Chiang T M, Postlethwaite A E, Beachey E H, Seyer J M, Kang A H
J Clin Invest. 1978 Nov;62(5):916-22. doi: 10.1172/JCI109219.
We previously showed that collagen, alpha-chains, and collagen-derived peptide fragments induce chemotactic migration of human fibroblasts in vitro. We now describe biochemical and immunological evidence showing there are binding sites for collagen peptides on fibroblast membranes.By the use of (14)C-labeled alpha1(I) chain, binding to intact fibroblasts was demonstrated. The process was reversible, and time- and fibroblast concentration-dependent. Scatchard plot analyses of the data obtained for the binding of alpha1(I) suggested that there are congruent with 16 x 10(6) binding sites per fibroblast with an association constant of 1.1 x 10(7)/M for alpha1(I). Dissociation of the bound radioactivity and subsequent chromatographic analysis on agarose A-1.5 m revealed that the alpha1 was unaltered. The binding of (14)C-labeled alpha1 was inhibited by each of the CNBr peptides derived from alpha1 chain of chick skin collagen and CNBr peptide mixtures of various genetic types of collagen chains. Immunofluorescence studies with anti-alpha1 antibody showed that alpha1-treated fibroblasts exhibited strong immunofluorescence. The intensity of fluorescence was markedly diminished by prior absorption of the antibody with alpha1. The alpha1-treated cells stained with preimmune sera did not show significant fluorescence.Dose-response curves of fibroblast chemotaxis induced by alpha1 and the binding of alpha1 by fibroblasts correlate closely. Furthermore, the potency of alpha1-CNBr peptides as chemotactic agents correlates with their ability to inhibit the binding of labeled alpha1(I). These data suggest the hypothesis that collagenderived peptides cause fibroblast chemotactic migration by acting on fibroblast membranes.
我们之前曾表明，胶原蛋白、α链和胶原蛋白衍生的肽片段在体外可诱导人成纤维细胞的趋化性迁移。我们现在描述生化和免疫学证据，表明成纤维细胞膜上存在胶原蛋白肽的结合位点。通过使用（14）C标记的α1（I）链，证实了其与完整成纤维细胞的结合。该过程是可逆的，且具有时间和成纤维细胞浓度依赖性。对α1（I）结合所获得数据的Scatchard图分析表明，每个成纤维细胞有16×10（6）个结合位点，α1（I）的缔合常数为1.1×10（7）/M。结合的放射性物质解离并随后在琼脂糖A - 1.5m上进行色谱分析，结果显示α1未发生改变。来自鸡皮肤胶原蛋白α1链的每个CNBr肽以及各种遗传类型胶原蛋白链的CNBr肽混合物均能抑制（14）C标记的α1的结合。用抗α1抗体进行的免疫荧光研究表明，经α1处理的成纤维细胞呈现强烈的免疫荧光。用α1预先吸收抗体后，荧光强度明显减弱。用免疫前血清染色的经α1处理的细胞未显示出明显的荧光。α1诱导的成纤维细胞趋化性的剂量反应曲线与成纤维细胞对α1的结合密切相关。此外，α1 - CNBr肽作为趋化剂的效力与其抑制标记的α1（I）结合的能力相关。这些数据提示了一个假说，即胶原蛋白衍生的肽通过作用于成纤维细胞膜导致成纤维细胞的趋化性迁移。
