Steinert P M, Marekov L N
Skin Biology Branch, NIAMSD, National Institutes of Health, Bethesda, Maryland 20892-2755, USA.
J Biol Chem. 1995 Jul 28;270(30):17702-11. doi: 10.1074/jbc.270.30.17702.
The cornified cell envelope (CE) is a 15-nm thick layer of insoluble protein deposited on the intracellular side of the cell membrane of terminally differentiated stratified squamous epithelia. The CE is thought to consist of a complex amalgam of proteins cross-linked by isodipeptide bonds formed by the action of transglutaminases, but little is known about how or in which order the several putative proteins are cross-linked together. In this paper, CEs purified from human foreskin epidermis were digested in two steps by proteinase K, which released as soluble peptides about 30% and then another 35% of CE protein mass, corresponding to approximately the outer third (cytoplasmic surface) and middle third, respectively. Following fractionation, 145 unique peptides containing two or more sequences cross-linked by isodipeptide bond(s) were sequenced. Based on these data, most (94% molar mass) of the outer third of CE structure consists of intra- and interchain cross-linked loricrin, admixed with SPR1 and SPR2 proteins as bridging cross-links between loricrin. Likewise, the middle third of CE structure consists largely of cross-linked loricrin and SPR proteins, but is mixed with the novel protein elafin which also forms cross-bridges between loricrin. In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. In addition, the data support a model for the intermediate to final stages of CE assembly: the proteins elafin, SPR1 and SPR2, and loricrin begin to be deposited on a preformed scaffold; later, elafin deposition decreases as loricrin and SPR accumulation continues to effect final assembly. The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events.
角质化细胞包膜（CE）是一层15纳米厚的不溶性蛋白质层，沉积在终末分化的复层鳞状上皮细胞膜的胞内侧。CE被认为是由转谷氨酰胺酶作用形成的异二肽键交联的蛋白质复合物，但对于几种假定的蛋白质如何交联在一起或交联的顺序知之甚少。在本文中，从人包皮表皮纯化的CE分两步用蛋白酶K消化，分别释放出约30%和另外35%的CE蛋白质量作为可溶性肽，分别对应于大约外侧三分之一（细胞质表面）和中间三分之一。分级分离后，对145个含有两个或更多通过异二肽键交联的序列的独特肽段进行了测序。基于这些数据，CE结构外侧三分之一的大部分（94%摩尔质量）由链内和链间交联的兜甲蛋白组成，与SPR1和SPR2蛋白混合，作为兜甲蛋白之间的桥接交联。同样，CE结构的中间三分之一主要由交联的兜甲蛋白和SPR蛋白组成，但与新蛋白弹性蛋白酶混合，弹性蛋白酶也在兜甲蛋白之间形成交联桥。此外，在两个水平上都发现了涉及兜甲蛋白与角蛋白1、2e、10或丝聚蛋白的交联。这些数据首次证实了这几种蛋白质确实是CE结构的交联蛋白成分。此外，这些数据支持了一个关于CE组装中间到最终阶段的模型：弹性蛋白酶、SPR1和SPR2以及兜甲蛋白开始沉积在一个预先形成的支架上；后来，随着兜甲蛋白和SPR积累继续影响最终组装，弹性蛋白酶沉积减少。涉及角蛋白的交联的发现进一步表明，在这些事件中，下方的细胞质角蛋白中间丝-丝聚蛋白网络锚定在正在形成的CE上。
