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

皮质细胞被膜是一层多组分的15 nm厚的 沉积在血浆内表面的高度不溶性蛋白质 终末分化上皮细胞的膜。 这 不溶性是由于蛋白质的交联 通过引入的Nepaly-（γ-谷氨酰）赖氨酸异二肽交联 转氨酶 为了确定表皮的结构 Corpus细胞包膜，我们已经设计了消化方案与胰蛋白酶 和蛋白酶K释放可溶性氨基酸肽 微测序 细胞被膜分离自完全Corpus人 用甲醇/KOH皂化前后的包皮表皮，或 培养的角质形成细胞。 我们现在已经测序了来自 我们可以得出结论：兜甲蛋白构成了细胞的主体 包膜，高达70%，主要位于外部（细胞质 表面）结构的三分之二; SPR 1和SPR 2蛋白质混合 贯穿兜甲蛋白相，并作为跨桥蛋白 在兜甲蛋白中，新的蛋白质弹性蛋白也与兜甲蛋白混合。 兜甲蛋白;在细胞被膜结构的内部， 对应于其组装的最早阶段，外皮蛋白和 半胱氨酸蛋白酶抑制剂A是彼此交联的丰富组分， 甲蛋白相;角蛋白中间丝链也交联 组分，但在这种情况下，它们间接交联， 桥粒的桥斑蛋白，由中间丝介导 相关蛋白质 全长人兜甲蛋白已在大肠杆菌中表达。 细菌并纯化至均质。 三种表皮表达 转氨酶在体外以不同的方式交联它。 基于 交联模式，数据表明，转氨酶3，和 在较小程度上，转氨酶1都需要交联 兜甲蛋白在体内。 我们表达了小鼠和人的SPR 1、SPR 2和SPR 3 用于随后的生物化学、体外交联的细菌中的蛋白质 结构分析。 为了明确其在 表皮，我们已经制备了构建体以制备兜甲蛋白缺失小鼠。

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.