Anti-ATP5B monoclonal antibody, clone 8F4G3

Objectives: Prothymosin (ProT) was reported to inhibit the neuronal necrosis by facilitating the plasma membrane localization of endocytosed glucose transporter 1/4 through an activation of putative G(i)-coupled receptor. The present study aims to identify a novel ProT target, which may lead to an activation of G(i)-coupled receptor.Methods: We used G(i)-rich lipid rafts fraction of retinal cell line N18-RE-105 cells for affinity cross-linking. The biological confirmation that F-0/F-1 ATPase is a target protein complex was performed by cell-free experiments using ELISA-based binding assay, surface plasmon resonance assay and quartz crystal microbalance assay, and cell-based experiments to measure extracellular ATP level in the HUVECs culture.Results: From the cross-linking study and above-mentioned protein-protein interaction assays, ATP5A1 and ATP5B, F-1 ATPase subunits were found to ProT binding target proteins. In the culture of HUVEC cells, furthermore, ProT increased the extracellular ATP levels in a reversible manner by anti-ATP5A1- and ATP5B-antibodies.Conclusion: The present study suggests that ProT may activate ecto-F-0/F-1 ATPase and produced ATP. This study leads to next subjects whether produced ATP and its metabolites, ADP or adenosine may activate corresponding G(i)-coupled receptors.

Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.