Studies on Calcium-ion Binding Proteins Involved in Protein Cross-linking in Skin Epidermis

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Formation of cornified envelop, which is composed of cross-linked structural proteins by transglutaminase, is inevitable for the barrier function of skin epidermis. Transglutaminase in the skin is mainly regulated by the concentration of calcium ion and proteolytic activation mechanisms.In this study, distribution of skin-specific transglutaminase (TGase 3) was investigated using monoclonal antibody. TGase 3 was found to be specifically expressed depending on the keratinocyte differentiation. In the differentiated keratinocytes, TGase 3 appeared to be localized in the cytoplasm. Additionally, using recombinant protein and monoclonal antibody, regulation of TGase 3 by CLSP (calmodulin binding skin protein), which is calcium-binding protein interacting with TGase 3, was analyzed. No regulatory effect on enzymatic activity was observed.On the while, using model organisms (slime mold ; Physarum polycephalum), role of TGase in wound healing in the epidermis was analyzed. TGase in Physarum (PpTGase), which we have cloned, is very similar to mammalian TGase in the structure. Recombinant biologically active PpTGase was expressed and purified. Cross-linking reaction of possible substrate calcium-binding protein (CBP40) was characterized. In in vitro reaction, dimer and oligomer molecules of CBP40 were observed and those molecules were also confirmed specifically in the wound slime mold, suggesting its role in the wound healing.