Brown-Augsburger P, Broekelmann T, Rosenbloom J, Mecham R P
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):149-55. doi: 10.1042/bj3180149.
Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associated proteins. In this study we have used cultured auricular chondrocytes as a model system to examine whether this interaction is critical for elastic fibre formation in vivo. Auricular chondrocytes, which deposit an abundant elastic fibre matrix, were cultured in the presence of Fab fragments of antibodies directed against the C-terminus (CTe) or an N-terminal domain (ATe) of tropoelastin. Immunofluorescent staining of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting microfibril structure. Cells grown under identical conditions in the presence of ATe, however, formed fibres that stained normally for both elastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody directed against a domain near the N-terminus of MAGP, did not organize tropoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescence staining patterns, fewer elastin-specific cross-links, indicative of insoluble elastin, were detected in the extracellular matrix of cells cultured in the presence of CTe. The medium from these cultures, however, contained more soluble elastin, consistent with an antibody-induced alteration of elastin assembly but not its synthesis. Northern analysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminus of tropoelastin interacts with microfibrils during the assembly of elastic fibres. Further, the results suggest that the interaction between tropoelastin and microfibrils might be mediated by a domain involving the N-terminal half of MAGP.
体外研究表明，原弹性蛋白的C末端通过与微原纤维相关蛋白相互作用介导弹性蛋白聚合。在本研究中，我们使用培养的耳软骨细胞作为模型系统，以检查这种相互作用对体内弹性纤维形成是否至关重要。在针对原弹性蛋白C末端（CTe）或N末端结构域（ATe）的抗体Fab片段存在的情况下，培养能沉积丰富弹性纤维基质的耳软骨细胞。对细胞沉积的细胞外基质进行免疫荧光染色显示，CTe抗体抑制弹性蛋白的沉积，而不影响微原纤维结构。然而，在ATe存在的相同条件下生长的细胞形成的纤维，对弹性蛋白和微原纤维蛋白的染色均正常。在微原纤维相关糖蛋白（MAGP）：21 - 35（一种针对MAGP N末端附近结构域的抗体）存在的情况下培养的软骨细胞，不能将原弹性蛋白组织成纤维。然而，对MAGP和原纤蛋白的免疫染色显示微原纤维正常。与免疫荧光染色模式一致，在CTe存在下培养的细胞的细胞外基质中，检测到较少的弹性蛋白特异性交联（指示不溶性弹性蛋白）。然而，这些培养物的培养基中含有更多的可溶性弹性蛋白，这与抗体诱导的弹性蛋白组装改变而非其合成一致。对抗体处理的培养物和对照培养物进行Northern分析，证实原弹性蛋白mRNA水平相当。这些结果证实，在弹性纤维组装过程中，原弹性蛋白的C末端与微原纤维相互作用。此外，结果表明，原弹性蛋白与微原纤维之间的相互作用可能由涉及MAGP N末端一半的结构域介导。
