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Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients feature show low levels of bisection, galactosylation and sialylation Manfred Wuhrer, Kathrin Stavenhagen, Carolien A. M. Koeleman, Maurice H. J. Selman, Lorraine Harper, Bart Jacobs, Caroline O. Savage, Roy Jefferis, André M. Deelder, and Matthew D. Morgan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr500780a • Publication Date (Web): 11 Mar 2015 Downloaded from http://pubs.acs.org on March 14, 2015

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Journal of Proteome Research

Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation and sialylation

Manfred Wuhrer1,2,3,*, Kathrin Stavenhagen1,2, Carolien A. M. Koeleman1, Maurice H. J. Selman1, Lorraine Harper4, Bart C. Jacobs5, Caroline O. S. Savage4, Roy Jefferis4, André M. Deelder1, Matthew Morgan4

1

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The

Netherlands 2 3

Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands Department of Molecular Cell Biology and Immunology, VU University Medical Center,

Amsterdam, The Netherlands 4

School of Immunity and Infection, College of Medical and Dental Sciences, University of

Birmingham, Birmingham, UK 5

Department of Immunology and Department of Neurology, Erasmus MC, University Medical

Center Rotterdam, Rotterdam, The Netherlands

AUTHOR INFORMATION Corresponding Author * Manfred Wuhrer, Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands; [email protected]; tel. +31 (0)20 5987527 Notes The authors declare no competing financial interest.

Key words: autoantibody, glycopeptide, glycosylation, immunoglobulin, mass spectrometry

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ABSTRACT Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA) which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham Vasculitis Activity Scores (BVAS), serum cytokines and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation and bisection was reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.


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INTRODUCTION Granulomatosis with polyangiitis (GPA) is an inflammatory autoimmune disease usually associated with circulating anti-proteinase 3 (PR3) specific anti-neutrophil cytoplasm antibodies (ANCA) and is one of the ANCA associated vasculitides (AAV). It frequently causes severe lung, upper airways and kidney damage and can affect any organ of the body. Men and women are affected equally and the peak age of onset is in the 7th decade.1 In Europe the annual incidence is less than 10 per 1 million population2, 3 and the prevalence estimated at around 145 per million population.4, 5 Current treatment consists of immunosuppression with corticosteroids with the addition of cyclophosphamide for severe disease and the further addition of plasma exchange for life or organ threatening disease (commonly dialysis dependent renal failure and/or pulmonary haemorrhage).6 The disease is characterized by periods of remission followed by relapse with approximately 50% of patients experiencing a relapse over five years. The pathogenesis is complex involving both the cellular and humoral components of the immune system. The development of ANCA, which are class switched immunoglobulin (Ig) G antibodies, implies a breach of self-tolerance and roles for T and B cells in their production. ANCA have been demonstrated in vitro to activate cytokine primed neutrophils leading to endothelial adhesion, superoxide production and degranulation causing tissue damage and inflammation.7 Rat and murine models of anti-myeloperoxidase (MPO) ANCA have demonstrated that MPO-ANCA can drive vasculitis in vivo.8-10 A recently described model of human anti-PR3 ANCA disease has demonstrated that PR3-ANCA can drive the development of glomerulonephritis and pulmonary haemorrhage.11 Human IgG is glycosylated at Asn297 in the Fc portion of the antibody. N-glycans carrying specific decorations (galactoses, fucose, sialic acid and bisecting N-acetylglucosamine (GlcNAc)) attached to the heptasaccharide core determine the ability of the IgG to interact with activating Fcγ receptors (FcγR) I, IIa and III and the inhibitory FcγRIIb.12, 13 Previous studies have demonstrated that, as in other autoimmune diseases, total IgG from patients with ANCA associated vasculitis is hypogalactosylated and that this aberrant glycosylation is limited to the Fc and not the Fab portion of the antibody.14-16 Recently, it was shown that anti-PR3 autoantibodies in GPA show reduced levels of autoantibody sialylation but the glycosylation status of anti-PR3 specific autoantibodies has not otherwise been investigated.17 3 ACS Paragon Plus Environment


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Here we report on the relative galactosylation, sialylation, fucosylation and bisection of total IgG1 and IgG2 and anti-PR3 specific IgG1 from patients with GPA and correlations with inflammatory cytokines and disease activity and progression.

EXPERIMENTAL SECTION A. Patient samples ANCA positive sera were obtained from patients with active disease. The cohort comprised 39 patients with newly diagnosed life or organ threatening disease requiring treatment including plasmapheresis. Plasmapheresis took place within 48 h of diagnosis and 1 liter of the effluent of the first treatment session was saved in aliquots at -20°C for later conversion to serum by the addition of calcium chloride as previously described.16 Control sera were obtained from 38 age and sex matched healthy individuals. Additionally, serum was collected from 10 patients with active disease (nine with new diagnosis; 1 patient with relapsed disease) within 48 h of starting immunosuppressive therapy. These patients had a greater range of disease severity with median Birmingham Vasculitis Activity Scores (BVAS) of 15; range 3-2.18 Only two patients had organ threatening disease. Patients in this group were recruited as part of the ACTIVE trial already reported and had clinical data collected over 12 months follow up.19 In addition they had a panel of serum cytokines measured at entry by multiplex assay (Biorad multiplex human cytokine assay as per manufacturer’s instructions). Ethical approval was in place for all samples collected and consent obtained from all donors.

B. Sample preparation IgG was purified from serum samples of all 49 GPA patients and 38 healthy controls (see Table 1 for details). Protein A-Sepharose beads (GE Healthcare, Eindhoven, The Netherlands) were washed three times with 10 volumes of PBS. 15 µl of beads per well were applied to a 96-well filter plate (Multiscreen Solvinert, 0.45 µm pore-size low-binding hydrophilic PTFE; Millipore, Billerica, MA). The volume was brought to 150 µl with PBS, and 2 µl of serum was applied per well. The plate was sealed with tape and incubated on a shaker for 1 h. The beads were


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washed 5x with 200 µl PBS under vacuum. After washing 2x with 200 µl water, immunoglobulins (IgG1, IgG2 and IgG4) were eluted with 100 µl of 100 mM formic acid (p. a. for mass spectrometry; Merck, Darmstadt, Germany) into a V-bottom microtiter plate (Nunc, Roskilde, Denmark). Samples were dried by vacuum centrifugation. Anti-PR3 antibodies were purified using Bindazyme human anti-PR3 ELISA plates (The Binding Site, Birmingham, UK).20 Sera of anti-PR3-positive GPA patients as well as anti-PR3-negative controls (20 μl) were mixed with 130 μl dilution buffer and incubated in antigen-coated ELISA plates for 1 h at 37°C. Supernatants were discarded and plates were washed three times with washing buffer followed by washing twice with 25 mM ammonium bicarbonate (Merck, Darmstadt, Germany). Anti-PR3 were then eluted by incubation for 15 min at RT with 50 µl 100 mM formic acid. Eluates were transferred to 96 well V-bottom microtiter plates and dried by vacuum centrifugation. For proteolytic cleavage of the dried eluates a 20 µg aliquot of trypsin (sequencing grade; Promega, Leiden, The Netherlands) was dissolved in 4 ml of 25 mM ammonium bicarbonate. Within 1 min after preparation, 40 µl of this mixture was added per well to the dried purified antibodies. Samples were shaken (1 min), incubated overnight at 37°C, and stored at -20°C until usage.

C. Mass spectrometric IgG Fc glycosylation analysis Mass spectrometric IgG Fc glycosylation analysis was performed by LC-MS as described previously,21 with minor modifications, see Supplementary Information for details. Assignment of the peptide moieties and thereby the subclasses was achieved using the established elution orders of tryptic Fc glycopeptides in reverse phase-LC as well as ion trap collision-induced dissociation.22-24 Structures of the attached glycans were assigned on the basis of literature data on serum IgG glycosylation of healthy individuals, autoimmune patients

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and, more

specifically, AAV patients.14-16 No discrimination was made between 3-antenna and 6-antenna galactosylation and sialylation.

D. Data processing and statistical analysis



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The detected IgG1 and IgG2 Fc glycopeptide signals (see Supporting Information Table 1) were quantified using QuantAnalysis (Bruker) via extracted ion chromatograms of double protonated and triple protonated glycopeptides species with an integration window from -0.3 to +1.5 of the theoretical m/z values. Automatically generated integration marks were adjusted manually where necessary. Signals of the different charge states were added per glycopeptide species. IgG1 and IgG2 glycopeptide signals were integrated per subclass, and normalized to the total of each subclass. Accordingly, the reported relative abundance of all IgG1 glycopeptides registered in one sample adds up to 100%, and the same holds true for IgG2. Statistical analysis was performed using GraphPad Prism 5 (La Jolla, CA) and SPSS version 21 (IBM, Armonk, NY). Paired or unpaired parametric or non-parametric tests were applied as appropriate to the data distribution. P values 0.05).

Galactosylation of the PR3 specific IgG1 is associated with inflammatory cytokines and longer time to remission For 10 of the 49 GPA patients included in this study longitudinal data on disease progression and serum cytokine concentrations were available. For these patients we found positive correlations between serum cytokine concentrations, galactosylation of anti-PR3 specific IgG1 and time to remission (Table 2). There were also significant correlations between anti-PR3 specific IgG1 sialylation and serum IL-1B (r = 0.65; p = 0.042) and IL-12 (r = 0.65; p = 0.042) but not IL-2, IL-15 or time to remission. The correlation coefficient between anti-PR3 specific IgG1 galactosylation and time to remission was 0.68 (p = 0.031). There were no correlations between galactosylation or sialylation of total IgG1 and serum cytokine concentrations or time to remission. There were no significant correlations between total or anti-PR3 specific IgG1 galactosylation or sialylation and BVAS.


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Interestingly in this group there was a strong correlation between total IgG1 galactosylation and sialylation (r = 0.73; p = 0.016) and a trend to a correlation between anti-PR3 specific IgG1 galactosylation and sialylation (r = 0.59; p = 0.078) although there was no correlation between total IgG1 and anti-PR3 IgG1 galactosylation (r = 0.15; p = 0.68) or sialylation (r = 0.05; p = 0.89). There were no significant correlations between IgG1 galactosylation or sialylation and time to remission or serum cytokines. There was a strong correlation between the presence of bisecting GlcNAc on PR3-specific IgG1 and disease activity as measured by BVAS (r = -0.75; p = 0.02) but no correlation with cytokine concentrations, time to remission or other PR3-specific IgG1 glycoforms.

DISCUSSION We are the first to use mass spectrometry to investigate the glycosylation of purified anti-PR3specific IgG from patients with GPA. We have demonstrated that these antigen-specific autoantibodies are hypogalactosylated and that their glycosylation correlates with measures of disease activity and inflammation. These correlations are restricted to the pathogenic antigenspecific antibodies and not to total patient IgG. Notably, these features were not revealed by previous studies which either used lectin binding or analyzed total IgG glycosylation and not the glycosylation of anti-PR3-specific IgG.14-17 We have shown a marked glycosylation difference between antigen-specific IgG and total patient IgG. Anti-PR3 specific IgG showed significantly less galactosylation and sialylation and fewer bisecting species than total IgG and there was little or correlation between the glycosylation of PR3 specific IgG and total IgG. This is the first time that a disease-associated decrease in bisecting GlcNAc has been demonstrated on IgGs. We have also demonstrated that the glycosylation changes in anti-PR3 specific IgG occur at the Fc portion. This is significant as it may influence their interactions with the CH2 domain of the neutrophil Fcγ receptors. The glycosylation status of anti-PR3 IgG may be important in determining its ability to activate neutrophils and cause tissue and organ damage. It should be noted that ANCA autoantibodies in GPA do have a pathogenic role and that their contribution to pathology seems to be for a large part Fc-receptor mediated.31 32 However, the link between serum anti-PR3 IgG titers and



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disease activity remains obscure as increases in antibody titers during remission do not accurately predict relapse.33 Neutrophils constitutively express the activatory Fcγ receptors FcγR2a and FcγR3b. The high affinity IgG FcγR1a is expressed in response to inflammatory cytokines such as IFNγ or G-CSF3436

and also on ex vivo neutrophils from AAV patients both with active disease and in

remission.37 Expression of the inhibitory receptor Fcγ2b has also recently been reported on neutrophils.38,

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Previous studies have demonstrated that anti-PR3 specific IgG activate

cytokine primed neutrophils from healthy donors via FcγR2a and FcγR3b40-43 and also via FcγR1a in ex vivo neutrophils from patients with active AAV. Notably, IgG1 Fc glycosylation is known to modulate the interaction with FcγR3b,44 therefore suggesting a role of anti-PR3 IgG1 Fc glycosylation in the pathology of GPA. A previous study demonstrated that desialylation of IgG from GPA patients in remission using sialidase (leaving galactose covalently attached to the Fc) significantly increased neutrophil superoxide generation.17 In the same study there was a negative correlation between anti-PR3 specific IgG sialylation and its ability to stimulate neutrophil superoxide release. Increased sialylation has been reported to confer anti-inflammatory properties to IgG and this has been proposed as the mechanism of action for therapeutic intravenous immunoglobulin in autoimmune inflammatory conditions with increased activation of the inhibitory FcγR2b.45 Consequently, the low degree of sialylation observed for both total IgG and anti-PR3 IgG may have a pro-inflammatory effect. Others have demonstrated a similar role for galactosylation of IgG1 in increasing activation of FcγR2b.46 The expression of FcγR2b on neutrophils of GPA patients has not been examined. Previous work suggests that complete deglycosylation of IgG “silences” Fcγ receptor activation47 although there is some evidence that aglycosylated multimeric IgG interactions can activate the high affinity FcγR1.48 Deglycosylation of IgG from AAV patients using endoglycosidase S significantly reduces its ability to induce cytokine primed healthy donor neutrophil degranulation and inhibits glomerular crescent formation in a murine model of antimyeloperoxidase ANCA vasculitis.49 As in previous studies we found that IgG1 and IgG2 Fc from GPA patients were hypogalactosylated and hyposialylated compared to healthy controls.15-17 Similar observations have been made for other autoimmune diseases26, 50 and studies in rheumatoid arthritis have 10 ACS Paragon Plus Environment

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demonstrated that hypogalactosylation of IgG occurs years before clinical onset of disease and after the initial detection of auto-antibodies and IgG becomes progressively more hypogalactosylated over time.51, 52 We found a correlation between Fc galactosylation and sialylation for total IgG1 and IgG2 of both controls and GPA patients (Figure 3A&B). This is in line with the expectations as the galactosylated structures are the biosynthetic precursors for sialylation. Interestingly we found little or no correlation between total IgG1 and PR3-specific IgG1 galactosylation and sialylation in GPA patients suggesting differential control of post-translational modification of IgG1 in Bcells. We have previously observed a similar phenomenon following vaccination with influenza where changes in glycosylation status were limited to antigen specific IgG and not total IgG.53 IgG glycosylation modification appears to be under the control of T helper cytokines and the interaction between T and B cells. We have previously demonstrated that inflammatory cytokines including IFNγ increase the galactosylation of IgG1.54 In animal studies of IgA nephropathy, where the aberrant glycosylation of IgA is implicated in disease pathogenesis, the balance of T helper (Th) 1 versus Th2 cytokines determines the relative galactosylation and sialylation of IgA.55, 56 This is consistent with our novel finding of a positive correlation between the galactosylation of PR3-specific IgG1, pro-inflammatory cytokine concentrations and time to remission and suggests that T cell activation drives the aberrant glycosylation of ANCA and the time to achieve remission. A previous study observed a negative correlation between BVAS and anti-PR3 specific IgG sialylation suggesting this was linked with the ability to activate neutrophils17 whereas we observed a strong negative correlation between the presence of bisecting GlcNAc on anti-PR3 IgG and BVAS but no correlation between BVAS and galactosylation or sialylation. It should be noted however that sialylation of anti-PR3 specific IgG in the previous study was determined by lectin binding assay rather than mass spectrometry. Binding of sialic acid-specific lectins to Ig seems not to reflect Fc glycosylation, but may rather be related to Fab sialylation,57,

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thereby precluding a direct comparison of the lectin-binding studies from literature17 with our mass spectrometric Fc glycosylation analyses of anti-PR3 IgG1. When compared to the IgG Fc glycosylation analyses in other autoimmune diseases, the effects described here for bisecting GlcNAc are novel. The only previous investigation of changes in the expression levels of bisected structures on IgG of autoimmune patients were obtained by 11 ACS Paragon Plus Environment


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lectin binding studies,59 and confirmation of these results by structural analysis of the glycan moieties is still lacking. In rheumatoid arthritis and myositis, patients show reduced IgG1 and IgG2 Fc galactosylation, whilst the levels of bisecting GlcNAc are preserved.26, 50, 60, 61 Previous analyses of IgG glycosylation changes in GPA did not show decreased levels of bisecting GlcNAc.15,

16

This apparent discrepancy may be because total IgG glycosylation (without

separation of Fc and Fab) was studied previously and levels of bisected structures on the Fab portions of IgG possibly obscured a reduction on the Fc portions.15 Increased bisected structures on Fab rather than Fc in both healthy controls and GPA patients was suggested by targeted analysis of Fc and Fab glycans, but the analyzed sample amounts were too small to facilitate statistical analysis.16 Hence, when analyzing protein glycosylation at the level of released glycans, site specific glycosylation changes might be obscured by glycosylation patterns of other sites which do not undergo these changes, indicating the relevance of sitespecific glycosylation analysis at the glycopeptide level. As this approach allows the unambiguous assignment of glycans to specific proteins and sites, it minimizes the interference from other sites as well as co-purified, contaminating proteins. Little is known about the functional consequences of reduced bisecting GlcNAc although some studies have demonstrated that increased expression of bisected species increases IgG1 interaction with FcγR3a (which is not implicated in ANCA activation of neutrophils) with no effect on FcγR2b interaction.62-65 Similarly, the control of expression of bisecting GlcNAc on IgG is poorly understood. There is some evidence that antigen stimulation leads to reduced bisecting GlcNAc on antigen-specific IgG1 following vaccination suggesting a role for T cells in determining this post-translational modification53 although we found no correlation between bisecting GlcNAc and cytokine concentrations. Altogether these findings are interesting as they demonstrate changes in the glycosylation of anti-PR3 specific IgG compatible with an ongoing antigen-specific response involving Th1 associated cytokines. The association between altered anti-PR3 specific IgG glycosylation, disease activity and neutrophil activation is worthy of further investigation. Changes in the anti-PR3 specific IgG glycosylation status over time as patients move from active disease to remission and to future relapse may identify a useful biomarker to monitor disease activity and predict risk of relapse. If changes in the glycosylation status of the antibody can be shown to directly influence the 12 ACS Paragon Plus Environment

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activation of neutrophils this may open up new therapeutic avenues as glycosylation can be altered in vivo. Key to this would be understanding the relative contributions of the different Fcγ receptors to ANCA induced neutrophil activation and the way that changes in Fc glycosylation alter these relationships.

ACKNOWLEDGEMENTS This work has been supported by funding from the European Union’s Seventh Framework Programme (FP7-Health-F5-2011) under grant agreement 278535 (HighGlycan). Dr. Morgan was supported by a grant from The Health Foundation.

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Figure legends Figure 1: NanoLC-ESI-MS analysis of IgG1 Fc glycosylation. Representative examples of IgG1 Fc glycosylation are shown from a healthy control (top panel), a GPA patient (middle panel) and anti-PR3 specific IgG1 from the same GPA patient (bottom panel).Glycopeptides were registered in triple-protonated form. Blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose; purple diamond, N-acetylneuraminic acid; *, irrelevant adduct; Contr., control.

Figure 2: Comparison of the IgG1 and IgG2/3 glycosylation features of GPA patients and controls. Fc glycosylation features were compared for total serum IgG of presumably healthy controls (C), total serum IgG of GPA patients (GPA), and anti-PR3 autoantibodies from the same patients (PR3). Both IgG1 (A, B) and IgG2 (C, D) were compared with regard to relative abundance of agalactosylated (Agal.) species and overall galactosylation (A, C) as well as with regard to sialylation, relative abundance of bisected species (Bisecting) and afucosylation (C, D). Median values are indicated by blue, horizontal bars. Significant differences are indicated by two asterisks (p

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