Ashworth J L, Kelly V, Rock M J, Shuttleworth C A, Kielty C M
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences and Department of Medicine, University of Manchester M13 9PT, UK.
J Cell Sci. 1999 Nov;112 ( Pt 22):4163-71. doi: 10.1242/jcs.112.22.4163.
The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.
原纤蛋白组装成微原纤维的分子机制尚不清楚。在本研究中，我们采用重组方法研究了人原纤蛋白-1的羧基末端加工和组装过程。原纤蛋白-1羧基末端的加工受到弗林蛋白酶切割位点下游紧邻位点的N-糖基化以及与钙网蛋白结合的强烈影响。在相同条件下，原纤蛋白-2的羧基末端加工效率低于原纤蛋白-1的羧基末端。对原纤蛋白-1氨基末端区域以及原纤蛋白-1未加工和经弗林蛋白酶加工的羧基末端区域进行大小分级分离，结果显示氨基末端形成了大量二硫键连接的聚集体。未加工的羧基末端原纤蛋白-1也有一些聚集现象，但经加工的羧基末端序列保持单体状态，除非存在外显子12 - 15编码的氨基末端序列。这些数据表明在氨基末端以及弗林蛋白酶切割位点下游的极端羧基末端序列中存在原纤蛋白-1分子识别序列，以及特定的氨基末端和羧基末端关联，这可能驱动经弗林蛋白酶加工的原纤蛋白分子在细胞外空间中进行重叠线性堆积。两种原纤蛋白异构体加工过程的差异可能反映了它们在细胞外空间组装能力的不同。
