title: Mapping of a Mycoplasma-Neutralizing Epitope on the Mycoplasmal p37 Protein.
abstract: 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.
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Min Kyu
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Won-Tae
authors.initials: WT
authors.lastname: Kim
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Hyun Min
authors.initials: HM
authors.lastname: Lee
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Hong Seo
authors.initials: HS
authors.lastname: Choi
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Yu Ra
authors.initials: YR
authors.lastname: Jo
authors.affiliation: Department of Laboratory Medicine, Hanyang University College of Medicine, Seoul, Korea.
authors.forename: Yangsoon
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Department of Surgery, Hanyang University College of Medicine, Seoul, Korea.
authors.forename: Jaemin
authors.initials: J
authors.lastname: Jeong
authors.affiliation: Department of Surgery, Hanyang University College of Medicine, Seoul, Korea.
authors.forename: Dongho
authors.initials: D
authors.lastname: Choi
authors.affiliation: Center for Colorectal Cancer, Research Institute and Hospital of National Cancer Center, Goyang-si, Korea.
authors.forename: Hee Jin
authors.initials: HJ
authors.lastname: Chang
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Dae Shick
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science, BK21 PLUS Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Korea.
authors.forename: Young-Joo
authors.initials: YJ
authors.lastname: Jang
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea.
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
mesh_terms: D000072283:A549 Cells; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D006528:Carcinoma, Hepatocellular / Q000097:blood; D045744:Cell Line, Tumor; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood; D008565:Membrane Proteins / Q000276:immunology*; D009174:Mycoplasma / Q000276:immunology*; D009175:Mycoplasma Infections / Q000276:immunology / Q000382:microbiology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D000939:Epitopes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; C000621139:p37 protein, Mycoplasma; D005982:Glutathione Transferase