Functional characterization of recombinant human 2GPI. (A) Color-coded domain structure of the recombinant variants used in this work (ie, LT-2GPI, hrβ2GPI, and ST-2GPI) highlighting the position and chemical composition of the N-terminal tag. The long tag (LT, yellow) is composed of three parts: i) a calcium-dependent epitope for the monoclonal antibody HPC4 (EDQVDPRLIDGK); ii) a site-specific biotinylation sequence (AviTag); and iii) a conventional enterokinase recognition site (DDDDK). Two flexible linkers (ie, GGGS) were introduced to separate the three functional units of the tag to avoid the formation of a secondary structure and to ensure exposure of the tag to the solvent. Removal of LT with enterokinase generates hr2GPI. The short label version of 2GPI contains only the HPC4 (purple) purification label.
(B) SDS-PAGE analysis of recombinant proteins (sample 1 = LT-2GPI; sample 2 = hr2GPI; sample 3 = ST-2GPI) and plasma-purified 2GPI (sample 4) before (left) and after (right) the elimination of glycosylations. NEB Protein II Deglycosylation Mix was used to remove O- and N-linked glycosylations under denaturing conditions. Chemical identification was verified by N-terminal sequencing and the results are as follows: band 1 = EDQVD; band 2 = TRB; lane 3 = EDQVD; band 4 = GRTC. (C) Representative sensograms of the interaction between LT-2GPI and liposomes (PC: PS 80:20) monitored by SPR.
The liposomes were immobilized on an L1 chip and soluble 2GPI (0-2 M) was used in the fluid phase (D) Dose-dependent curves that quantify the interaction of LT-2GPI (red circles), hr2GPI (blue circles) and ST-2GPI (green circles) and p2GPI (gray circles) with SPR controlled liposomes. The affinity values (Kd) are 0.19 ± 0.05 µM for LT-2GPI, 0.33 ± 0.08 µM for hr2GPI; 0.22 ± 0.08 M for ST-2GPI and 0.32 ± 0.09 M for p2GPI. No significant binding was observed with liposomes made entirely of PC (light gray circles). Each experiment was repeated at least three times, using two different lots of proteins.
(E) Reactivity of immobilized LT-2GPI (red bars), hr2GPI (blue bars) and ST-2GPI (green bars) and p2GPI (gray bars) against anti-2GPI IgG antibodies followed by ELISA. Comparisons between 2 groups were made using a two-sample t-test. The results were considered significant at p