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 46 kDa polypeptide called involucrin which is incorporated into the envelope by a calcium-dependent transglutaminase. Involucrin is likely to account for the majority of glutamyl-lysine linkages that hold the envelope together. In spite of its importance, no information is available regarding which glutamines within involucrin are targeted for crosslinking by transglutaminase or which sections of the involucrin 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 goal of the experiments described in this proposal is to understand the role of involucrin in the envelope assembly process and how this impacts on the disease state. To provide a tool for these studies, we cloned and sequenced the complete human involucrin gene. Our sequence revealed 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. In the present experiments we propose to utilize a unique approach to study envelope formation and express the cloned involucrin gene in a variety of cell types to gain new knowledge regarding 1) which glutamine residues within the involucrin peptide are the substrates for the calcium-dependent transglutaminase, 2) whether involucrin can participate in envelope formation in rat keratinocytes and 3) to determine the role of involucrin in stabilizing the cornified envelope.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM043751-01A1
Application #
3302773
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1991-01-01
Project End
1994-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Lambert, A; Ekambaram, M; Robinson, N et al. (2000) Transglutaminase reactivity of human involucrin. Skin Pharmacol Appl Skin Physiol 13:17-30
Agarwal, C; Efimova, T; Welter, J F et al. (1999) CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester. J Biol Chem 274:6190-4
LaCelle, P T; Lambert, A; Ekambaram, M C et al. (1998) In vitro cross-linking of recombinant human involucrin. Skin Pharmacol Appl Skin Physiol 11:214-26
Robinson, N A; Eckert, R L (1998) Identification of transglutaminase-reactive residues in S100A11. J Biol Chem 273:2721-8
Eckert, R L; Crish, J F; Robinson, N A (1997) The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation. Physiol Rev 77:397-424
Eckert, R L; Crish, J F; Banks, E B et al. (1997) The epidermis: genes on - genes off. J Invest Dermatol 109:501-9
Robinson, N A; Lapic, S; Welter, J F et al. (1997) S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes. J Biol Chem 272:12035-46
Robinson, N A; LaCelle, P T; Eckert, R L (1996) Involucrin is a covalently crosslinked constituent of highly purified epidermal corneocytes: evidence for a common pattern of involucrin crosslinking in vivo and in vitro. J Invest Dermatol 107:101-7
Eckert, R L; Welter, J F (1996) Transcription factor regulation of epidermal keratinocyte gene expression. Mol Biol Rep 23:59-70
Yaffe, M B; Murthy, S; Eckert, R L (1993) Evidence that involucrin is a covalently linked constituent of highly purified cultured keratinocyte cornified envelopes. J Invest Dermatol 100:3-9

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