Lise Hafkenscheid

The Emerging Importance of IgG Fab Glycosylation in Immunity

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15–25% of serum IgG contains glycans within the variable domains. These so-called “Fab glycans” are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

Structural analysis of variable domain glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis reveals the presence of highly sialylated glycans.

Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab′)2 fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region

Significance Structural variation of antibodies is generally defined in terms of amino acid composition, neglecting posttranslational modifications such as N-linked glycosylation. Little is known about the role of the glycans that are present in about 15% of variable domains. However, recent studies suggest that variable domain glycans exhibit distinct patterns according to (patho)physiological conditions, and can have immunomodulatory effects. Here we highlight a physiological role for variable domain glycans that is predetermined in the germline antibody repertoire: We show that variable domain N-linked glycans are acquired during somatic hypermutation at positions predisposed in the germline and may be positively selected during affinity maturation, representing an additional mechanism of secondary antibody diversification that contributes to the extent of the B-cell antibody repertoire. A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N-linked glycans, a process conditional on the introduction of consensus amino acid motifs (N-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

The extensive glycosylation of the ACPA variable domain observed for ACPA-IgG is absent from ACPA-IgM

Recently, we described the presence of highly sialylated N -linked glycans in the antigen-binding fragment (Fab) of almost all anti-citrullinated protein antibody (ACPA) IgG molecules.1 2 These glycans could not be found on several other autoantibody systems analysed. Given the low affinity of ACPA,3 this observation raises the intriguing possibility that citrullinated antigen-specific B cells could be selected based on the presence of glycans in the variable domain, rather than on affinity for their cognate antigen. N -glycosylation requires the presence of specific consensus sequences in the amino acid backbone of proteins.4 However, only few human germline Ig variable region genes encode for such sequences.5 So far, we could identify several N -glycosylation sites in ACPA-IgG Fab-domains using mass spectrometry, but none of these were encoded in the germline sequence.1 This suggests that the extensive presence of N -glycans in ACPA-IgG Fab-domains results from somatic mutations. Moreover, it indicates that the ACPA response matures under the influence of T-cell help, presumably in germinal centres, and makes it conceivable that the introduction of N -glycosylation sites …

ACPA IgG galactosylation associates with disease activity in pregnant patients with rheumatoid arthritis

Objectives Patients with autoantibody-positive rheumatoid arthritis (RA) are less likely to experience pregnancy-induced improvement of RA disease activity (DAS28-C reactive protein (CRP)) compared with patients with autoantibody-negative RA. Anti-citrullinated protein antibodies (ACPAs) are the most specific autoantibodies for RA. We previously demonstrated that disease improvement is associated with changes in total IgG glycosylation, which regulate antibody effector function. Therefore, we sought to analyse the ACPA-IgG glycosylation profile during pregnancy with the aim to understand the lower change of pregnancy-induced improvement of the disease in patients with autoantibody-positive RA. Methods ACPA-IgGs were purified from ACPA-positive patient sera (n=112) of the Pregnancy-induced Amelioration of Rheumatoid Arthritis cohort, a prospective study designed to investigate pregnancy-associated improvement of RA. The fragment crystallisable (Fc)glycosylation profile of ACPA-IgGs was characterised by mass spectrometry and compared with that of total IgG derived from the same patients or from ACPA-negative patients. Results All ACPA-IgG subclasses display significant changes in the level of galactosylation and sialylation during pregnancy, although less pronounced than in total IgG. The pregnancy-induced increase in ACPA-IgG galactosylation, but not sialylation, associates with lower DAS28-CRP. In ACPA-positive patients, no such association was found with changes in the galactosylation of total IgG, whereas in ACPA-negative patients changes in disease activity correlated well with changes in the galactosylation of total IgG. Conclusions In ACPA-positive RA, the pregnancy-induced change in galactosylation of ACPA-IgG, and not that of total IgG, associates with changes in disease activity. These data may indicate that in ACPA-positive patients the galactosylation of ACPA-IgG is of more pathogenic relevance than that of total IgG.

HappyTools: A software for high-throughput HPLC data processing and quantitation

High-performance liquid chromatography (HPLC) is widely used for absolute quantitation. The advent of new columns and HPLC technology has enabled higher sample throughput, and hence, larger scale studies that perform quantitation on different sample types (e.g. healthy controls vs. patients with rheumatoid arthritis) using HPLC are becoming feasible. However, there remains a lack of methods that can analyse the increased number of HPLC samples. To address this in part, the modular toolkit HappyTools has been developed for the high-throughput targeted quantitation of HPLC measurements. HappyTools enables the user to create an automated workflow that includes retention time (tr) calibration, data extraction and the calculation of several quality criteria for data curation. HappyTools has been tested on a biopharmaceutical standard and previously published clinical samples. The results show comparable accuracy between HappyTools, Waters Empower and ThermoFisher Chromeleon. However, HappyTools offered superior precision and throughput when compared with Waters Empower and ThermoFisher Chromeleon. HappyTools is released under the Apache 2.0 license, both the source code and a Windows binary can be freely downloaded from https://github.com/Tarskin/HappyTools.

FRI0083 Reduced increase of ACPA IGG-FC galactosylation during pregnancy in comparison to total IGG: an explanation why autoantibody positive RA-patients improve less during pregnancy?

Background Rheumatoid arthritis (RA) disease activity (DAS28-CRP) improves less during pregnancy in autoantibody positive patients.1 The most specific autoantibodies for RA are anti-citrullinated protein antibodies (ACPAs), which mainly occur as the immunoglobulin (Ig) G isotype. An association with DAS28-CRP and the pregnancy-associated improvement is well established for the Fc glycosylation of total IgG, in particular for galactosylation (Gal) and sialylation (SA).2 The Fc glycosylation of ACPAs – mainly present as IgG – has been reported to be different from the total IgG Fc glycosylation.3 Objectives We sought to determine whether the change in ACPA IgG glycosylation during pregnancy is different from that of total IgG, and whether this relates to the improvement of RA during pregnancy. Methods ACPA positive patient sera (n=152) were obtained within the framework of the PARA cohort, a prospective study designed to investigate pregnancy-associated improvement of RA. ACPA IgG was isolated using microscale affinity chromatography. Trypsin digested ACPA IgG was measured using nano-liquid chromatography mass spectrometry, and compared to total IgG. Results Pregnancy-associated changes in the levels of glycosylation were observed for all ACPA IgG subclasses. Pregnancy-associated glycosylation changes were less pronounced during pregnancy and after delivery in ACPA IgG (Gal +5%; SA +0.5%) compared to total IgG (Gal +11%; SA +2.5%; Figure 1), but – for total IgG – not different between ACPA+ and ACPA- patients. No association of the change in DAS28-CRP with the change in ACPA IgG or total IgG galactosylation was observed for ACPA+ patients, whereas a strong association of total IgG galactosylation was observed for ACPA- patients. Conclusions During pregnancy the increase in galactosylation of ACPA IgG was less pronounced than that of total IgG, whereas the increase in the galactosylation of total IgG was not different between ACPA+ and ACPA- patients. Since it is known that changes in IgG galactosylation are associated with improvement of RA during pregnancy and since ACPA is thought to be of pathogenic significance in RA, our data might provide an explanation why ACPA+ RA patients are less likely to improve during pregnancy. References Ince-Askan H, Hazes JM, Dolhain RJ. Identifying clinical factors associated with low disease activity and remission of rheumatoid arthritis during pregnancy. Arthritis Care Res (Hoboken) 2016 doi: 10.1002/acr.23143. Bondt A, Selman MHJ, Deelder AM, et al. Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation. Journal of Proteome Research 2013;12(10):4522–31. doi: 10.1021/pr400589m. Scherer HU, van der Woude D, Ioan-Facsinay A, et al. Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis Rheum 2010;62(6):1620–29. doi: 10.1002/art.27414. Acknowledgements This project is funded by the Dutch Arthritis Foundation (NR 10–1-411) and by the European Unions Seventh Framework Program (FP7-Health-F5–2011) under grant agreement no°278535 (HighGlycan). We thank Dr Jan Wouter Drijfhout (LUMC, Leiden) for providing the CCP2 peptide. Disclosure of Interest None declared

OP0177 N-glycosylation sites in the variable domain of b cell receptors specific for citrullinated antigens

Background Recent structural analysis of anti citrullinated protein antibodies (ACPA) in serum and synovial fluid of patients with rheumatoid arthritis (RA) revealed that the vast majority (>90%) of secreted ACPA IgG molecules carry N-glycans in the variable (Fab) domain. This remarkable degree of Fab-glycosylation is absent from ACPA-depleted control IgG and from autoantibodies in other diseases. So far, it is unclear why ACPA carry this feature and which biological effects these glycans mediate in the context of RA. Of note, however, N-glycosylation requires a specific amino acid consensus sequence in the protein backbone, which is very rare in germline-encoded variable region genes. Objectives To study the B cell receptor (BCR) repertoire of ACPA-expressing B cells to determine the frequency, origin and localisation of N-glycosylation sites in ACPA Fab domains. Methods Citrullinated antigen-specific and non citrulline-reactive control B cells were identified in peripheral blood of ACPA-positive RA patients by antigen-specific tetramer staining and isolated by fluorescence activated cell sorting. Cells were either sorted in pools and directly lysed, or sorted as single cells and cultured for two weeks followed by the detection of ACPA-positive culture wells by ELISA. Full-length immunoglobulin (Ig) rearrangements were identified by anchoring reverse transcription of Ig sequences, amplification by nested PCR and either next generation sequencing (NGS, PacBio platform) or, for single cell transcripts, Sanger sequencing (scSeq). Sequence reads were analysed using IMGT V-QUEST tools. Results The mean number of nucleotide mutations in heavy chains (HC) of IgG BCRs derived from ACPA-expressing B cells was high (33 in NGS, 48 in scSeq samples; similarity to germline: 88% in NGS, 84% in scSeq). NGS identified 12 unique IgG clones derived from 4 donors, of which 10 (83%) had at least one N-glycosylation site in the HC or light chain (LC). scSeq identified 86 unique IgG clones derived from 6 donors, of which 68 (79%) had N-glycosylation sites. For 57/86 IgG clones, we could determine the combination of HC and LC sequences. In these, only 7 (12%) clones had no sites, while 19 (33%) had one, 23 (41%) had two, 5 (9%) had three and 3 (4%) clones had four sites. 57 sites were found in the HC and 34 in the LC. All N-glycosylation sites were created by somatic mutations and not encoded in the germline sequence. Several sites were located in antigen-engaging regions. No correlation was found between the number of N-glycosylation sites and the number of somatic mutations. Conclusions We demonstrate that B cell surface-expressed ACPA-IgG molecules carry a remarkably high frequency of N-glycosylation sites in the Fab domain, all of which are generated by somatic mutation. This could indicate that ACPA-expressing B cells acquire a selective survival advantage by introducing N-glycosylation consensus sequences in the Fab domain, a process that is likely to occur under the influence of T cell help and that could facilitate the break of tolerance to citrullinated antigens. Disclosure of Interest None declared