Part I
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 i...
Part I
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.
Part II
Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer death worldwide, and frequent treatment failure is due to frequent intra- and extra-hepatic metastases. Previously, I showed the presence of mycoplasma-infected circulating tumor cells (CTCs) in the blood of HCC patients. To study how mycoplasma infection affects HCC progression, in this study, I investigated the characteristics of mycoplasma-infected tumor tissues and CTCs in HCC patients. The mycoplasmal protein p37 was detected in 133 (65%) out of 204 HCC tissues and showed significant correlations with higher histologic stages and vascular invasion. p37 expression was associated with poor disease-free survival in HCC patients. Mycoplasma-infected CTCs were detected in 40 (89%) out of 45 HCC patients in the range 0.1-14.8 per blood ml. Mycoplasma-infected cells were also detected in 4 (40%) out of 10 healthy donors in the range 0-1.3 per blood ml, and all of them were epithelial cell adhesion molecule (EpCAM)-positive, irrespective of vimentin positivity. Most (95%) of mycoplasma-free CTCs showed intermediate phenotype with neither EpCAM nor vimentin expression, but most (76%) of mycoplasma-infected CTCs were vimentin-positive or EpCAM-positive, suggesting that mycoplasma infection enriches vimentin- or EpCAM-positive CTCs. Chronic mycoplasma infection increased the migratory potential of HCC cells. Immunoprecipitation further revealed that the p37 protein interacts with EpCAM, whose expression is associated with enhanced epithelial-mesenchymal transition and metastasis in many cancers. The results suggest that mycoplasma infection promotes HCC cell migration and metastasis through the interaction between the mycoplasmal p37 protein and host EpCAM.
Part I
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.
Part II
Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer death worldwide, and frequent treatment failure is due to frequent intra- and extra-hepatic metastases. Previously, I showed the presence of mycoplasma-infected circulating tumor cells (CTCs) in the blood of HCC patients. To study how mycoplasma infection affects HCC progression, in this study, I investigated the characteristics of mycoplasma-infected tumor tissues and CTCs in HCC patients. The mycoplasmal protein p37 was detected in 133 (65%) out of 204 HCC tissues and showed significant correlations with higher histologic stages and vascular invasion. p37 expression was associated with poor disease-free survival in HCC patients. Mycoplasma-infected CTCs were detected in 40 (89%) out of 45 HCC patients in the range 0.1-14.8 per blood ml. Mycoplasma-infected cells were also detected in 4 (40%) out of 10 healthy donors in the range 0-1.3 per blood ml, and all of them were epithelial cell adhesion molecule (EpCAM)-positive, irrespective of vimentin positivity. Most (95%) of mycoplasma-free CTCs showed intermediate phenotype with neither EpCAM nor vimentin expression, but most (76%) of mycoplasma-infected CTCs were vimentin-positive or EpCAM-positive, suggesting that mycoplasma infection enriches vimentin- or EpCAM-positive CTCs. Chronic mycoplasma infection increased the migratory potential of HCC cells. Immunoprecipitation further revealed that the p37 protein interacts with EpCAM, whose expression is associated with enhanced epithelial-mesenchymal transition and metastasis in many cancers. The results suggest that mycoplasma infection promotes HCC cell migration and metastasis through the interaction between the mycoplasmal p37 protein and host EpCAM.
주제어
#Mycoplasma Neutralization antibody CA27 p37 protein Epitope
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