Kirsch T, Wuthier R E
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208.
J Biol Chem. 1994 Apr 15;269(15):11462-9.
Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.
基质小泡（MV）是一种能在体外迅速积累钙离子并诱导矿物质形成的微观结构，与肥大软骨中的II型和X型胶原蛋白以及蛋白聚糖相关联。然而，这些基质蛋白对MV功能的作用尚不清楚。这促使我们研究II型和X型胶原蛋白结合对MV摄取钙离子的影响。从鸡生长板软骨中分离出的MV，在合成软骨淋巴液中用纯细菌胶原酶和1 M氯化钠处理，以选择性地完全去除相关的II型和X型胶原蛋白。这些去除胶原蛋白的小泡对45Ca2+的摄取明显减少。用去污剂进一步处理会破坏膜结构，但能恢复钙离子摄取，这表明在胶原酶和1 M氯化钠处理后，小泡膜结构和小泡腔内的成核核心仍然完整。将天然的II型或X型胶原蛋白重新添加到经胶原酶和1 M氯化钠处理的MV中，会刺激它们的钙离子摄取达到与未处理小泡相似的水平。用胃蛋白酶处理的II型和X型胶原蛋白在刺激钙离子摄取方面效果较差，表明这些胶原蛋白的非三螺旋结构域参与其中。对这些胶原蛋白进行胃蛋白酶处理也会降低它们与膜联蛋白V（膜附着蛋白CII）的结合，膜联蛋白V是在MV中发现的三种膜联蛋白之一，这表明膜联蛋白V参与介导II型和X型胶原蛋白与MV表面的结合。此外，用胰凝乳蛋白酶处理经胶原酶和1 M氯化钠处理的MV，胰凝乳蛋白酶会破坏膜联蛋白V以及许多其他MV蛋白，从而阻止这些胶原蛋白对钙离子摄取的刺激作用。因此，II型和X型胶原蛋白与MV之间的相互作用激活了钙离子流入MV的过程，可能在小泡钙化中起重要作用。
