Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43

Abstract

Objective: Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of beta(2)-glycoprotein I (beta(2)GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues.

Methods: We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays.

Results: E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean +/- SD inhibition 14 +/- 18.5% versus 44.1 +/- 31.7% for G40E and 62.9 +/- 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase.

Conclusion: The binding of aPL to beta(2)GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.

Figure 1

Binding of polyclonal IgG from patients with antiphospholipid syndrome (APS) to Escherichia coli–expressed recombinant human domain I in the solid phase. APS, n = 22; systemic lupus erythematosus (SLE) controls, n = 20; healthy controls, n = 10. For APS patients versus SLE/autoimmune controls, P = 0.0004; for APS patients versus healthy controls, P < 0.0001; for SLE/autoimmune controls versus healthy controls, P = 0.39. Bars show the mean.

Figure 2

Binding of polyclonal IgG from patients with antiphospholipid syndrome (APS) to mutant recombinant human domain I in the solid phase. A panel of purified IgG samples derived from 10 patients with APS was selected. Enhanced binding as compared with wild-type recombinant human domain I was greatest with the domain I mutant in which both D residues were mutated (Mut 8+9) and the extended domain I (DI ext) mutant. The greatest reduction in binding compared with wild-type domain I was observed with the single-point D8 and D9 mutants and the R39 mutant. Bars show the mean.

Figure 3

Binding of polyclonal IgG from patients with antiphospholipid syndrome to mutant recombinant human domain I (DI) in the fluid phase. The graph shows the degree to which wild-type and mutant recombinant human domain I (at 3.25 μM) can inhibit polyclonal IgG antiphospholipid antibodies (aPL) from binding to whole β₂-glycoprotein I coated on cardiolipin. Variable inhibition was observed with wild-type recombinant human domain I and extended domain I (DI ext). The domain I mutant in which both D residues were mutated (Mut 8+9) had the effect of enhancing binding to the majority of aPL tested. In contrast, the R39S mutation resulted in a significant reduction in binding as compared with wild-type (P = 0.001). G40E had a variable effect. Values are the mean and SD of 8 samples.

Figure 4

Computer models generated by the Swiss-Model automated protein structure homology-modeling server. a, Molecular structure of the wild-type recombinant domain I molecule. b, Electrostatic charge on the surface of the wild-type recombinant domain I molecule. c, Molecular structure of R39S. d, Electrostatic charge on the R39S molecular surface. Residues R39 and R43 are shown in dark blue, G40E in purple, the domain I–II interlinker region in gray, and residues D8 and D9 in red. Mutating R39 to S39 (light blue) results in marked alteration of surface epitope conformation, with no significant effect on the overall protein tertiary structure.