During terminal differentiation epidermal keratinocytes manifest a programmed set of morphological and biochemical changes that result in the production of two major structures: 1) an envelope of covalently linked protein enclosing 2) a constellation of keratin intermediate filaments. A major precursor of the envelope is a soluble protein called involucrin (hINV) which is incorporated into the envelope by a calcium- dependent transglutaminase. hINV is likely to account for the majority of glutamyl-lysine linkages that hold the envelope together. In spite of its importance, inadequate information is available regarding which glutamines within involucrin are targeted for crosslinking by transglutaminase or which sections of the hINV molecule are essential for high strength envelope formation. Active envelope formation is essential for survival and abnormal envelope formation is a feature of several epidermal diseases. The ultimate aim of the experiments described in this proposal is to understand the role of hINV in the envelope assembly process and how this impacts on the disease state. To provide tools for these studies, we cloned and sequenced the complete hINV gene, structurally characterized the protein and produced the normal and mutant hINV proteins in bacteria. Our results show that the molecule is an extended alpha-helix composed of highly similar, tandemly linked repeats of ten amino acids. Each repeat contains three glutamine residues, each of which is a potential crosslink site. The proposed studies are designed to gain a better understanding of cornified envelope structure and the role of hINV in envelope formation and are a logical extension of the studies completed during the initial two years of grant support. In the present experiments we propose to 1) identify which proteins become crosslinked to hINV during cornified envelope formation, 2) construct a series of hINV mutants to determine why GLN496, among the more than 100 glutamine residues present in the hINV protein, is the preferred site for initial crosslink formation and 3) determine the effects of expression of selected mutant hINV proteins on epidermal function in transgenic mice.