Isolation and characterization of a cDNA for osteopontin-k: A kidney cell adhesion molecule with high homology to osteopontins

Oxford University Press (OUP) - Tập 7 Số 6 - Trang 693-699 - 1992
Joseph F. Crivello1, E. Delvin2
1Department of Physiology and Neurobiology, University of Connecticut, Storrs
2Genetics Unit, Shriners Children Hospital, Montreal, Quebec, Canada

Tóm tắt

Abstract Screening of a bovine renal cDNA library with MAbs(1) resulted in the isolation of a 1447 bp cDNA. This cDNA (pBk2.1) was sequenced and shown to contain an open reading frame with a putative protein of 261 amino acids, with a molecular weight of 29,573 (minute leader sequence) and a hydrophobic leader sequence of 16 amino acids. pBk2.1 was shown to share a high level of nucleic acid sequence homology over portions of its sequence to human, porcine, mouse, and rat osteopontins (40–60%). The peptide (osteopontin-k) had a potential glycosylation site (Asn-X-Ser/Thr), a GRGDS receptor binding region, a high level of asparagine residues, and a high abundance of acid amino acids characteristic of osteopontin-like cell adhesion molecules. The N-terminal amino acid region of pBk2.1 (the first 82 amino acids) and 42 amino acids at the C terminus had the highest level of homology with the osteopontins at 86%. The middle portion of the peptide had greatly reduced homology, ranging from 50% (amino acids 83–174) to 12% (amino acids 175–219). There were also deletions and additions of sequence in osteopontin-k that were not found in the other osteopontins. The homologies suggest that these proteins are highly related and may be derived from a common gene by alternative splicing. A 678 bp cRNA probe constructed from pBk2.1, containing a region with low homology to the osteopontins (amino acids 183–219 with <20% homology, plus amino acids 220–261 and untranslated sequence), was used in northern blots and RNAse protection assays. In a northern blot, this cRNA probe detected a 1.8–2.0 kb mRNA in bovine kidney. The greatest signal was seen in kidney, followed by liver and heart. Normal trabecular bone had a weak signal, with no signal in pancrease or skeletal muscle. For more accurate quantitation of mRNA levels, RNAse protection assays were used. These experiments demonstrated that this mRNA is expressed at highest levels in bovine kidney but at very low levels in osteosarcoma cells and at lower levels in liver, heart, and skeletal muscle. Treatment of bovine proximal tubule cells with 100 nM 1,25-(OH)2D3 resulted in a increase in osteopontin-k mRNA expression (2.7-fold). PTH had no effect on the expression of osteopontin-k mRNA. These results suggest that pBk2.1 represents a predominantly renal-specific cell adhesion molecule within the family of osteopontins that may be under hormonal regulation by the vitamin D system.

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Tài liệu tham khảo

Bort, 1988, Characterization of monoclonal antibodies specific for the bovine renal mitochondrial vitamin D hydroxylases., Endocrinology, 123, 2491, 10.1210/endo-123-5-2491

Craig, 1989, Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis., J Biol Chem, 264, 9682, 10.1016/S0021-9258(18)60584-8

Noda, 1989, Transcriptional regulation of osteopontin production in rat osteoblast-like cells by PTH., J Cell Biol, 108, 713, 10.1083/jcb.108.2.713

Reinholt, 1990, Osteopontin–apossible anchor of osteoclasts to bone., Proc Natl Acad Sci USA, 87, 4473, 10.1073/pnas.87.12.4473

Nagata, 1989, Sulfation of secreted phosphoprotein I (Sppi, Osteopontin) is associated with mineralized bone formation., Biochem Biophys Res Comm, 165, 234, 10.1016/0006-291X(89)91059-0

Kubota, 1989, Multiple forms of SPPI synthesized by normal and transformed rat bone cell populations: Regulation by TGF-β., Biochem Biophys Res Commun, 162, 1453, 10.1016/0006-291X(89)90837-1

Noda, 1990, LIF suppresses proliferation, alkaline phosphatase activity, and type I collagen mRNA level and enhances osteopontin mRNA level in murine osteoblast-like cells., Endocrinology, 127, 185, 10.1210/endo-127-1-185

Senger, 1989, Elevated expression of secreted phosphoprotein-I (osteopontin 2Ar) as a consequence of neoplastic transformation., Anticancer Res, 9, 1291

Qi, 1990, Isolation and characterization of the gene for secreted phosphoprotein-I (SPPI, osteopontin)., J Dent Res, 69, 114

Qi, 1990, Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein-I (SSPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein–demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI., J Biol Chem, 265, 7583, 10.1016/S0021-9258(19)39154-9

Nemir, 1989, Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties., J Biol Chem, 264, 8202, 10.1016/S0021-9258(19)84697-5

Miyazaki, 1989, Nucleotide sequence of cDNA for mouse osteopontin-like protein., Nucleic Acids Res, 17, 3298, 10.1093/nar/17.8.3298

Miyazaki, 1990, The mouse osteopontin gene–expression in monocytic lineages and complete nucleotide sequence., J Biol Chem, 265, 4432, 10.1016/S0021-9258(18)77320-1

Wrana, 1989, Full length cDNA sequence of porcine phosphoprotein-I (APP-I, osteopontin)., Nucleic Acids Res, 17, 10119, 10.1093/nar/17.23.10119

Young, 1990, cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin., Genomics, 7, 491, 10.1016/0888-7543(90)90191-V

Little, 1987, DNA Cloning: A Practical Approach, 1

Mierendorf, 1987, Direct sequencing of denatured plasmid DNA., Methods Enzymol, 152, 556, 10.1016/0076-6879(87)52061-4

Barnes, 1987, Sequencing DNA with dideoxyribonucleotides as chain terminators: Hints and strategies for big projects., Methods Enzymol, 152, 538, 10.1016/0076-6879(87)52060-2

Crivello, 1985, Bovine renal vitamin D3 hydroxylases. Regulation of activities by inhibitors and antioxidants., Endocrinology, 117, 447, 10.1210/endo-117-2-447

Crivello, 1986, Interaction of bovine renal mitochondrial cytochrome P450 with antioxidants., Arch Biochem Biophys, 248, 551, 10.1016/0003-9861(86)90508-4

Rodan, 1989, Opposing effects of FGF and PT on alkaline phosphatase, osteopontin, osteocalcin, and type I collagen mRNA levels in ROS 17/2.8 cells., J Biol Chem, 264, 19934, 10.1016/S0021-9258(19)47201-3

Chomczynski, 1987, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction., Anal Biochem, 162, 156, 10.1016/0003-2697(87)90021-2

Kerr, 1990, Human osteopontin 2: Evidence that osteopontin(s) is a multigene family (abstr)., J Bone Miner Res, 5, S75

Sambrook, 1990, Molecular Cloning: A Laboratory Manual

Keifer, 1989, The cDNA and derived amino acid sequence for human osteopontin., Nucleic Acids Res, 17, 3306, 10.1093/nar/17.8.3306

Oldberg, 1986, Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence., Proc Natl Acad Sci USA, 83, 8819, 10.1073/pnas.83.23.8819

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Oxford University Press (OUP)

Tập 7 Số 6

693-699

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