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

角化包膜的形成是皮肤表皮屏障功能的必由之路，角化包膜由转谷氨酰胺酶交联的结构蛋白组成。皮肤中的转谷氨酰胺酶主要受钙离子浓度和蛋白水解酶激活机制的调节，本研究利用单抗研究了皮肤特异性转谷氨酰胺酶(TGase-3)的分布。TGase 3的表达依赖于角质形成细胞的分化。在分化的角质形成细胞中，TGase 3似乎定位于细胞质。此外，利用重组蛋白和单抗分析了与TGase 3相互作用的钙结合蛋白CLSP对TGase 3的调节作用。同时，利用模式生物(粘菌、多头绒泡菌)，分析了TGase在表皮创面愈合中的作用。我们克隆的绒毛霉菌TGase(PpTGase)在结构上与哺乳动物的TGase非常相似。表达并纯化了具有生物活性的重组PpTGase。对可能底物钙结合蛋白(CBP40)的交联反应进行了表征。在体外反应中，观察到了CBP40的二聚体和寡聚体分子，并在伤口黏液霉菌中特异性地证实了这些分子，提示其在伤口愈合中起到了作用。

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.