Expression and function of RGD-CAP conatining RGD-motif in cartilage

获得以下结果以阐明RGD-CAP作为介质在与软骨细胞的生长和发育有关的细胞和基质的相互作用中的作用。(1)从鸡胚软骨细胞cDNA文库中克隆了含有RGD序列的胶原相关蛋白RGD-CAP（collagen-associated protein containing the RGD sequence）。推导的氨基酸序列（680个氨基酸残基）包含4个内部重复结构域和2个高度保守的序列（H1和H2）在每个重复。该蛋白含有一个整合素识别位点，在四个重复结构的第四个结构域中的RGD基序。(2)我们使用RGD-CAP包被的培养皿检测了重组鸡RGD-CAP对软骨细胞和成纤维细胞粘附的影响。当兔软骨细胞或人成纤维细胞接种在用RGD-CAP包被的塑料培养皿上时，与对照培养皿相比，细胞粘附增强。(3)RGD-CAP在II型或III型胶原存在下产生协同的细胞粘附刺激，在I型或V型胶原存在下产生相加的刺激。(4)RGD-CAP对细胞粘附的作用需要二价阳离子（Mg^<2+>或Mn^<2+>），EDTA和GRGDS肽可以降低RGD-CAP对细胞粘附的作用。(5)针对人整联蛋白α 1、α 5或β 1亚基而非α 2、α 3或β 2的单克隆抗体可降低RGD-CAP诱导的细胞粘附。这些发现表明，RGD-CAP是一种新的整合素α 1 β 1/α 5 β 1的配体，并且RGD-CAP上的RGD序列以外的位点也参与细胞粘附。(6)含有两个重复结构域的RGD-CAP片段也能够有效地增强软骨细胞的粘附，表明两个结构域的重复的重要性。

The following results were obtained to clarify the role of RGD-CAP as the mediator in the interaction of cells and matrix concerning the growth and development of chondrocytes.(1) A cDNA clone for RGD-CAP (collagen-associated protein containing the RGD sequence) was isolated from chick embryo chondrocyte cDNA library. The deduced amino acid sequence (680 amino acid residues) contained four internal repeat domains and two highly conserved sequences (Hi and H2) in each repeat. The protein contains an integrin recognition site, the RGD motif in the fourth domain of four repetitive structures.(2) We examined the effect of recombinant chick RGD-CAP on the adhesion of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit chondrocytes or human fibroblasts were seeded on plastic dishes coated with RGD- CAP, cell adhesion was enhanced compared with control dishes.(3) RGD-CAP produced a synergistic stimulation of cell adhesion in thepresence of type II or III collagen, and an additive stimulation in the presence of type I or V collagen.(4) The effect of RGD-CAP on cell adhesion required divalent cations (Mg^<2+> or Mn^<2+>) , and was reduced by EDTA and by the GRGDS peptide.(5) Monoclonal antibodies to the human integrin alpha1, alpha5, or beta1 subunit but not alpha2, alpha3, or beta2 decreased the RGD-CAP-induced cell adhesion. These findings suggest that RGD-CAP is a novel ligand for integrin alpha1beta1/alpha5beta1, and that a site other than the RGD sequence on RGD-CAP is also involved in cell adhesion.(6) An RGD-CAP fragment containing two repeating domains was also capable of enhancing efficiently the adhesion of chondrocytes indicating the importance of the repetition of two domains.