Mutants of β2-glycoprotein I: Their features and potent applications

β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K²⁸⁷C²⁸⁸ was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18-30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.