Raghunath M, Putnam E A, Ritty T, Hamstra D, Park E S, Tschödrich-Rotter M, Peters R, Rehemtulla A, Milewicz D M
Department of Physiological Chemistry and Pathobiochemistry and of Medical Physics and Biophysics, University of Münster, Münster, Germany.
J Cell Sci. 1999 Apr;112 ( Pt 7):1093-100. doi: 10.1242/jcs.112.7.1093.
Fibrillin-1, the main component of 10-12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin.
原纤蛋白-1是细胞外基质10 - 12纳米微原纤维的主要成分，以原纤蛋白形式合成并经蛋白水解加工成为纤连蛋白。通过该识别位点的自发突变阻止原纤蛋白转化，已将推定的切割位点定位到原纤蛋白-1的羧基末端结构域，位于氨基酸精氨酸2731和丝氨酸2732之间。该位点包含弗林蛋白酶/PACE家族前体蛋白转化酶的碱性氨基酸识别序列（R-G-R-K-R-R）。在本研究中，我们使用一种微型原纤蛋白来证实成纤维细胞和重组表达的弗林蛋白酶/PACE、PACE4、PC1/3和PC2在羧基末端结构域的切割作用。对共有识别基序中的氨基酸进行定点诱变可阻止转化，从而确定可裂解键并表征切割所需的碱性氨基酸。使用PACE/弗林蛋白酶抑制剂，我们发现野生型原纤蛋白在转化为纤连蛋白之前不会整合到细胞外基质中。因此，原纤蛋白-1是首个被证明为枯草杆菌蛋白酶样蛋白酶底物的细胞外基质蛋白，原纤蛋白向纤连蛋白的转化通过排除未切割的原纤蛋白来控制微原纤维生成。
