Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes.
The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin (from the old english "sciell" for shell).
Kvedar
J C
JC
Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston 02129.
Manabe
M
M
Phillips
S B
SB
Ross
B S
BS
Baden
H P
HP
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002352:Carrier Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004848:Epithelium; D000939:Epitopes / Q000032:analysis; D006197:Hair / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009262:Nails / Q000166:cytology; D011506:Proteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D011506:Proteins; C111281:SCEL protein, human