Use of Single Point Mutations in Domain I of β2-Glycoprotein I to Determine Fine Antigenic Specificity of Antiphospholipid Autoantibodies

Abstract

Autoantibodies against β2-glycoprotein I (β2GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in β2GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole β2GPI containing single point mutations in the first domain. The binding to β2GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40–43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-β2GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-β2GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40–43 contributes to a dominant target epitope.

Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human β2‐glycoprotein I: Mutation studies including residues R39 to R43

“…Altering residue R39 to serine had a greater effect on reducing binding to aPL in the fluid phase than either the G40E or R43G changes that have previously been studied (16,17). The R39S change reduced binding to aPL in both the solid-phase and fluid-phase ELISAs, whereas G40E reduced binding only in the fluid phase.…”

“…In region R39-R43, we studied the mutations G40E and R43G, which were previously identified as being important for binding aPL derived from patients with APS (14,16,17). However, we studied the effects of these mutations in isolated domain I of ␤ 2 GPI, whereas previous studies incorporated these mutation within whole ␤ 2 GPI.…”

“…The recombinant human domain I proteins chosen to test binding in the fluid phase were wild-type recombinant human domain I, 2 proteins that demonstrated good binding in the solid-phase ELISA (Mut 8ϩ9 and extended domain I), a poor binder (R39S), and 1 mutant that had been shown in the context of whole ␤ 2 GPI to cause marked reduction in binding to clinically relevant aPL (16,17) (G40E), although our solid-phase ELISA had shown an opposite effect of this mutation. Figure 3 shows that results for the fluid phase differed from those for the solid phase.…”

“…These hypotheses were generated through the study of the known domain I structure (19), previous binding studies performed by other investigators using variants of whole ␤ 2 GPI containing mutations in domain I (16,17), and mutation studies performed on monoclonal aPL in our laboratory (25,26). In comparing our results with those of previous groups of investigators, it is important to stress that we studied isolated recombinant human domain I, whereas the investigators in most previous studies examined whole ␤ 2 GPI containing mutations in domain I.…”

“…The ability of anti-␤ 2 GPI antibodies purified from patients with autoimmune disease to bind domain I of ␤ 2 GPI has been shown to be strongly correlated with the occurrence of thrombosis in those patients (16). Studies on expressed mutants of whole ␤ 2 GPI with incorporated single-point substitution mutations within domain I identified the residues G40 and R43 as being important in conferring binding to aPL (14,17). De Laat et al (16) identified a population of anti-␤ 2 GPI antibodies derived from patients with thrombosis that bind domain I exclusively.…”