TRUNCATED PROFIBRILLIN OF A MARFAN PATIENT IS OF APPARENT SIMILAR SIZE AS FIBRILLIN - INTRACELLULAR RETENTION LEADS TO OVER-N-GLYCOSYLATION
TRUNCATED PROFIBRILLIN OF A MARFAN PATIENT IS OF APPARENT SIMILAR SIZE AS FIBRILLIN - INTRACELLULAR RETENTION LEADS TO OVER-N-GLYCOSYLATION
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DOI：
10.1006/jmbi.1995.0270
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发表时间：
1995-05-19
影响因子：
5.6
通讯作者：
STEINMANN, B
中科院分区：
文献类型：
--
作者：
RAGHUNATH, M;KIELTY, CM;STEINMANN, B
我们研究了一名患有严重马凡综合征的个体的培养成纤维细胞中的 profibrillin-1 (proFib) 合成和微纤维形成，该患者的一个 FBN1 等位基因中含有过早终止密码子 (W2756ter)。对这些细胞产生的细胞外基质的旋转阴影分析表明，仅存在极少数完整的微纤维，这些微纤维在珠间区域内显示出明显的混乱。代谢脉冲追踪研究在细胞内鉴定出一群截短的 proFib 分子，其分泌速度比源自正常等位基因的正常 proFib 更慢。与原纤维蛋白 (Fib) 相比，培养基中野生型 proFib 的含量显着减少。我们的研究结果表明：（1）截短的 proFib 被分泌并干扰微原纤维组装； (2) 该突变可能靠近 proFib C 末端的假定切割位点，该切割位点是 proFib 转化为 Fib 所必需的； (3) 截短的 proFib 由于细胞内滞留而被过度 N-糖基化，而不是由于 proFib 不完全切割且 N-糖基化位点持续存在； (4) 并非 proFib 中所有潜在的 N-糖基化位点似乎都被正常使用，因为我们可以通过布雷菲德菌素 A 介导的细胞内捕获在正常细胞中产生过度糖基化的 proFib，并随后在去除该化合物后在培养基中出现过度糖基化的 Fib。可以想象，翻译后过度修饰可能对于调节马凡综合征 FBN1 突变的表型很重要。
We studied profibrillin-1 (proFib) synthesis and microfibril formation in cultured fibroblasts from an individual with severe Marfan syndrome harboring a premature stop codon (W2756ter) in one FBN1 allele. Rotary shadowing analysis of extracellular matrix produced by these cells revealed the presence of only a very few intact microfibrils which showed marked disorganisation within the interbeaded domains. Metabolic pulse-chase studies identified intracellularly a population of truncated proFib molecules which were secreted more slowly than the normal proFib derived from the normal allele. Culture media contained strikingly reduced amounts of wild-type proFib in comparison to fibrillin (Fib). Our findings imply that (1) the truncated proFib is secreted and disturbs microfibril assembly; (2) the mutation is probably close to a putative cleavage site in the proFib C terminus necessary for the conversion of proFib to Fib; (3) the truncated proFib is over-N-glycosylated due to intracellular retention rather than incomplete cleavage of proFib with persistence of N-glycosylated sites; (4) not all potential N-glycosylation sites in proFib seem to be normally used, since we could produce over-N-glycosylated proFib in normal cells by brefeldin A mediated intracellular captivation and subsequent appearance of over-glycosylated Fib in culture medium upon removal of the compound. It is conceivable that post-translational over-modification might be important for modulating the phenotype of FBN1 mutations in Marfan syndrome.