Ashworth J L, Murphy G, Rock M J, Sherratt M J, Shapiro S D, Shuttleworth C A, Kielty C M
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Biochem J. 1999 May 15;340 ( Pt 1)(Pt 1):171-81.
Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.
原纤维蛋白是细胞外基质中直径为10 - 12纳米的弹性微原纤维的主要结构成分。我们之前已经表明，原纤维蛋白分子和组装好的微原纤维都易受丝氨酸蛋白酶的降解。在本研究中，我们调查了六种基质金属蛋白酶（MMP - 2、MMP - 3、MMP - 9、MMP - 12、MMP - 13和MMP - 14）对原纤维蛋白分子以及从睫状小带分离出的完整富含原纤维蛋白的微原纤维的潜在分解代谢作用。使用新合成的重组原纤维蛋白分子，确定了原纤维蛋白 - 1内的主要切割位点。特别是，这六种不同的基质金属蛋白酶从分子的N端区域产生了一个约45 kDa的主要降解产物，而对截短的、未加工的和经弗林蛋白酶加工的C端进行处理也产生了大的降解产物。在一个预测会破坏钙结合的钙结合表皮生长因子样结构域中引入一个导致晶状体异位的单氨基酸替代（E2447K；氨基酸的单字母符号），显著改变了原纤维蛋白 - 1 C端的降解模式。然而，在一个上游钙结合表皮生长因子样结构域中具有类似E→K替代的突变原纤维蛋白 - 1的片段化模式与野生型分子无法区分。超微结构检查突出显示，从睫状小带分离出的富含原纤维蛋白的微原纤维被基质金属蛋白酶严重破坏。这是首次证明原纤维蛋白分子和富含原纤维蛋白的微原纤维会被基质金属蛋白酶降解，并且某些氨基酸替代会改变片段化模式。这些研究对生理性和病理性原纤维蛋白分解代谢以及衰老和疾病中结缔组织弹性丧失具有重要意义。
