Abstract
We have isolated and characterized a full-length cDNA clone encoding human loricrin. Curiously, this protein displays major differences from the recently described mouse loricrin (Mehrel, T., Hohl, D., Nakazawa, H., Rothnagel, J.A., Longley, M.A., Bundman, D., Cheng, C.K., Lichti, U., Bisher, M.E., Steven, A. C., Steinert, P.M., Yuspa, S.H., and Roop, D.R. (1990) Cell 61, 1103-1112). Although both proteins are glycine-serine-cysteine-rich, the sequences have not been conserved. However, analysis of the sequences reveals a common motif of quasi-peptide repeats of an aliphatic or aromatic amino acid residue followed by several glycine and/or serine and cysteine residues. These sequences are interspersed and flanked by short glutamine- or glutamine/lysine-rich peptides. Thus loricrins consist of a family of cell envelope proteins of highly variable sequences that nevertheless retain common structural elements. We show that unlike all other putative protein components of the cell envelope, loricrins are highly insoluble, due at least in part to cross-linking by disulfide bonds. Furthermore, we have isolated four peptides from purified human cell envelopes that contain recognizable loricrin sequences and which are cross-linked by the N epsilon-(gamma-glutamyl)lysine isodipeptide bond. The presence of such bonds thus affords an explanation for the extraordinary insolubility of loricrin by cross-linking to the cell envelope and can also explain the low steady-state levels of monomeric loricrin in cytoskeletal extracts of epidermis. This study represents the first report of this isodipeptide cross-link in a protein component of the cornified cell envelope. We propose a model for the structure of loricrin in which (i) the unusual glycine-serine-rich sequences adopt a flexible loop conformation, indexed on the recurrent aliphatic residues; (ii) inter- or intramolecular isodipeptide and disulfide cross-links induce or stabilize folding of loricrin so as to form a more compact rosette-like structure; and (iii) the presence of the flexible glycine-rich loops necessarily will impact a flexible character to the cell envelope and entire epithelium.
Highlights
From the $Dermatology Branch and the §LaboratoorfyCellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutesof Health, Bethesda, Maryland20892
At about the same putative proteincomponents of the cell envelope,lori- time, a setof proteins is deposited on the inside of the plasma crins arehighly insoluble, dueat least in part tocross- membrane, which collectively constitute a structure of 10-20 linking by disulfide bonds
This study represents the first report of this isodipeptide crosslink ina protein component of the cornified cell envelope
Summary
Daniel Hohl$$ll, Thomas Mehrelg 88, Ulrike Lichtig, Maria L. Comparisons of the mouse and human loricrins reveal similarities and differences that provide important new inmin at 95 "C.CEs were collected by centrifugation at 5000 X g for 15 min and re-extracted once the same way In our experience, such preparations of swollen CEs still contain major amounts of keratin proteins, that were removed as follows. 8 and pL30.8-2were used to hybrid select poly(A)-enriched RNA labeled spots were eluted and a portion analyzed for amino acid from human foreskin epidermis (26), followed by translation in uitro, composition data. Those containing both lysine and glutamate, and to confirm that theselected RNA encodes a protein similarto thein t.huspossible candidates for cross-linked peptides, were characterized vivoproduct (22). Riboprobes (24) modified by using silane-treated slides (28) and incubation in humid chambers during hybridization and ribonuclease treatment (29)
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