Direct evidence that involucrin is a major early isopeptide cross-linked component of the keratinocyte cornified cell envelope.
Involucrin was the first protein to be identified as a likely constituent of the insoluble cornified cell envelope (CE) of stratified squamous epithelia. However, to date, direct isolation from CEs of involucrin cross-linked by way of the transglutaminase-induced isopeptide bond has not been reported. We have treated human foreskin CEs with methanol/KOH (saponification) to hydrolyze off much of the lipids. By immunogold electron microscopy, this exposed large amounts of involucrin epitopes as well as of desmoplakin, a desmosomal structural protein. About 20% of the total CE protein could be solubilized by proteolytic digestion after saponification, of which involucrin was the most abundant. Subsequent amino acid sequencing revealed many peptides involving involucrin cross-linked either to itself or to a variety of other known CE protein components, including cystatin alpha, desmoplakin, elafin, keratins, members of the small proline-rich superfamily, loricrin, and unknown proteins related to the desmoplakin family. Specific glutamines or lysines of involucrin were used to cross-link the different proteins, such as glutamines 495 and 496 to desmoplakin, glutamine 288 to keratins, and lysines 468, 485, and 508 and glutamines 465 and 489 for interchain involucrin cross-links. Many identical peptides were obtained from immature CEs isolated from the inner living cell layers of foreskin epidermis. The multiple cross-linked partners of involucrin provide experimental confirmation that involucrin is an important early scaffold protein in the CE. Further, these data suggest that there is significant redundancy in the structural organization of the CE.
Steinert
P M
PM
Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752, USA. pemast@helix.nih.gov
Marekov
L N
LN
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000276:immunology; D051180:Desmoplakins; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011498:Protein Precursors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D003598:Cytoskeletal Proteins; C495294:DSP protein, human; D051180:Desmoplakins; D000939:Epitopes; D011498:Protein Precursors; C032253:involucrin; D014357:Trypsin