Date Published: February 28, 2019
Publisher: Public Library of Science
Author(s): Olivier M. Lardinois, Leesa J. Deterding, Jacob J. Hess, Caroline J. Poulton, Candace D. Henderson, J. Charles Jennette, Patrick H. Nachman, Ronald J. Falk, Joseph J. Barchi.
Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity.
Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments.
IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion.
Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.
Anti-neutrophil cytoplasmic autoantibody (ANCA)-vasculitis (AAV) is a systemic autoimmune disease characterized by focal necrotizing lesions affecting arterioles, capillaries, and venules. This disease may affect many organs, and the kidneys and the lungs are commonly involved [1, 2]. The two major ANCA antigens are myeloperoxidase (MPO)  and proteinase 3 (PR3) , which are constituents of neutrophil primary granules  and monocyte lysosomes . PR3-ANCA are found in the majority of patients with granulomatosis with polyangiitis (GPA), and some patients with and microscopic polyangiitis (MPA). MPO-ANCA are found in most patients with MPA as well as in some patients with GPA or with eosinophilic GPA (EGPA). There is increasing support to categorize patients based on their ANCA serotype, i.e., PR3-ANCA and MPO-ANCA, as opposed to the traditional disease classification, i.e., GPA, MPA, and EGPA, as significant clinical differences exist between these two small-vessel vasculitides [7–17].
Detailed information about polyclonal IgG purification, generation of IgG-F(ab’)2 and IgG-Fc fragments, pull-down experiments using SNA and MPO-coated beads, enzyme-linked lectin assay (ELLA), anti-MPO enzyme-linked immunosorbent assay (ELISA), and Lectin blot analysis are given in the supplemental material and methods (See S1 Text in the Supporting Information).
The present study uses multiple approaches to examine changes in IgG Fc and Fab glycosylation with disease activity in patients with AAV. Glycosylation analyses of IgG from AAV patients have been performed before primarily on total IgG derived from peripheral blood [36, 48] or on anti-PR3 specific IgG [44, 52]. The only previous investigation on the glycosylation states of anti-MPO specific IgG was performed using lectin-binding assays . The glycosylation state of anti-MPO-specific autoantibodies has not otherwise been investigated.