Domains 16 and 17 of tropoelastin in elastic fibre formation.
Domains 16 and 17 of tropoelastin in elastic fibre formation.
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DOI：
10.1042/bj20061145
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发表时间：
2007-02
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通讯作者：
H. Wachi;Fumiaki Sato;Junji Nakazawa;Risa Nonaka;Z. Szabó;Z. Urban;T. Yasunaga;I. Maeda;K. Okamoto;B. Starcher;Dean Y Li;R. Mecham;Y. Seyama
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文献类型：
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作者：
H. Wachi;Fumiaki Sato;Junji Nakazawa;Risa Nonaka;Z. Szabó;Z. Urban;T. Yasunaga;I. Maeda;K. Okamoto;B. Starcher;Dean Y Li;R. Mecham;Y. Seyama
自然发生的突变可用于识别对蛋白质功能重要的结构域。我们研究了弹性蛋白基因 800-3G>C 的突变，这是 SVAS（瓣上主动脉瓣狭窄）的常见疾病等位基因。我们在来自两个不同 SVAS 家族的原代皮肤成纤维细胞中发现，这种突变会导致外显子 16-17 的跳跃，并产生稳定的 mRNA。缺乏结构域 16-17 (Delta16-17) 的原弹性蛋白可有效合成并由转染的视网膜色素上皮细胞分泌，但与正常相比，如免疫荧光染色和锁链素测定所示，在细胞外基质中的沉积不足。固相结合测定表明 Delta16-17 与 fibrillin-1 和 fibulin-5 存在正常的分子相互作用。然而，如凝聚温度升高所示，Delta16-17 的自缔合减弱。此外，负染色电子显微镜证实Delta16-17在形成原纤维聚合物方面存在缺陷。结构域16与结构域30具有高度同源性，可以形成促进纤维形成的β-折叠结构。综上所述，我们得出结论，结构域 16-17 对于原弹性蛋白的自缔合和弹性纤维的形成很重要。这项研究首次发现弹性蛋白域通过促进同型相互作用在弹性纤维形成中发挥重要作用。
Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800-3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16-17 and results in a stable mRNA. Tropoelastin lacking domains 16-17 (Delta16-17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Delta16-17 with fibrillin-1 and fibulin-5. However, self-association of Delta16-17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Delta16-17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a beta-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16-17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.