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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043751-07
Application #
2022365
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1991-01-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Physiology
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

Showing the most recent 10 out of 14 publications