Biosynthesis and Complex Formation of Cartilage Specific Functional Matrix/Chondromodulin-I

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The complete primary amino acid sequence of chondromodulin-I (ChM-I) has been known for bovine counterpart. In the present study, we tried to clone out human ChM-I cDNA from which the primary amino acid sequence would be deduced. While cartilage tissue is widely found in embryonic tissue, there is only a little cartilage tissue found in postnatal animals. Most of cartilage in embryo is replated by bone at the end of embryonic development. Thus, there is a considerable difficulty in obtaining fresh cartilage tissue of the amount enough for construction of cDNA library. To avoid this difficulty, we isolated RNA from human chondrosarcoma of a highly differentiated type from which cDNA library was constructed. The full coding region of human ChM-I cDNA was successfully amplified from the cDNA by PCR.Short5' untranslated stretch was cloned out from human genomic DNA library. Sequencing of the human ChM-I cDNA fragment indicated that human ChM-I has three base/one amino acid deletion in comp … More arison with the bovine counterpart. Sequence identity between human and bovine Chm-I precursor was determined to be 89.6% based on the nucleotide sequences and 91.9% based on the deduced amino acid sequences. N-Glycosylation site in the mature ChM-I was conserved in the human counterpart, but one of two O-glycosylated Thr residues in bovine ChM-I was missing. In contrast, C-terminal half of the mature ChM-I which contains 8 cysteine residues was completely identical except for the one amino acid substitution (His to Tyr). Then, we attempted to express human ChM-I cDNA in COS cells. The yield of the expressed recombinant ChM-I was unexpectedly low. However, the yield was improved by replacing the 5' franking sequences of the ATG start codon from GGCTTC to GGCACC.We could identify mature human ChM-I in the conditioned medium from the transfected COS cell culture as a diffuse 25 kDa band by SDS-PAGE analysis. These results suggested that we could successfully establish the experimental model to study biosynthetic and secretory pathway of mature ChM-I. Less