Abstract
We previously showed that collagen, α-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 14C-labeled α1(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 α1(I) suggested that there are ≅ 16 × 106 binding sites per fibroblast with an association constant of 1.1 × 107/M for α1(I). Dissociation of the bound radioactivity and subsequent chromatographic analysis on agarose A-1.5 m revealed that the α1 was unaltered. The binding of 14C-labeled α1 was inhibited by each of the CNBr peptides derived from α1 chain of chick skin collagen and CNBr peptide mixtures of various genetic types of collagen chains.
Immunofluorescence studies with anti-α1 antibody showed that α1-treated fibroblasts exhibited strong immunofluorescence. The intensity of fluorescence was markedly diminished by prior absorption of the antibody with α1. The α1-treated cells stained with preimmune sera did not show significant fluorescence.
Dose-response curves of fibroblast chemotaxis induced by α1 and the binding of α1 by fibroblasts correlate closely. Furthermore, the potency of α1-CNBr peptides as chemotactic agents correlates with their ability to inhibit the binding of labeled α1(I). These data suggest the hypothesis that collagenderived peptides cause fibroblast chemotactic migration by acting on fibroblast membranes.
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Selected References
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