Homology modeling threedimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

AnxB1, a novel annexin previously isolated from Cysticercus cellulose, shows high thrombi affinity and anticoagulant activity in vivo. In order to investigate the relationship between structure and biological function, a predicted three-dimensional (3D) model of AnxB1 was generated by homology modeling. This model contains four homologous internal-domains and the C/ga trace of domain I, II and IV shows high similarity. Based on the structure characterization, four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E. coli. Two of the mutants, GST-M3 and GST-M4 reserved high anticoagulant activity (p<0.01 vs. GST). Furthermore, compared with the wild type GST-AnxB1, the immunogenicity of GST-M3 and GST-M4 was reduced significantly (p<0.01) and the molecular weight was lowered to 27 kD and 34 kD, respectively. These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.