EXPRESSION, STRUCTURE AND FUNCTION OF LORICRIN, A MAJOR CELL ENVELOPE PROTEIN

角化的细胞包膜是由15纳米厚的多组分组成的一层 沉积在血浆内表面的高度不溶的蛋白质 终末分化的上皮细胞的膜。这 不溶性是由于蛋白质通过 引入的尼泊尔-(γ-谷氨酰基)赖氨酸异二肽交联物 转谷氨酰胺酶。为了确定表皮的结构 角化的细胞包膜，我们设计了用胰酶消化的方案 和蛋白水解酶K释放氨基酸的可溶性多肽 微测序。从完全角化的人身上分离出细胞膜 甲醇/氢氧化钾皂化前后的包皮表皮，或 培养角质形成细胞。我们现在已经对大约500个肽进行了测序 我们可以得出这样的结论：氯氰酸构成了细胞的大部分 被膜，高达70%，主要定位于外层(细胞质 表面)2/3的结构；SPR1和SPR2蛋白混合在一起 在氯化蛋白阶段，并作为交叉桥蛋白 在氯氰菊酯中；新的蛋白质elafin也与 在细胞被膜结构的内部， 与其组装的最早阶段相对应，总蛋白和 胱抑素a是相互交联的丰富成分， 氯化物相；角蛋白中间细丝链也是交联的 组件，但在本例中，它们间接地交叉链接到 桥粒的桥粒蛋白，由中间丝介导 相关蛋白。全长的人类氯氯蛋白已经在 细菌，并提纯到同质。三种表皮表达的基因 转谷氨酰胺酶在体外以不同的方式将其交联。基于 数据表明，谷氨酰胺转氨酶3和 在较小程度上，转谷氨酰胺酶1和转谷氨酰胺酶1都需要 活体内的氯氰菊酯。我们表达了小鼠和人的SPR1、SPR2和SPR3 细菌中的蛋白质用于随后的生化、体外交联 和结构分析。为了定义其在 表皮，我们已经构建了制备Clicrin基因缺失小鼠的结构。

The cornified cell envelope is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of terminally differentiating epithelial cells. This insolubility is due to crosslinking of the proteins by Nepsilon-(gamma-glutamyl) lysine isodipeptide crosslinks introduced by transglutaminases. In order to determine the structure of the epidermal cornified cell envelope, we have devised digestion protocols with trypsin and proteinase K to release soluble peptides for amino acid microsequencing. Cell envelopes were isolated from fully cornified human foreskin epidermis before and after saponification with methanol/KOH, or cultured keratinocytes. We have now sequenced about 500 peptides from which we can conclude that: loricrin constitutes the bulk of the cell envelope, up to 70%, and is localized primarily on the outer (cytoplasmic surface) two-thirds of the structure; SPR1 and SPR2 proteins are admixed throughout the loricrin phase, and serve as crossbridging proteins amongst the loricrin; the novel protein elafin is also admixed with the loricrin; in the inner portions of the cell envelope structure, corresponding to the earliest stages of its assembly, involucrin and cystatin a are abundant components crosslinked to each other and the loricrin phase; keratin intermediate filament chains are also crosslinked components, but in this case, they are crosslinked indirectly to desmoplakin of the desmosomes, mediated by an intermediate filament associated protein. Full-length human loricrin has been expressed in bacteria and purified to homogeneity. Three epidermally-expressed transglutaminases crosslink it differently in vitro. Based on the patterns of crosslinking, the data suggest that transglutaminase 3, and to a lesser extent transglutaminase 1, are both required to crosslink the loricrin in vivo. We have expressed mouse and human SPR1, SPR2 and SPR3 proteins in bacteria for subsequent biochemical, in vitro crosslinking and structural analyses. In order to define its precise function in the epidermis, we have made constructs to prepare loricrin null mice.