Glycosylation of autoantibodies: Insights into the mechanisms of immune thrombocytopenia

Abstract

Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets (PLTs). IgG effector functions depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. AAbs were purified from ITP patients (n=15) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and activated complement. N-glycan cleavage significantly reduced both effects. In vivo survival of human PLTs was assessed after co-transfusion with native or N-glycan cleaved AAbs in a NOD/SCID mouse model. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL(5h))75% vs 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL(5h) 82% vs 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL(5h) 42%, p<0.001). N-glycosylation of human ITP AAbs appears to be required for platelet phagocytosis and complement activation, reducing platelet survival in vivo. Posttranslational modification of AAbs may constitute an important determinant for the clinical manifestation of ITP.

Impact of Glycosylation and Species Origin on the Uptake and Permeation of IgGs through the Nasal Airway Mucosa

“…For example, an IgG3 variant with hinge-region deletions that prevent binding to FCGR but not to FcRn was still transported across the human placenta [ 83 ]. Likewise, deglycosylated IgGs were transported in mice [ 84 ]. Furthermore, a comparison of glycosylation patterns between fetal and maternal IgG by Stapelton et al in 2018 concluded that IgG transport was not glycosylation selective [ 85 ].…”

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia

“…A NOD/SCID mouse model was used to investigate the elimination of human platelets by anti-GPV autoantibodies in ITP patients. 24 In brief, NOD/SCID mice (NOD.CB17-Prkdcscid/J; Stock No. complexes, 001303) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) via Charles River, Research Models and Services (Sulzfeld, Germany).…”

The Neonatal Fc Receptor (FcRn): A Misnomer?

“…Pointing against involvement of FcγRs is the fact that an IgG3 variant with hinge-region deletions that prevents binding to all FcγR but retains FcRn binding was still transported to the fetus (154). Likewise, aglycosylated IgG variants that are unable to interact with FcγR, but bind FcRn, were transported in mice (155). Comparison of glycosylation patterns between fetal and maternal IgG showed that IgG transport was not glycosylation selective (143).…”