Isolation of two immunologically related transglutaminase substrates from cultured human keratinocytes.
Transglutaminase substrates A and B were identified in soluble extracts of cultured keratinocytes and human epidermis by their reactions with dansyl cadaverine in the presence of Ca++ ion. Substrate B was present in substantial amounts in both extracts whereas A, easily seen in cell extracts, was decreased and sometimes not detected in tissue extracts. Substrates A and B from cultured cells were separated by Sephadex G-75 chromatography and isolated by preparative sodium dodecyl sulfate (SDS) gel electrophoresis by which A had a mol wt of 125,000 and B had a mol wt of 12,000. Amino acid analysis of A, B, and cornified envelope were similar but not identical. The isopeptide bond is not a significant structural feature of A inasmuch as its content is less than 0.25 bonds/molecule. Antibodies raised to A cross-reacted with B and vice versa and A showed partial identity to B when reacted with anti-B. Anti-A reacted with epidermis being adsorbed by the edges of cornifying cells but only weakly by cells of the Malpighian layer. Anti-B also reacted with cornifying, but its reaction was more intense with the cytoplasm of Malpighian cells. Substrate A appears to be incorporated into cornified envelope immediately after its appearance in cells of the granular layer and seems similar to a protein isolated by a different method. Substrate B, convertible by transglutaminase to higher molecular weight species, may also participate in cornified envelope assembly and shares some structural similarities to A.
D000217:Acyltransferases / Q000378:metabolism*; D002478:Cells, Cultured; D003429:Cross Reactions; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D008970:Molecular Weight; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification*; D012867:Skin / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases
D011506:Proteins; D007633:Keratins; D000217:Acyltransferases; D011503:Transglutaminases