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

软骨源性生长调节因子软骨调节素-I(ChM-I)的分子克隆及ChM-I cDNA的功能表达

基本信息

批准号：
03680148
负责人：
HIRAKI Yuji
金额：
$ 1.22万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

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

我们在软骨中发现了一种生长促进因子，在碱性 FGF 存在的情况下，可协同刺激培养的兔生长板软骨细胞的 DNA 合成。在本研究中，我们成功地从胎牛骨骺软骨中纯化出这种活性成分，并将其命名为软骨调节蛋白。第 121 章长氨基酸的 ChM-I 被编码为由 335 个氨基酸残基组成的较大膜蛋白前体的 C 端部分，结合 N 端氨基酸序列分析，在成熟的 ChM-I 中鉴定出了三个可能的糖基化位点。一、今年，我们尝试在哺乳动物表达系统中表达ChM-I前体cDNA，以研究其生物合成机制并评估表达的重组分子。含有 ChM-I 前体 cDNA 序列的完整编码区的 pcDL-SRalpha296 然后，将载体转染到培养物中的 COS-1 细胞中，回收条件培养基，通过肝素亲和柱浓缩，并进行分析。抗ChM-I抗体的免疫印迹实验表明重组ChM-I被糖基化并且通过SDS-PAGE分析具有相似的分子大小25kDa。我们通过反相HPLC将重组ChM-I从条件培养基中纯化至同质。重组ChM-I的N端氨基酸序列与天然存在的ChM-I相同。Thr^9的氨基酸残基。这些结果有力地支持了我们的预测，即成熟的 ChM-I 被加工并分泌出细胞。如上所述纯化的重组 ChM-I 受到刺激。在存在和不存在碱性FGF的情况下培养的生长板软骨细胞的蛋白聚糖合成和细胞的DNA合成因此重组ChM-I保留了与源自胎牛软骨的天然ChM-I相似的生物活性。