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

参与皮肤表皮蛋白交联的钙离子结合蛋白的研究

• 批准号: 14560063
• 负责人: HITOMI Kiyotaka
• 金额: $ 2.18万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560063/
• 关键词: epidermis; cross-linking; calcium; transglutaminase; Physarum; プロテアーゼ

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.

皮肤表皮的屏障功能不可避免地要形成由转氨酶交联的结构蛋白组成的皮质包膜。皮肤中的转氨酶主要受钙离子浓度和蛋白水解激活机制的调节，本研究利用单克隆抗体研究了皮肤特异性转氨酶（TGase 3）的分布。发现TGase 3根据角质形成细胞分化特异性表达。在分化的角质形成细胞中，TGase 3似乎定位于细胞质中。此外，使用重组蛋白和单克隆抗体，分析了通过CLSP（钙调蛋白结合皮肤蛋白）调节TGase 3，CLSP是与TGase 3相互作用的钙结合蛋白。同时，利用模式生物（黏菌;多头绒泡菌），分析了TGase在表皮创伤愈合中的作用。我们克隆的绒泡菌TGase（PpTGase）在结构上与哺乳动物TGase非常相似。表达并纯化了具有生物活性的重组PpTGase。对可能的底物钙结合蛋白（CBP 40）的交联反应进行了表征。在体外反应中，观察到CBP 40的二聚体和寡聚体分子，并且这些分子在伤口黏菌中也被特异性地证实，表明其在伤口愈合中的作用。

项目成果

Hitomi K., Wada F.: "Molecular cloning and characterization of transglutaminase in slime mold"Minerva Biotechnologica (Journal of Biotechnology and Molecular biology). 14. 196-197 (2002)

Hitomi K.、Wada F.：“粘菌中转谷氨酰胺酶的分子克隆和表征”Minerva Biotechnologica（生物技术和分子生物学杂志）。

Hitomi K., et al.: "Analysis of epidermal-type transglutaminase in human stratified epithelia and cultured keratinocytes using monospecific antibodies."Journal of Dermatological Science. 32. 95-103 (2003)

Hitomi K. 等人：“使用单特异性抗体分析人复层上皮和培养的角质形成细胞中的表皮型转谷氨酰胺酶。”皮肤病学杂志。

Hitomi K.et al.: "Analysis of epidermal-type transglutaminase in human stratified epithelia and cultured keratinocytes using monospecific antibodies"Journal of Dermatological Science. 32. 95-103 (2003)

Hitomi K.等人：“使用单特异性抗体分析人复层上皮和培养的角质形成细胞中的表皮型转谷氨酰胺酶”皮肤病学杂志。

Wada F et al.: "Identification of mammalian-type transglutaminase in Physarum polycephalum : Evidences from cDNA sequence and involvement of GTP in the transamidating activity"Europeam. Journal of Biochemistry. 269. 3451-3460 (2002)

Wada F 等人：“多头绒泡菌中哺乳动物型转谷氨酰胺酶的鉴定：来自 cDNA 序列的证据以及 GTP 参与转酰胺基活性”欧洲。

Hitomi K.: "Molecular Evolution of Transglutaminase : from bacteria to animal"Developments in Biophysics and Biochemistry (Research Signpost). 3. 223-234 (2003)

Hitomi K.：“转谷氨酰胺酶的分子进化：从细菌到动物”生物物理学和生物化学的发展（研究路标）。