Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.
Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.
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DOI：
10.1002/art.22306
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发表时间：
2007-01
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致病性抗磷脂抗体（aPL）结合β2-糖蛋白I （β2GPI）的自身抗原n端结构域（结构域I），其中残基G40-R43是重要的。然而，与结构域1的其他区域同源的肽也被证明可以结合aPL。此外，没有关于改变R39的影响的已发表的报告，R39的表面暴露比G40-R43残留物更大。我们使用了一种新的、高效的方法，通过大肠杆菌生产和纯化人类结构域I来创建结构域I的多个突变体，然后使用固相和液相两种方法筛选这些结构域I突变体，以结合从抗磷脂综合征患者中纯化的一系列多克隆IgG。大肠杆菌表达纯化结构域I选择性结合IgG来源于抗磷脂综合征患者。在R39-R43区，R39S突变在减少液相与一组aPL结合方面的影响最大（平均±SD抑制率为14±18.5%，而G40E为44.1±31.7%，野生型结构域I为62.9±25.7%）。相反，将D8和D9分别改变为S8和G9，在流体阶段可以增强与aPL的结合。添加剩余的结构域I-II互连体导致在固相而不是流体相中比野生型的结合增强。aPL与β2GPI结构域I的结合是复杂的，可能涉及不连续的表位，除G40-R43外，还包括R39，结构域I - ii互连体，可能还有D8和D9。与野生型结构域I相比，与aPL结合增强的结构域I变异可能有助于开发新的治疗方法。
Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of β2-glycoprotein I (β2GPI), 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. 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. 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. The binding of aPL to β2GPI 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.