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

软骨调节素-I（ChM-I）的完整一级氨基酸序列已知为牛对应物。在本研究中，我们试图克隆出人ChM-Ⅰ cDNA，从中推导出一级氨基酸序列。虽然软骨组织广泛存在于胚胎组织中，但在出生后的动物中仅发现少量软骨组织。胚胎期软骨在胚胎发育末期大部分被骨覆盖。因此，获得足够量的新鲜软骨组织以构建cDNA文库存在相当大的困难。为了避免这一困难，我们从高度分化型的人软骨肉瘤中分离RNA，构建cDNA文库。从人ChM-I基因cDNA中成功扩增出ChM-I全长编码区，并从人基因组DNA文库中克隆出ChM-I短5 '端非翻译片段。对人ChM-Ⅰ cDNA片段的测序结果表明，人ChM-Ⅰ与人ChM-Ⅰ相比有3个碱基/1个氨基酸的缺失， ...更多信息 与牛的对应物发生了反应。基于核苷酸序列和基于推导的氨基酸序列，人和牛Chm-I前体之间的序列同一性被确定为89.6%和91.9%。成熟ChM-I中的N-糖基化位点在人ChM-I中是保守的，但牛ChM-I中的两个O-糖基化Thr残基中的一个缺失。相比之下，C-末端的一半，成熟的ChM-I，其中包含8个半胱氨酸残基是完全相同的，除了一个氨基酸的取代（His到Tyr）。然后，我们尝试在COS细胞中表达人ChM-1 cDNA。表达的重组ChM-I的产率出乎意料地低。然而，通过将ATG起始密码子的5'侧翼序列从GGCTTC替换为GGCACC，产量得到提高。我们可以通过SDS-PAGE分析鉴定来自转染的COS细胞培养物的条件培养基中的成熟人ChM-I，其为弥散的25 kDa条带。这些结果表明，我们可以成功地建立实验模型，研究成熟ChM-I的生物合成和分泌途径。少

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