Screening, purification, and identification of annexin B1 mutants with high phosphatidylserine-binding activity and reduced immunogenicity.
Annexin B1 has many potential biomedical applications based on its high affinity for negatively charged phospholipid (phosphatidylserine, PS) in the presence of physiological concentrations of calcium. Low immunogenicity is prerequisite for the in vivo application of a nonhuman protein as a novel-imaging agent. In the present study, three sequence-deleted mutants with different numbers of functional domains were designed and expressed according to the predicted three-dimensional structure of annexin B1. The mutants of annexin B1, as well as the wild-type annexin B1, were expressed as Glutathione-S-transferase (GST)-fusion proteins. Two mutants with their purity above 80% could be obtained after one-step primary purification procedure on basis of the PS-binding activity. The immunogenicity of the two mutants was evaluated in mice by detecting the titers of elicited antigen-specific IgG. A member of three mutants of annexin B1, M12, which involved N-terminal amino-acid sequence and double functional domain I and II of annexin B1, was finally selected to detect apoptosis that is due to its lowest immunogenicity among the candidate mutants. Flourescein isothiocyanate-labeled M12 could bind the outer membranes of apoptotic cells and discriminate apoptotic cells in the early stage from necrotic cells when used with propidium iodide. (99m)Tc-labeled M12 could recognize the apoptotic hepatocytes induced by anti-Fas antibody treatment. Our data in vitro and in vivo demonstrated that M12 could be applied as a promising agent for the detection of apoptosis.
Wang
Fang
F
Department of Medical Genetics, Second Millitary Medical University, Shanghai, 200433, China.
Luo
Quan-Yong
QY
He
Ying
Y
Sun
Shu-Han
SH
D000818:Animals; D017302:Annexins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D009154:Mutation; D010718:Phosphatidylserines / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D017302:Annexins; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; C411023:anti-Fas monoclonal antibody; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate