Analysis of epidermal-type transglutaminase (transglutaminase 3) in human stratified epithelia and cultured keratinocytes using monoclonal antibodies.
BACKGROUND
Epidermal-type transglutaminase (TGase 3) is involved in the cross-linking of structural proteins in the epidermis, which results in the formation of the cornified envelope. TGase 3 is activated by limited proteolysis of a 77 kDa zymogen during keratinocyte differentiation.


OBJECTIVE
To characterize the expression of TGase 3 in human epidermis and cultured keratinocytes, we established specific monoclonal antibodies against the TGase 3.


METHODS
Recombinant proteins for human TGase 3 produced in bacteria and baculovirus-infected insect cells were purified as an antigen. Hybridomas are established and used for characterization of expression in epidermis and keratinocytes.


RESULTS
Four antibodies were generated against recombinant human TGase 3, which reacted with the 77 kDa zymogen and in some cases either the 47 or 30 kDa active proteolytic fragments. In human epidermis and cultured keratinocytes, only the zymogen form of TGase 3 was detected. Immunohistochemical analysis of the skin revealed that the enzyme is present in the cells of the granular and cornified layers consistent with its role in cornified envelope formation. In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions.


CONCLUSIONS
Using monoclonal antibody against human TGase 3, we showed the expression of TGase 3 in upper layers of epidermis. TGase 3 displayed a diffuse cytoplasmic distribution in vitro consistent with its proposed role in the early phase of cornified cell envelope assembly in the cytoplasm.
Hitomi
Kiyotaka
K
Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, 464-8601, Nagoya, Japan. hitomi@agr.nagoya-u.ac.jp
Presland
Richard B
RB
Nakayama
Tomomi
T
Fleckman
Philip
P
Dale
Beverly A
BA
Maki
Masatoshi
M
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology*; D004847:Epithelial Cells / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D012867:Skin / Q000201:enzymology; D014018:Tissue Distribution; D011503:Transglutaminases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; C498712:TGM3 protein, human; D011503:Transglutaminases