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. The insolubility is due in large part to the crosslinking of the proteins by transglutaminases. We have studied two of the 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. Structural studies are in progress. We have characterized the differential expression properties of the SPR1 and SPR2 proteins in mouse epidermis and epithelial tissues. We have used controlled proteolysis to dissect apart the epidermal cornified cell envelope formed in foreskin epidermis in vivo and in keratinocyte cultures in vitro. By alkaline hydrolysis in methanol to remove covalently bound ceramide lipids of the former, we have been able to explore the innermost aspects of this structure, corresponding to the initial stages of its assembly. Our data suggest that assembly is initiated at the site where keratin filaments and many proteins meet desmosomes.
