A novel approach for the detection of proteolytically activated transglutaminase 1 in epidermis using cleavage site-directed antibodies.
It has been suggested that transglutaminase 1 is proteolytically activated upon the terminal differentiation of the keratinocyte, but the mechanisms are not well understood. We have established two mouse hybridoma cell lines producing monoclonal antibodies that specifically detect proteolytically cleaved transglutaminase 1. One detects the amino-terminus of the fragment produced by cleavage between Arginine 93 and Glycine 94, and the other detects the amino-terminus of the fragment produced by cleavage between Arginine 573 and Glycine 574. Using these two antibodies, immunohistochemical analyses of the epidermis revealed that the cleavages of the transglutaminase 1 protein occur early in the terminal differentiation of keratinocytes in the basal layer of the epidermis, that the cleavage between Arginine 573 and Glycine 574 (producing the 574G fragment) precedes the cleavage between Arginine 93 and Glycine 94 (producing the 94G fragment), that the 94G fragment is localized to the plasma membrane of keratinocytes and has cross-linking activity, whereas the 574G fragment is dispersed in the cytosol and does not have detectable levels of activity on in situ transglutaminase assay, and that 1-alpha-25-dihydroxycholecalciferol or all-trans retinoic acid treatment and ultraviolet B exposure disturb the localization of the transglutaminase 1 fragments with changes in the morphology of differentiating keratinocytes. All these results demonstrate that the antibodies generated in this work are useful to dissect the mechanism by which transglutaminase 1 is activated, and would provide us with novel insights into the biogenesis of the epidermis.
Iizuka
Ryoko
R
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan. ryoko-iiduka@yakult.co.jp
Chiba
Katsuyoshi
K
Imajoh-Ohmi
Shinobu
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology*; D000918:Antibody Specificity; D002454:Cell Differentiation; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D021621:Imaging, Three-Dimensional; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011503:Transglutaminases / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D011503:Transglutaminases; C105804:transglutaminase 1