not meant to exclude artificial or synthetic mixtures with other compounds.
  • the term is also not meant to exclude the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification or compounding with a pharmaceutically acceptable preparation.
  • biologically active polypeptide means the naturally occurring polypeptide per se as well as biologically active analogues thereof, including synthetically produced polypeptides and analogues thereof, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof.
  • biologically active polypeptide also encompasses biologically active fragments thereof, as well as “biologically active sequence analogues” thereof. Different forms of the peptide may exist. These variations may be characterized by difference in the nucleotide sequence of the structural gene coding for proteins of identical biological function.
  • biologically active sequence analogue includes nonnaturally occurring analogues having single or multiple amino acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogues resulting in derivatives which retain one or more of the native biologically active properties are included within the scope of this invention.
  • nucleotides are indicated by their bases using the following standard one-letter abbreviations: Guanine G Adenine A Thymine T Cytosine C Unknown N
  • amino acid residues are indicated using the following standard one-letter abbreviations:
  • amino acid as used herein is meant to denote the above recited natural amino acids and functional equivalents thereof.
  • This invention provides isolated nucleic acid molecules encoding a common calcium sensor protein of parathyroid, placental and kidney tubule cells and comprising a coding sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 83, SEQ ID No. 85, SEQ ID No. 87, and SEQ ID No. 89.
  • this invention provides a vector comprising an isolated nucleic acid molecule encoding the calcium sensor protein or a fragment thereof which encodes functional regions of the sensor.
  • the invention provides a method of preparing calcium sensor protein which comprises inserting a nuleic acid encoding the calcium sensor or a fragment thereof in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the calcium sensor protein produced by the resulting cell, and purifying the calcium sensor protein so recovered.
  • This method for preparing a calcium sensor protein or fragment thereof uses recombinant DNA technology methods which are well known in the art.
  • the calcium sensor protein or a fragment thereof may be prepared using standard solid phase methodology of peptide synthesis.
  • the present invention also provides antisense nucleic acids which can be used to down regulate or block the expression of the calcium sensor protein either in vitro, ex vivo or in vivo.
  • the down regulation of gene expression can be made at both translational or transcriptional levels.
  • Antisense nucleic acids of the invention are more preferentially RNA fragments capable of specifically hybridizing with all or part of the sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 83, SEQ ID No. 85, SEQ ID No. 87, and SEQ ID No. 89 or the corresponding messenger RNA.
  • These antisense can be synthetic oligonucleotides prepared based on the sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No.
  • SEQ ID No. 11 SEQ ID No. 83, SEQ ID No. 85, SEQ ID No. 87, and SEQ ID No. 89, optionally modified to improve their stability of selectivity, as disclosed for instance in EP 92574.
  • They can also be DNA sequences whose expression in the cell produces RNA complementary to all or part of the calcium sensor protein mRNA.
  • These antisenses can be prepared by expression of all or part of the sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 83, SEQ ID No. 85, SEQ ID No. 87, and SEQ ID No. 89 in the opposite orientation (EP 140 308) .
  • Tissue specimens Samples of human parathyroid glands were obtained at surgery of patients with primary HPT. Other human tissue specimens (kidney, epididymis, liver, pancreas, adrenal gland, small gut, spleen, lung and striated muscle) were sampled from organs removed at surgery. rHuman placental tissue was collected in conjunction with uncomplicated pregnancies at full term. All specimens were immediately quick-frozen in isopentane and stored at -70°C.
  • the 500 kDa calcium sensor protein was isolated and purified, from altogether 25 human placentas, by immunosorbent and ion exchange chromatographies, following a previously described protocol (7) .
  • the procedure utilizes two different monoclonal antiparathyroid antibodies (1,7), Ell and Gil, known to bind different epitopes of the calcium sensing protein; Ell has displayed no functional effect, while Gil efficiently blocks calcium regulation in both parathyroid and placental cells (1,7).
  • the calcium sensor protein preparation was subjected to gel chromatography on a Zorbax GF25 gel column (9.2 x 250 mm), prior to enzymatic digestion.
  • the biologically active calcium sensor protein of the present invention has been isolated as described. It can also be prepared by chemical synthesis in a recombinant ENA biosystem.
  • Biologically active fragments of the calcium sensor protein can also be prepared using synthetic or recombinant technologies which are known in the art.
  • Oligonucleotide synthesis Oligonucleotides were synthesized using an .ABI 381 oligonucleotide synthesizer (Applied Biosys- terns) . The following oligonucleotide mixture was utilized as a probe for screening of the placental cDNA library:
  • the first nine nucleotides contain an EcoR I and a BamH I site, respectively, and the remaining nucleotides correspond to amino acid residues 1 to 6 of peptides 293 and to residues 8 to 13 of peptide 292.
  • PCR reaction Part of the ⁇ gt 11 cDNA clone CAS-1 was to amplified by PCR using two degenerated probes corresponding to portions of peptides 292 and 293. The following conditions were used: 170 ng template DNA, 1 pmol of each oligonucleotide mixture as primers, dNTP 3mM, Taq-polymerase 0.75 u. The reaction was carried out in 20 ⁇ l of lOmM Tris-HCl, pH 8.0, 1.5 mM MgCl2- 50mM KC1 in a Perkin-Elmer 9600 PCR-machine
  • a placental ⁇ ZAP-II cDNA library was screened with the PCR-fragment from the cDNA clone CAS-1 labeled by random priming as the probe. The screening was carried out as above. 2 x 10 ⁇ plaques distributed on ten 20 x 25 cm agar plates were screened. Nucleotide sequence determination. The insert of the phage clone CAS-2 was released from the phage vector in the Bluescript+ vector using a helper phage (Stratagene, La Jolla, Ca.). Nucleotide sequence reactions were carried out according to the cycle sequencing procedure, utilizing a kit from Applied Biosystems.
  • G36a GCGTTTTCTXTTT CTTTCCTT 13,026 SEQ ID No. ,104
  • RT reverse trancriptase
  • PCR amplification of first-strand cDNA was performed in a Perkin-Elmer 9600 Thermal Cycler using the following program: 1 cycle of denaturation at 94°C for 2 min., followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 51°C for 10 sec, , and extension at 72°C for 3 min., after which, the products of the reactions were separated by electrophoresis and gel purified (QIAGEN) .
  • PCR reagents were purchased from Perkin- Elmer and used according to manufacturer's suggestions.
  • PCR fragments were then nucleotide sequenced using a dideoxynucleotide chain-termination method (Perkin-Elmer Prism Dye Deoxy Terminator Cycle Sequencing Kit) , and an ABI 373 automated DNA sequencer (Applied Biosystems) . PCR fragments from four separate reactions were sequenced on both strands to confirm sequence data. Computer generated DNA sequence analysis was performed using Auto-Assembler and Factura (Applied Biosystems) , and MacVector and AssemblyLIGN (Eastman Kodak Company) software programs.
  • Hybridizations were performed at 42°C for 18-24 h in 50% formamide, 4 x saline sodium citrate (SSC; 300 mM NaCI, 30 mM
  • a peptide corresponding to one putative CAS SH3 binding region (ATPPPSPSLPAKPKPPSRR) (SEQ ID No. 18) was synthesized on an ABI model 430A synthesizer using FastMoc t ⁇ n chemistry.
  • the peptide was HPLC purified and analyzed by mass spectroscopy. 5 mg of the peptide was coupled to 500 ul of Amino Link (Pierce) agarose as described by the supplier. Efficiency of coupling was checked by RP-HPLC of peptide solution before and after coupling and spectrophotometrically at a wavelength of 220 nm. Both methods indicated a coupling efficiency of >70%.
  • the coupled peptide was reacted with 5 ug aliquots of various GST-SH3 fusion proteins at room temperature for 1 hour before the resin was washed extensively with T BS.
  • the resin was boiled in SDS loading dye and electrophoresed on an SDS-PAGE gel. Binding ability of the various SH3 proteins for the peptide was judged by the relative intensity of the Coomassie blue-stainable bands on the SDS gel. GST protein alone was used alone as a control.
  • the calcium sensor protein was purified from placental tissue by means of Pectin chromatography, immunosorbent chromatography utilizing the immobilized monoclonal anti- parathyroid antibodies, and finally ion exchange chromatography (1,7) .
  • the same antibodies were used in a sandwich ELISA to monitor the purification (7) .
  • the whole final preparation consisting of 200 ⁇ g of the 500 kDa protein chain (7), was made 6 M with regard to guanidine-HCl and applied to a gel chromatography column, equilibrated with 2 M guanidine-HCl, 0.1 M Tris-Cl, pH 8.5. The column was eluted with the same buffer. Virtually all protein material emerged close to the void volume at the expected position for a protein with a molecular mass of 500 kDa. Separate fractions containing this material were combined and endoproteinase Lys C (1 ⁇ g) was added. The digestion was allowed to proceed over night at 37°C.
  • the fragmented protein was reduced by incubation with 0.1% ⁇ - mercaptoethanol at 37°C for 30 min and subsequently alkylated with 4-vinyl pyridine (0.3%) at room temperature for 2 h.
  • the peptide mixture was then applied to a reversed phase C4 column equilibrated in 5% acetonitrile in 0.2% trifluoroacetic acid.
  • Peptides were eluted by a linear gradient of 5 60% acetonitrile in 0.02% trifluoracetic acid (Fig 1). Due to the large number of peptides, the elution pattern was complex.
  • An oligonucleotide mixture (48 bp) was constructed to encode amino acid residues 2 to 17 of the sequenced peptide 292.
  • five inosine bases were inserted at degenerated positions where no guidance could be obtained from the codon usage in humans. At nine positions, where two bases were possible, one of the bases was suggested with a likelihood exceeding 70% from codon usage, and was therefore used in the oligonucleotide mixture.
  • the mixed oligonucleotide was radioactively labelled and used as a probe to screen a human placental ⁇ gt 11 cENA library. Approximately 2 x 10 ⁇ plaques were screened and a single positive clone, CAS-1, was found. The insert of this clone was estimated to 2.3 kb, by restriction mapping. To obtain a recognizable sequence of the clone in a rapid way, an attempt was made to PCR amplify part of the sequence using degenerated oliogonucleotides corresponding to part of peptides 292 and 293 as primers. A distinct DNA fragment of approximately 430 bp was obtained assuming that the peptide 292 is located carboxy- terminal to peptide 293.
  • the fragment was partially sequenced using the oligonucleotide mixture corresponding to peptide 293 as the primer. In one reading frame from the obtained sequence, the sequence VGRHI could be deduced, in excellent agreement with the carboxyterminal 5 amino residues of peptide 293.
  • a human placental ⁇ Z.AP-II cDNA library reported to contain clones with large inserts was screened with the PCR fragment as the probe. From 2 x 10 6 plaques a single clone, CAS-2, was found. The insert of this clone, estimated to 2.8 kb, was released in the Bluescript + vector, using a helper phage.
  • the CAS-2 sequence was extended using standard methodology. Reverse transcriptase PCR amplification and standard 32p-labeled probe screening of human lambda kidney cDNA libraries were used to complete the cloning of the CAS cDNA (SEQ ID No. 83) . Probe fragments were designed off appropriate clones, starting with clone pCAS-2 ( Figure 11) , to allow isolation of overlapping but 5 '-extended clones from these libraries. This cDNA walking procedure was used for the isolation of all cDNA clones except clones pMeg2, pHPlC ⁇ , pHPlBl, and pM4Bl.
  • the 500 kDa placental calcium sensor belongs to the LDL- receptor superfamily.
  • FIG. 3 shows an alignment of placental 500 kDa protein sequence to the sequence of the Heymann antigen (SEQ ID No. 5) as well as to two other members of the same protein superfamily, the LDL-receptor (SEQ ID No. 6) and the LDLreceptor-related protein (identical to the (2- macroglobulin receptor, (11,15,16), SEQ ID No. 7).
  • the sequence identity between the placental calcium-sensor and the Heymann antigen gp330 was estimated to be 82% in the region of comparison (236 amino acid residues) .
  • a complete sequence of the human calcium sensor protein is shown in SEQ ID No. 83.
  • the identity between rat gp330 and the human homolog is 77%.
  • the structure of human gp330 is shown in Figure 10.
  • the protein is 4655 amino acids in length and comprises an N- terminal signal peptide of 25 amino acids, a 4398 amino acid extracellular domain, a transmembrane region of 23 amino acids and a C-terminal domain of 209 amino acids.
  • the structure of human gp330 closely correlates with that of the rat homolog ( Figure 3 of ref. 67).
  • the antiparathyroid antibodies (Ell and Gil) were found to stain not only parathyroid, placental and proximal kidney tubule cells but also epididymal cells, as previously demonstrated for antibodies reactive with the Heymann antigen (17-20) .
  • RNA total RNA (approximately 10 ⁇ g/lane) from human kidney, placenta and parathyroid glands with the identified 2.8 kb clone as the probe, revealed one major hybridizing RNA species of approximately 15,000 bases in all these tissues (Fig 5) .
  • Src-homology regions 2 and 3 are conserved sequence motifs consisting of approximately 100 and 60 amino acid residues, respectively, and are found in many eukaryotic proteins with diverse function (42-44) .
  • SH3 domains have been li identified in several cytoskeleton-associated proteins, such as p80/p85, myosinlb, spectrin, neutrophil NADPH oxidase-associated proteins p47 and p67, and in several yeast proteins important for morphogenesis (i.e., Bemlp and ABP-1) , mating (FUS1) or for regulation of ras activity (cdc25 and ste6 (for review see Mussachio et al. (45)).
  • SH3 domains play a role in multimeric protein complex formation at or near cytoplasmic membranes.
  • Some proteins that contain both SH2 and SH3 domains perform the function of adaptor molecules by joining activated receptor tyrosine kinases with p21 ras guanine nucleotide-releasing protein (GNRP) .
  • GNRP guanine nucleotide-releasing protein
  • Grb2 and its homologues bind to phosphotyrosine on activated membrane-anchored receptor tyrosine kinases through their SH2 domain and to SOS through their amino- and carboxyterminal SH3 domains (46-50) .
  • SH2/SH3-containing and SH2/SH3-binding proteins are involved in a highly conserved signal transduction pathways from activated receptors.
  • the peptide was incubated with various purified GST-SH3 fusion proteins and the relative binding strengths of the fusion proteins was assayed by SDS-PAGE ( Figure 8) .
  • the data clearly indicate that several of the SH3-region containing proteins had an affinity for the peptide containing CAS-PEPl, with the following relative order of decreasing affinities: LANE 6: SH3- / * ?
  • PI3K SH3 of p85 subunit of phosphoinositol-3 kinase, (54,55)
  • LANE 7 SH3-PLC-gamma, (phospholipa ⁇ e-C gamma, (56)) > LANE 2: SH3-FYN (src-family soluble tyrosine kinase, (57), > LANE 4: SH3-GRB2, (growth factor receptor binding protein N-terminal SH3) and LANE 5 (C-terminal SH3 of GRB2) (58,59).
  • PI3K contains two SH2 regions and one SH3 region.
  • PI3K is relatively new to the family of signal transducing molecules, but appears to be involved with insulin signaling through the glucose transporter, and is believed to associate directly with the ras protein.
  • PliC-gamma is a well known signaling molecule also containing two SH2 regions and one SH3 region, and is known to hydrolyze membrane lipids to other powerful downstream signaling molecules (eg. IP3 and diacylglycerol) when stimulated by ligand activated growth factor receptors.
  • FYN is a highly characterized member of the src-family of soluble tyrosine kinases known to be intimately associated with cell growth and differentiation. FYN contains one SH2 and one SH3 region, is also known to be stimulated by ligand activated growth factor receptors. GRB2 contains two SH3 regions and one SH2 region, and is known as an adaptor molecule in that it has no known intrinsic enzymatic capabilities. GRB2 molecules are also stimulated by ligand activated growth factor receptors.
  • SH3-GAP GTP-ase activating protein, LANE 3, (60, 61)
  • SH3-NCF neutral cytotoxic factor-type 1, LANE 8, or -type 2, lane 9, (62, 63)
  • CAS-PEPl does not bind a control GST fusion protein as shown in lane 1 of Fig. 8.
  • the cytoplasmic domain of CAS-2 also comprises a p85-SH2 binding region.
  • SH2 containing proteins all require phosphorylated tyrosine residues for an interaction, it is well established that the amino acid residues surrounding the tyrosine residue dictate the specificity and strength of the interaction (64) .
  • Figure 9 defines those amino acid sequence requirements that are necessary for interaction with the SH2 region of the p85 regulatory subunit of PI3K.
  • the evidence demonstrates that the cytoplasmic domain of the calcium sensor protein of the invention contains three consensus SH3 binding regions and one potential SH2 recognition region of the type recognized by the SH2 region of p85 and supports an involvment of SH2 and SH3 mediated signal transduction for biological activity of the calcium sensor protein, possibly through PI3K.
  • the potential interaction of PI3K with the calcium sensor protein is even more interesting in light of recent evidence linking the CAS-2 protein to calcium sensing in human parathyroid tissue, given that calcium sensing appears to involve G-protein activation, PKC activation, and inositol phosphate generation, all of which are activities that can be associated with PI3K signal transduction cascades.
  • these regions provide useful tools in assays for the identification of compounds that either stimulate or inhibit the signal transduction pathways used by the calcium sensor protein.
  • agonists or antagonists which mimic or inhibit the activity of the calcium sensor protein SH2/SH3 regions will be useful for the treatment of diseases that are intimately associated with the sensor, such as primary hyperparathyroidism (HPT) (52) and osteoporosis.
  • HPT primary hyperparathyroidism
  • Rat Heymann nephritis antigen, gp330 belongs to the LDL receptor superfamily of large, multifunctional glycoproteins (68, 69, 70) . Identification of the calcium sensor protein as the human homolog of rat gp330 enables new diagnostic and therapeutic agents for human disease.
  • human gp330 may be used in assays for detecting autoantibodies associated with human membranous glomerulonephritis.
  • suitable assays include im unoassays, such as ELISA.
  • synthetic peptides based on the human gp330 sequence may be used to localize immunodominent B- or T-lymphocyte recognition sites.
  • the invention enables detection of gp330 specific autoantibodies and helper, cytotoxic or suppressor T-cells.
  • the invention permits identification of patients who may develop idiopathic autoimmune membranous glomerulonephritis and patients susceptible to autoimmune membranous glomerulonephritis following a renal allograft.
  • Human gp330 is useful for treatment of human membranous glomerulonephritis according to a variety of methods, For example, gp330 may be coupled to a polyphenol followed by immunization of a patient according to U.S. Patent 4,702,907, the entire contents of which are incorporated herein by reference. Treatment in this manner results in selective immunosupression of antibodies specific for gp330. As an alternative method of treatment, it is also possible to selectively remove gp330-reactive autoantibodies from sera by immobilizing gp330, or fragment thereof, on a solid support and pass the sera over the support, thereby effectively removing autoantibodies characteristic of human membranous glomerulonephritis.
  • human gp330 or a fragment thereof, can be directly administered to a patient in order to perturb formation of immune complexes.
  • Synthetic peptides based on the sequence of human gp330 are also useful therapetically.
  • Administration of immunogenic peptides inhibits activation or function of gp330 specific helper and cytotoxic T-cells.
  • the structure of human gp330 includes 16 growth factor repeats separated by 8 YWTD spacer regions and 1 epidermal growth factor repeat in the immediate extracellular juxtamembrane region ( Figure 11) . Therefore, administration of gp330, or a fragment thereof having growth factor activity, is useful in the treatment of wounds, such as burns and abrasions. Epidermal growth factor is also a potent inhibitor of gastric acid secretion. Therefore, gp330, or a fragment thereof having epidermal growth factor activity, is useful for treatment or prevention of gastric ulcers. Determination of effective amounts of therapeutic agent for administration is within the skill of the practitioner.
  • parathyroid As key regulator of the calcium homeostasis has been related to its extraordinarily capacity to sense and respond to variation in the extracellular Ca 2+ ion concentration.
  • Essential for recognition of changes in external calcium is a cation receptor or sensor of the parathyroid cell membrane, the presence of which was implicated by a series of in vitro studies on parathyroid cell regulation
  • the parathyroid calcium sensor or receptor is known to have features in common with most other classical receptors for cellular activation, although it exhibits the unusual ability to bind and be activated by divalent cations. Cation binding triggers biphasic rise in [Ca 2+ i] and concomittant activation of phospholipase C, possibly via a coupled G-protein, with a resulting accumulation of inositol phosphates (2,5,9,10).
  • the Heymann antigen has been revealed as the dominating antigen causing membranous, autoimmune glomerulonephritis in the rat after immunization with a crude tubular protein fraction (17,19).
  • Using anti-gp 330 antibodies a protein with an estimated molecular size larger than 400 kDa has been identified in man (20) .
  • the sequence identity of 77% between the human placental 500 kDa calcium sensor protein and the rat Heymann nephritis antigen indicates that they represent related forms of the calcium sensor protein in two different species.
  • LDL-receptor the LDL-receptor, the LDL-receptor-related protein and the Heymann antigen, have been thought to function as receptors for proteins, but all exhibit functionally important Ca 2+ -binding ability (16,27,28) .
  • Ca 2+ binding is necessary for the interaction of the LDL-receptor with apo-B (27) .
  • the LDL-receptor related protein ⁇ 2-macroglobulin receptor
  • Ca + is necessary for binding of activated 0.2-macroglobulin to the receptor (16) .
  • the rat Heymann antigen was shown by a blotting technique to interact with Ca 2+ (28) .
  • the Ca + binding motifs of the calcium sensor protein remain to be identified.
  • the sensor protein (as well as the Heymann antigen) contains EGF-like modules, like other members of the LDL-receptor superfamily (11,16,27), which may represent putative Ca + binding sites.
  • each EGF-like module is known to bind one Ca 2+ ion (29-34) , and the EGF-like modules have also been demonstrated to mediate Ca 2 "- " dependent protein/protein interaction (35) .
  • a 43 kDa membrane protein (o»2-macroglobulin receptor- associated protein, or Heparin-binding protein) (28,36) is known to interact both with the LDL-receptor-related protein and with the rat Heymann antigen in a Ca 2 "-"dependent manner (28) .
  • No physiological function has yet been assigned to this protein, but it appears also in tissues where the Heymann antigen and the LDL-receptorrelated proteins are not expressed (28) .
  • An interesting observation is the presence of a putative leucine- zipper motif in the aminoterminal part of the 43 kDa protein (36), considering that such motifs have been suggested to influence the opening and closure of membrane ion channels (37) .
  • the 43 kDa protein interacts with the Heymann antigen, it can be assumed to form a complex also with the calcium sensor protein in a Ca 2+ "dependent manner. Interaction with the 43 kDa protein might be important for the transmission of Ca 2+ induced conformational changes within the extracellular portion of the molecule to the cell interior. It is also possible that additional proteins interact with the calcium sensor in a Ca "-"dependent manner, and that such an interaction is important for the modulation of the sensor response.
  • the calcium sensor protein of the placenta may be involved in maintenance of a feto-maternal Ca * gradient and placental Ca 2+ transport, possibly by mediating calcium regulation of the parathyroid hormone related peptide (PTHrP) production and/or 1,25 (OH)2D3 metabolism (8) . Its presence already within the blastocyst (unpublished observation) may indicate a function also as adhesion molecule, or implicate involvement in differentiation or growth regulation, as suggested for the Heymann antigen (38) . The function of a calcium sensor within the kidney tubule brush border is less well explored.
  • the enzyme 1- ⁇ -hydroxylase present in the placenta and proximal kidney tubule is regulated by extracellular calcium, and the calcium sensor might accordingly regulate 1,25 (OH)2D3 metabolism, but it may possibly also influence Ca 2+ reabsorption from the glomerular filtrate (7-9) .
  • No. 89 can be used for isolating the genomic sequence encoding the calcium sensor.
  • an analysis of overlapping cDNA clones in conjunction with PCR techniques is used.
  • the genomic sequence can be obtained from the analysis of overlapping genomic cosmid and/or lambda phage clones.
  • NAME CRUMLEY, Greg R.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 610-454-3816 (B) TELEFAX: 610-454-3808
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO '
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GGC AAT GGG CAT TGC ATT CCA CAT GAC AAT GTG TGT GAT GAT GCC GAT 720 Gly Asn Gly His Cys He Pro His Asp Asn Val Cys Asp Asp Ala Asp 225 230 235 240 GAC TGT GGT GAC TGG TCC GAT GAA CTG GGT TGC AAT AAA GGA AAA GAA 768 Asp Cys Gly Asp Trp Ser Asp Glu Leu Gly Cys Asn Lys Gly Lys Glu 245 250 255
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAACATGCCT AGCTGATGAG TTCAAGTGTG ATGGTGGGAG GTGCATCCCA AGCGAATGGA 1200 TCTGTGACGG TGATAATGAC TGTGGGGATA TGAGTGACGA GGATAAAAGG CACCAGTGTC 1260
  • GTGCATTCCC CAGTCTTGGG TCTGTGATGG CGATGTGGAT TGTACTGACG GCTACATGAG 1380
  • TGTATCCCAA AGATATTGCG AGGTGTGACC GGCACAATGA CTGTGGTGAC TATAGCGACG 1500 AGAGGGCTGC TTATACCTAG ACTTGCCAAC AGAATCAGTT TCCTGTCAGA ACGGGCGCTG 1560
  • CATGCTGGCC CAGCACTGTG TGGATGCCAA
  • CAACACCTTC TGCTTTGATA ATCCCAGAGG 2400
  • ACTTGCCCTT CACCCTCAAT ATGGGTACCT CTACTGGGCA GACTGGGGTC ACCGCGCATA 2460
  • GCACTGCCTC ACCCTTTCGC TATTACCATT TTTGAAGACA CTATTTATTG GACAGATTGG 2700 AATACAAGGA CAGTGGAAAA GGGAAACAAA TATGATGGAT CAAATAGACA GACACTGGTG 2760
  • TTGTACCCAA TTAAATGAGG AGGATTTATC TGCTCCTGTA CAGCTGGGTT CGAAACCAAT 4440
  • CTTTGAAGAC CAGTTATACT GGATATCTAA GGAAAAGGGA GAAGTATGGA AACAAAATAA 5340 ATTTGGGCAA GGAAAGAAAG AGAAAACGCT GGTAGTGAAC CCTTGGCTCA CTCAAGTTCG 5400
  • CAGATCAGGG GCAGATCTTA ACATGGATAT TGGAGTGTCT GGTTTTGGAC CTGAGACTGC 5940 TATTGACAGG TCAATGGCAA TGAGTGAAGA CTTTGTCATG GAAATGGGGA AGCAGCCCAT 6000
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal

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  • Biochemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur l'isolement d'un clone de l'ADNc codant pour le détecteur calcique du placenta humain et les Northern blots subséquents confirmant l'expression de l'ARNm dans les cellules des tubules parathyroïdes et rénaux également chez l'homme. On a pu démontrer une grande similarité de séquence avec les antigènes de la néphrite de Heymann du rat qui sont des glycoprotéines de la bordure en brosse des tubules rénaux pouvant se fixer au calcium. L'immunohistochimie élabore une distribution des tissus de la protéine détectrice du calcium similaire à celle décrite antérieurement pour l'antigène de Heymann. Il est avancé que la protéine détectrice du calcium identifiée constitue un détecteur universel des fluctuations du calcium extracellulaire et joue un rôle clef pour la régulation du calcium par l'intermédiaire de différents systèmes d'organes. La protéine détectrice du calcium appartient à la superfamille LDL des glycoprotéines considérées comme agissant essentiellement comme récepteurs de protéine, avec cependant une forte capacité fonctionnelle de liaison au calcium.
PCT/US1995/015203 1993-05-23 1995-11-22 Proteine humaine detectrice du calcium, fragments de cette proteine et adn codant pour elle WO1996015801A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP51705696A JP2002500501A (ja) 1994-11-23 1995-11-22 ヒトカルシウムセンサータンパク質、その断片及びそれをコードするｄｎａ
AU42867/96A AU726886B2 (en) 1994-11-23 1995-11-22 Human calcium sensor protein, fragments thereof and DNA encoding same
EP95941448A EP0792159A4 (fr) 1994-11-23 1995-11-22 Proteine humaine detectrice du calcium, fragments de cette proteine et adn codant pour elle
US08/652,877 US6187548B1 (en) 1993-05-23 1996-05-23 Methods using human calcium sensor protein, fragments thereof and DNA encoding same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US34483694A 1994-11-23 1994-11-23
US08/344,836 1994-11-23
US48731495A 1995-06-07 1995-06-07
US08/487,314 1995-06-07

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WO1996015801A9 WO1996015801A9 (fr) 1996-07-18

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EP1462119A1 (fr) * 2003-03-24 2004-09-29 Schering AG Modulateurs de la capture de composés radiothérapeutiques et/ou radiodiagnostiques par l'intermédiaire de la megalin des cellules rénales et leur utilisation en thérapie et diagnostique
WO2004084953A1 (fr) * 2003-03-24 2004-10-07 Schering Ag Modulateurs du recaptage a mediation assuree par la megaline de substances de radiotherapie et/ou radiodiagnostic dans des cellules du rein ainsi que leur utilisation en therapie et dans des diagnostics

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EP0358977A1 (fr) * 1988-08-23 1990-03-21 The General Hospital Corporation Antigène de nephritis cloné
US5180819A (en) * 1989-12-22 1993-01-19 The Trustees Of Columbia University In The City Of New York Purified myeloblastin, nucleic acid molecule encoding same, and uses thereof
US5208144A (en) * 1988-08-23 1993-05-04 The General Hospital Corporation Method for detection of human dna containing the gene encoding low density lipoprotein receptor

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US6187548B1 (en) * 1993-05-23 2001-02-13 Rhone-Poulenc Rorer Pharmaceuticals Inc. Methods using human calcium sensor protein, fragments thereof and DNA encoding same
SE504108C2 (sv) * 1993-05-24 1996-11-11 Goeran Aakerstroem Human Kalcium sensor

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EP0358977A1 (fr) * 1988-08-23 1990-03-21 The General Hospital Corporation Antigène de nephritis cloné
US5208144A (en) * 1988-08-23 1993-05-04 The General Hospital Corporation Method for detection of human dna containing the gene encoding low density lipoprotein receptor
US5180819A (en) * 1989-12-22 1993-01-19 The Trustees Of Columbia University In The City Of New York Purified myeloblastin, nucleic acid molecule encoding same, and uses thereof

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Title
GENOMICS, Volume 2, Number 1, issued 01 July 1994, KORENBERG et al., "Chromosomal Localization of Human Genes for the LDL Receptor Family Member Glycoprotein 330 (LRP 2) and Its Associated Protein RAP (LRPAP1)", pages 88-93. *
KIDNEY INTERNATIONAL, Volume 35, issued 1989, KANALAS et al., "Isolation of 330kD Human Kidney Protein Similar to the Rat Heymann Nephritis Autoantigen (GP330)", page 351. *
See also references of EP0792159A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1462119A1 (fr) * 2003-03-24 2004-09-29 Schering AG Modulateurs de la capture de composés radiothérapeutiques et/ou radiodiagnostiques par l'intermédiaire de la megalin des cellules rénales et leur utilisation en thérapie et diagnostique
WO2004084953A1 (fr) * 2003-03-24 2004-10-07 Schering Ag Modulateurs du recaptage a mediation assuree par la megaline de substances de radiotherapie et/ou radiodiagnostic dans des cellules du rein ainsi que leur utilisation en therapie et dans des diagnostics

Also Published As

Publication number Publication date
EP0792159A1 (fr) 1997-09-03
EP0792159A4 (fr) 2003-01-02
AU4286796A (en) 1996-06-17
CA2205648A1 (fr) 1996-05-30
AU726886B2 (en) 2000-11-23

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