Kim Min Kyu, Kim Won-Tae, Lee Hyun Min, Choi Hong Seo, Jo Yu Ra, Lee Yangsoon, Jeong Jaemin, Choi Dongho, Chang Hee Jin, Kim Dae Shick, Jang Young-Joo, Ryu Chun Jeih
Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
Department of Laboratory Medicine, Hanyang University College of Medicine, Seoul, Korea.
PLoS One. 2016 Dec 30;11(12):e0169091. doi: 10.1371/journal.pone.0169091. eCollection 2016.
Many studies have shown that the mycoplasmal membrane protein p37 enhances cancer cell migration, invasion, and metastasis. Previously, we generated 6 monoclonal antibodies (MAbs) against the mycoplasmal protein p37 and showed the presence of mycoplasma-infected circulating tumor cells in the blood of hepatocellular carcinoma patients by using CA27, one of the six MAbs. When mycoplasmas were incubated with cancer cells in the presence of CA27, mycoplasma infection was completely inhibited, suggesting that CA27 is a neutralizing antibody inhibiting mycoplasma infection. To examine the neutralizing epitope of CA27, we generated a series of glutathione S-transferase (GST)-fused p37 deletion mutant proteins in which p37 was partly deleted. To express p37-coding sequences in E.coli, mycoplasmal TGA codons were substituted with TGG in the p37 deletion mutant genes. GST-fused p37 deletion mutant proteins were then screened to identify the epitope targeted by CA27. Western blots showed that CA27 bound to the residues 216-246 on the middle part of the p37 protein while it did not bind to the residues 183-219 and 216-240. Fine mapping showed that CA27 was able to bind to the residues 226-246, but its binding activity was relatively weakened as compared to that to the residues 216-246, suggesting that the residues 226-246 is essential for optimal binding activity of CA27. Interestingly, the treatment of the purified GST-tagged epitopes with urea showed that CA27 binding to the epitope was sodium dodecyl sulfate-resistant but urea-sensitive. The same 226-246 residues were also recognized by two other anti-p37 MAbs, suggesting that the epitope is immunodominant. The identification of the novel neutralizing epitope may provide new insight into the interaction between the p37 protein and host receptors.
许多研究表明，支原体膜蛋白p37可增强癌细胞的迁移、侵袭和转移能力。此前，我们制备了6种针对支原体蛋白p37的单克隆抗体（MAb），并利用这6种MAb之一的CA27，在肝癌患者血液中检测到了支原体感染的循环肿瘤细胞。当在CA27存在的情况下将支原体与癌细胞共同孵育时，支原体感染被完全抑制，这表明CA27是一种抑制支原体感染的中和抗体。为了研究CA27的中和表位，我们构建了一系列谷胱甘肽S-转移酶（GST）融合的p37缺失突变蛋白，其中p37被部分缺失。为了在大肠杆菌中表达p37编码序列，在p37缺失突变基因中，支原体的TGA密码子被替换为TGG。然后对GST融合的p37缺失突变蛋白进行筛选，以鉴定CA27靶向的表位。蛋白质免疫印迹分析表明，CA27与p37蛋白中部的216 - 246位氨基酸残基结合，而不与183 - 219位和216 - 240位氨基酸残基结合。精细定位显示，CA27能够与226 - 246位氨基酸残基结合，但其结合活性与216 - 246位氨基酸残基相比相对减弱，这表明226 - 246位氨基酸残基对于CA27的最佳结合活性至关重要。有趣的是，用尿素处理纯化的GST标记表位后发现，CA27与该表位的结合对十二烷基硫酸钠具有抗性，但对尿素敏感。另外两种抗p37单克隆抗体也识别相同的226 - 246位氨基酸残基，这表明该表位具有免疫显性。新型中和表位的鉴定可能为p37蛋白与宿主受体之间的相互作用提供新的见解。
