Molecular cloning of a cartilage-derived growth modulating factor, chondromodulin-I(ChM-I) and functional expression of ChM-I cDNA

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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 … More 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