Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine.
A method providing more sensitive detection of transglutaminase substrates was developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when [14C]putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r2 = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.
Kvedar
J C
JC
Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston 02129.
Pion
I A
IA
Bilodeau
E B
EB
Baden
H P
HP
Greco
M A
MA
D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D015153:Blotting, Western; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000276:immunology; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology; D000939:Epitopes; D006801:Humans; D007231:Infant, Newborn; D007425:Intracellular Membranes / Q000737:chemistry; D015603:Keratinocytes / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009924:Organ Culture Techniques; D011498:Protein Precursors / Q000032:analysis* / Q000276:immunology; D011700:Putrescine; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011498:Protein Precursors; C032253:involucrin; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine; D011700:Putrescine