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

锥形细胞包膜是一层多组分，厚度为15nm

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.