Perris R, Kuo H J, Glanville R W, Leibold S, Bronner-Fraser M
Reference Center for Oncology, Experimental Division 2, Aviano (PN) Italy.
Exp Cell Res. 1993 Nov;209(1):103-17. doi: 10.1006/excr.1993.1290.
Collagen type VI (Col VI) is a primary constituent of the extracellular matrix encountered by migrating avian neural crest cells in situ and is effective in promoting attachment and motility of these cells in vitro. In this study, we have explored the molecular mechanisms of neural crest-Col VI interaction by using quantitative assays for cell attachment and migration in vitro, proteolytic fragments of the collagen, and a panel of domain-specific monoclonal antibodies. Removal of the predominant portion of the amino-terminal globular domains of Col VI tetramers by pepsin digestion (P6 fragment) resulted in a > fivefold decrease in their cell adhesion and motility-promoting activity. Further digestion of P6 with bacterial collagenase, which causes a complete loss of the amino-terminal domains plus an adjacent triple-helical segment, did not affect adhesion but reduced migration down to 40% of that seen on undigested P6. Untreated and pepsin-digested Col VI monomers were significantly less effective than their tetrameric counterparts and a M(r) 200,000 fragment, generated from pepsin-digested monomers by a second pepsin treatment, only retained 40% of the motility-promoting activity while preserving the adhesive capacity. A mixture of amino- and carboxyl-terminal globular domains supported both cell attachment and migration. While neural crest cells adhered equally well to the individual intact alpha 1 (VI)/alpha 2(VI) and alpha 3(VI) chains, they migrated most extensively on the alpha 3(VI) chain. Conversely, pepsin-digested individual alpha chains were significantly less effective in promoting cell adhesion and locomotion. Selective preincubation of Col VI microfilaments and isolated tetramers with a panel of monoclonal antibodies against triple helix, carboxyl-terminal, and amino-terminal epitopes of the different constituent chains differentially perturbed neural crest cell attachment and migration. Sites differentially involved in neural crest cell attachment and migration seemed to be present at the carboxyl termini of the alpha 1(VI) and alpha 2(VI) chains and at the amino-terminus of the alpha 3(VI) chain. The results suggest that neural crest cells interact with Col VI through multiple and cooperative binding sites present within its triple-helical and globular domains. The differential involvement and efficiency of these sites in stimulating neural crest cell adhesion and migration is strongly determined by the supramolecular organization of the collagen and requires inter- and intramolecular structural integrity. Since neural crest cell attachment and migration on Col VI was completely inhibited by anti-beta 1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell--Col VI interaction.
VI型胶原蛋白（Col VI）是迁移中的禽类神经嵴细胞在原位所遇到的细胞外基质的主要成分，并且在体外能有效促进这些细胞的附着和运动。在本研究中，我们通过体外细胞附着和迁移的定量测定、胶原蛋白的蛋白水解片段以及一组结构域特异性单克隆抗体，探索了神经嵴 - Col VI相互作用的分子机制。用胃蛋白酶消化（P6片段）去除Col VI四聚体氨基末端球状结构域的主要部分，导致其细胞黏附及运动促进活性降低了五倍以上。用细菌胶原酶进一步消化P6，这会导致氨基末端结构域以及相邻的三螺旋片段完全丧失，并不影响黏附，但使迁移能力降至未消化P6的40%。未处理的和经胃蛋白酶消化的Col VI单体比其四聚体对应物的效果明显要差，并且通过第二次胃蛋白酶处理从经胃蛋白酶消化的单体产生的一个200,000分子量的片段，仅保留了40%的运动促进活性，同时保留了黏附能力。氨基末端和羧基末端球状结构域的混合物既支持细胞附着也支持细胞迁移。虽然神经嵴细胞对完整的α1(VI)/α2(VI)和α3(VI)链个体的黏附效果相同，但它们在α3(VI)链上的迁移最为广泛。相反，经胃蛋白酶消化的单个α链在促进细胞黏附和运动方面的效果明显较差。用一组针对不同组成链的三螺旋、羧基末端和氨基末端表位的单克隆抗体对Col VI微丝和分离的四聚体进行选择性预孵育，会不同程度地干扰神经嵴细胞的附着和迁移。似乎在α1(VI)和α2(VI)链的羧基末端以及α3(VI)链的氨基末端存在不同程度参与神经嵴细胞附着和迁移的位点。结果表明，神经嵴细胞通过其存在于三螺旋和球状结构域内的多个协同结合位点与Col VI相互作用。这些位点在刺激神经嵴细胞黏附和迁移中的不同参与程度和效率，很大程度上由胶原蛋白的超分子组织决定，并且需要分子间和分子内的结构完整性。由于抗β1整合素抗体完全抑制了神经嵴细胞在Col VI上的附着和迁移，有证据表明这类整合素对于神经嵴细胞 - Col VI相互作用至关重要。
