Molecular cloning of a cartilage-derived growth modulating factor, chondromodulin-I(ChM-I) and functional expression of ChM-I cDNA
| Project/Area Number |
03680148
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Osaka University |
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Principal Investigator |
HIRAKI Yuji Osaka Univ. Fac. of Dentistry, Dept. of Biochem., Assistant Professor, 歯学部, 講師 (40144498)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Fujio Osaka Univ. Fac. of Dentistry, Dept. of Biochem., Professor, 歯学部, 教授 (40028717)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Bone Formation / Growth Factor / Extracellular Matrix / Fibroblast Growth Factor / Cellular Differentiation / Chondromodulin-I / Chondrosurfactant / Chondrocyte / ChondromodulinーI |
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| Research Abstract |
We found a growth promoting factor in cartilage which synergistically stimulates DNA synthesis of cultured rabbit growth-plate chondrocytes in the presence of basic FGF. In this study, we succeeded in purifying this active principle into homogeneity from fetal bovine epiphyseal cartilage, and named it chondromodulin-I (ChM-I). The nucleotide sequence of ChM-I precursor cDNA cloned from the fetal bovine cartilage cDNA library indicated that 121 amino acid-long ChM-I is coded as a C-terminal portion of its larger membrane-protein precursor consisting of 335 amino acid residues. Together with N-terminal amino acid sequence analysis, three possible glycosylation sites were identified in the mature ChM-I.
This year, we attempted to express ChM-I precursor cDNA in mammalian expression system for studying mechanism of its biosynthesis and evaluation of the expressed recombinant molecules. First we constructed a expression vector derived from pcDL-SRalpha296 which harbored a full coding region
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of ChM-I precursor cDNA sequence. Then, the vector was transfected into COS-1 cells in culture. The conditioned medium was recovered, concentrated by a heparin-affinity column, and analyzed by immunoblotting of anti-ChM-I antibody. The experiment indicated expression of recombinant ChM-I which was glycosylated and had a similar molecular size of 25 kDa by SDS-PAGE analysis. We purified recombinant ChM-I into homogeneity from the conditioned medium by reverse-phase HPLC. The N-terminal amino acid sequence of recombinant ChM-I was identical to the naturally occurring ChM-I. The amino acid residue of Thr^9 was suggested to be glycosylated as found in the natural ChM-I. These results strongly supported the our prediction that mature ChM-I was processed and secreted out of the cells.
Recombinant ChM-I purified as described above stimulated proteoglycan synthesis of cultured growth-plate chondrocytes and DNA synthesis of the cells in the presence and absence of basic FGF. Thus recombinant ChM-I retained the biological activities similar to natural ChM-I derived from fetal bovine cartilage. Less
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Report
(3 results)
Research Products
(23 results)
