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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

我们发现了一种生长促进因子在软骨中协同刺激DNA合成的培养兔生长板软骨细胞在碱性FGF的存在下。在这项研究中，我们成功地纯化成同质的胎牛骨骺软骨，并命名为软骨调节素-I（ChM-I）的活性成分。从胎牛软骨cDNA文库中克隆的ChM-I前体cDNA的核苷酸序列表明，121个氨基酸长的ChM-I被编码为其由335个氨基酸残基组成的较大的膜蛋白前体的C-末端部分。结合N-末端氨基酸序列分析，在成熟的ChM-Ⅰ中发现了3个可能的糖基化位点。本研究试图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，并对表达的重组分子进行评价。首先，我们构建了含有完整编码区的pcDL-SR α 296表达载体 ...更多信息 ChM-I前体cDNA序列。然后，将该载体转染到培养的COS-1细胞中。回收条件培养基，通过肝素亲和柱浓缩，并通过抗ChM-I抗体的免疫印迹分析。实验表明表达了重组ChM-1，经SDS-PAGE分析，其糖基化且具有相似的25 kDa的分子大小。我们通过反相HPLC从条件培养基中纯化重组ChM-I至均一。重组ChM-I的N-末端氨基酸序列与天然存在的ChM-I相同。Thr ^[9]的氨基酸残基被认为是糖基化的，就像在天然ChM-Ⅰ中发现的那样。这些结果有力地支持了我们的预测，即成熟的ChM-Ⅰ被加工并分泌到细胞外，如上所述纯化的重组ChM-Ⅰ刺激培养的生长板软骨细胞的蛋白多糖合成，并在碱性FGF存在和不存在的情况下刺激细胞的DNA合成。因此，重组ChM-I保留了类似于来自胎牛软骨的天然ChM-I的生物活性。少