An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh
An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh
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DOI：
10.1074/jbc.m705731200
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发表时间：
2007-12-28
影响因子：
4.8
通讯作者：
Kawabata, Shun-ichiro
中科院分区：
文献类型：
--
作者：
Matsuda, Yasuyuki;Koshiba, Takumi;Kawabata, Shun-ichiro
在哺乳动物中，皮质细胞被膜在上皮细胞的质膜下形成，并提供由转氨酶（TGase）交联的不溶性蛋白质组成的重要物理屏障。在中国鲎中，TGase储存在血细胞中，并响应细菌脂多糖的模拟而分泌。在这里，我们的特点是TGase底物命名为caraxin，被确定在马蹄蟹角质层。其中一个同源物caraxin-1具有独特的结构域结构，由N-和C-末端七肽重复序列和具有五肽串联重复结构的中心结构域组成。Western blotting结果显示caraxin-1在表皮下特异性定位。此外，我们确定了五肽基序是几丁质结合单位。超离心分析表明，caraxin-1以310-350 kDa的寡聚体形式存在，基于单体的分子量，其类似于20-mer。低聚物通过TGase交联以形成具有蜂窝结构的精细网格，电子显微镜下发现其不同于由脂多糖诱导的血细胞胞吐触发的凝血网格。我们确定了几个交联网站的N-和C-末端结构域的caraxin-1。在caraxin-1中的位置36和118处将Leu替换为Pro减少了α-螺旋含量，这破坏了TGase依赖的网格，从而表明N-和C-末端结构域对于正确的网格形成的重要性。在节肢动物中，TGase依赖性蛋白质交联可能参与在表皮下表皮的宿主防御的初始阶段，如在哺乳动物皮肤的情况下。
In mammals, the cornified cell envelope forms beneath the plasma membrane in epithelia and provides a vital physical barrier consisting of insoluble proteins cross-linked by transglutaminase (TGase). In the horseshoe crab Tachypleus tridentatus, TGase is stored in hemocytes and secreted in response to the simulation of bacterial lipopolysaccharides. Here we characterized a TGase substrate designated as caraxin that was identified in horseshoe crab cuticle. One of the homologs, caraxin-1, possessed a unique domain structure consisting of N- and C-terminal heptad repeats and a central domain with a tandem-repeated structure of a pentapeptide. Western blotting showed the specific localization of caraxin-1 in sub-cuticular epidermis. Moreover, we identified the pentapeptide motif to be a chitin-binding unit. Analytical ultracentrifugation revealed that caraxin-1 exists as an oligomer with 310-350 kDa, which is similar to 20-mer based on the molecular mass of the monomer. The oligomers were cross-linked by TGase to form an elaborate mesh with honeycomb structures, which was electron-microscopically found to be different from the clotting mesh triggered by lipopolysaccharide-induced hemocyte exocytosis. We determined several cross-linking sites in the N- and C-terminal domains of caraxin-1. The replacements of Leu to Pro at positions 36 and 118 in caraxin-1 reduced the alpha-helix content, which destroyed the TGase-dependent mesh, thus indicating the importance of the N- and C-terminal domains for the proper mesh formation. In arthropods, TGase-dependent protein cross-linking may be involved in the initial stage of host defense at the sub-cuticular epidermis, as in the case of mammalian skin.