Matsunaga Nozomu, Shimizu Haruka, Fujimoto Kanako, Watanabe Kanako, Yamasaki Tsutomu, Hatano Naoya, Tamai Eiji, Katayama Seiichi, Hitsumoto Yasuo
Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama-shi, Okayama 700-0005, Japan.
Pharmaceutical Department, Shujitsu University, 1-6-1 Nishigawara, Naka-ku, Okayaka-shi, Okayama 703-8516, Japan.
Anaerobe. 2018 Jun;51:124-130. doi: 10.1016/j.anaerobe.2018.05.001. Epub 2018 May 9.
During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.
在鉴定产气荚膜梭菌细胞表面纤连蛋白（Fn）结合蛋白（Fbps）的研究过程中，我们确定甘油醛-3-磷酸脱氢酶（GAPDH）为候选Fbp。GAPDH是一种糖酵解酶，存在于多种原核生物和真核生物中。使用配体印迹分析和酶联免疫吸附测定（ELISA）研究了重组产气荚膜梭菌GAPDH（rGAPDH）的Fn结合活性。rGAPDH与纤溶酶原强烈结合，但不与层粘连蛋白或明胶结合。尽管GAPDH没有信号序列，但它在许多微生物的细胞表面表达。使用ELISA和流式细胞术分析产气荚膜梭菌细胞表面GAPDH的存在情况；纯化的rGAPDH与产气荚膜梭菌细胞表面结合。据报道，自溶素参与GAPDH与细胞表面的结合，我们通过ELISA和配体印迹测定评估了rGAPDH与产气荚膜梭菌自溶素Acp之间的相互作用。这些测定表明，rGAPDH与Acp的催化结构域结合，但不与细胞壁结合结构域结合。这些结果表明，自溶素介导GAPDH在产气荚膜梭菌细胞表面的表达，并表明GAPDH在结合Fn和纤溶酶原方面可能具有兼职功能。
