The epidermis performs two important functions--to provide strong physical protection and to serve as a permeability barrier. To perform the first function the epidermis synthesizes large amounts of keratin proteins which form the predominant (>90%) component in cornified cells. These keratins belong to a multigene family. They can be divided into an acidic (also known as Type I) and a basic (Type II) subfamily, and it has been shown that any acidic keratin can combine with any basic keratin to form 10 nm filaments. To gain insights into the nature of the forces responsible for filament formation, we will mutate systemically several selected amino acids in K18 keratin by site-directed mutagenesis, and test the mutant keratins' ability to polymerize with the normal partner, keratin K8. The test will be performed by transfecting the normal or mutant K18 cDNA into L cells artificially constructed to express K8 keratin, followed by immunofluorescent staining and EM analysis. It is expected that some of the mutant keratin K18 will produce abnormal keratin filaments with altered diameter, degree of lateral aggregation, or even being arrested at different stages of filament assembly. These results will shed light on the subunit interaction and dynamic aspects of keratin filament formation. To perform the other major function, i.e., as a permeability barrier, the epidermis elaborates in the granular layer numerous lamellar granules (200 nm) which discharge their contents into the intercellular space. Since these granules are difficult to purify, relatively little is known about their detailed biochemical composition. Using a panel of monoclonal antibodies, we have identified a 25K protein in lamellar granules. Experiments are designed to characterize this novel epidermal differentiation marker both on a protein and a cDNA/genomic DNA level. Results from these studies may yield new insights into an important regulatory step during terminal stages of keratinization, as well as on the formation of epidermal permeability barrier.
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