Characteristics of purified anti-β2GPI IgG N-glycosylation associate with thrombotic, obstetric and catastrophic antiphospholipid syndrome

Abstract

Objective Anti-β-2 glycoprotein I (anti-β2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-β2GP1 IgG with clinical features of APS. Methods We purify anti-β2GPI IgG and total IgG from 82 APS patients including 9 catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyze all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. Results Both purified anti-β2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with HC IgG. Anti-β2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aβ2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aβ2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-β2GPI IgG subclasses, increased bisection and core fucosylation of anti-β2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). Conclusion We comprehensively characterize the N-Glycans landscape of both anti-β2GP1 and total IgG in APS. Altered N-glycan profiles of anti-β2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-β2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.

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“… 54 Levels of the oxidized β2GPI antigen are proportional to the extent of antiphospholipid syndrome (APS) and thrombotic risk. 55 However, the molecular etiology by which oxidized modified proteins lead to lupus flares remains largely unknown, and as many have surmised, might be affiliated with their heightened immunogenicity in vivo.…”

Diagnosis and Management of Catastrophic Antiphospholipid Syndrome and the Potential Impact of the 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria

“…These changes can alter autoantibody functions, such as affecting C1q binding, C5a-mediated inflammation, and antibody-dependent cell cytotoxicity in CAPS. Indeed, altered IgG Fc N-glycosylation of aβ2GP1 displays a pro-inflammatory pattern in APS [ 33 ].…”

Activation of Platelet mTORC2/Akt Pathway by Anti-β2GP1 Antibody Promotes Thrombosis in Antiphospholipid Syndrome

“…Total IgG was purified from plasma obtained from HC using affinity chromatography as described. 39 The anti-β2GP1 antibody was affinity purified using human β2GP1 covalently linked to NHS (N-hydroxysuccinimide)-activated agarose beads (GE Healthcare) as described. 39 Following elution, the antibodies were concentrated and dialyzed against PBS before incubation or infusion.…”

“…39 The anti-β2GP1 antibody was affinity purified using human β2GP1 covalently linked to NHS (N-hydroxysuccinimide)-activated agarose beads (GE Healthcare) as described. 39 Following elution, the antibodies were concentrated and dialyzed against PBS before incubation or infusion. The purified antibodies were determined to be free of endotoxin (Genscript ToxinSensor) according to the manufacturer's instructions.…”