EXPRESSION, STRUCTURE AND FUNCTION OF THE CORNIFIED CELL ENVELOPE

复层鳞状上皮屏障功能的主要组成部分 是皮质细胞的包膜。 这是一个15纳米厚的多组分 一层高度不溶的蛋白质沉积在 细胞的质膜。 在表皮的情况下，5纳米厚的 一层神经酰胺脂质（脂质包膜）附着在外部 面 蛋白质包膜的不溶性在很大程度上是由于 通过转氨酶交联组成蛋白质。 我们已经详细研究了其中的两种蛋白质，兜甲蛋白和小 脯氨酸丰富（SPR）家族。 我们已经表达了人兜甲蛋白， 细菌，并用它来表征其结构，生化 性质和体外表皮转氨酶交联。 类似地，我们在细菌中表达了几种SPR蛋白， 表征了它们的生物化学和体外交联特性。 SPR蛋白质的溶液核磁共振结构研究正在进行中。 初步数据表明，中心肽重复结构域采用新的 3-环样蛋白折叠。 我们已经描述了微分 SPR 1和SPR 2蛋白在小鼠表皮中的表达特性 和上皮组织。我们的数据表明SPR蛋白似乎 在细胞被膜结构中起交联蛋白的作用。 我们 用可控的蛋白水解来分离皮质细胞 在体包皮表皮和表皮中形成的包膜 体外培养角质形成细胞。 大部分的蛋白质包膜 由兜甲菌素（70-80%）（与少量（2-20%）的 SPR。 在甲醇中通过碱性水解除去共价键合 包皮细胞包膜的神经酰胺脂质，我们已经能够探索 这个结构的最里面的方面，对应于最初的 其组装阶段。 我们的数据表明，细胞被膜组装是 起始于角蛋白丝和许多相关的 蛋白质与桥粒相遇。 我们发现脂质的神经酰胺 包膜由不同分子异质群体组成， 大小，并共价连接到外皮蛋白，以及可能的其他 蛋白质，在蛋白质包膜上。

A major component of barrier function in stratified squamous epithelia is the cornified cell envelope. This is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid enevlope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the crosslinking of the constituent proteins by transglutaminases. We have studied two of these proteins in detail, loricrin and the small proline rich (SPR) families. We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and crosslinking by epidermal transglutaminases in vitro. Similarly, we have expressed in bacteria several SPR proteins and characterized their biochemical and crosslinking properties in vitro. Solution nmr structural studies on SPR proteins are in progress. Preliminary data suggest the central peptide repeat domains adopt novel 3-loop- like protein folds. We have characterized the differential expression properties of the SPR1 and SPR2 proteins in mouse epidermis and epithelial tissues. Our data suggest the SPR proteins seem to function as crossbridging proteins in the cell envelope structure. We have used controlled proteolysis to dissect apart the cornified cell envelope formed in foreskin epidermis in vivo and in epidermal keratinocyte cultures in vitro. The bulk of the protein envelope consists of loricrin (70-80%( admixed with smaller amounts (2-20%) of SPRs. By alkaline hydrolysis in methanol to remove covalently bound ceramide lipids of foreskin cell envelopes, we have been able to explore the innermost aspects of this structure, corresponding to the initial stages of its assembly. Our data suggest that cell envelope assembly is initiated at the site where keratin filaments and many associated proteins meet desmosomes. We have found that the ceramides of the lipid envelope consist of a heterogeneous population of molecules of varying size, and are covalently attached to involucrin, as well as perhaps other proteins, on the protein envelope.