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Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

  • Albert Bondt
    Affiliations
    From the Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;

    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
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  • Yoann Rombouts
    Affiliations
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;

    Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
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  • Maurice H.J. Selman
    Affiliations
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
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  • Paul J. Hensbergen
    Affiliations
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
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  • Karli R. Reiding
    Affiliations
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
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  • Johanna M.W. Hazes
    Affiliations
    From the Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;
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  • Radboud J.E.M. Dolhain
    Affiliations
    From the Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;
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  • Manfred Wuhrer
    Correspondence
    To whom correspondence should be addressed: Dr. Manfred Wuhrer, PO Box 9600, 2300 RC Leiden, Tel.: 31-0-71-52-66396, [email protected]umc.nl
    Affiliations
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;

    Division of BioAnalytical Chemistry, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands;

    Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands
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  • Author Footnotes
    * This project is funded by the Dutch Arthritis Foundation (NR 10–1-411) and by the European Union's Seventh Framework Program (FP7-Health-F5–2011) under Grant No. 278535 (HighGlycan). Maurice H. J. Selman thanks Hoffmann la Roche for financial support.
    This article contains supplemental material.
Open AccessPublished:July 08, 2014DOI:https://doi.org/10.1074/mcp.M114.039537
      The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.
      Immunoglobulins are key players of the human immune system. Immunoglobulin G (IgG)
      The abbreviations used are:
      IgG
      immunoglobulin G
      ACN
      acetonitrile
      ConA
      concanavalin A
      Fab
      antigen binding fragment
      Fc
      crystallizable fragment
      GlcNAc
      N-acetylglucosamine
      HILIC
      hydrophilic interaction liquid chromatography
      SPE
      solid phase extraction.
      1The abbreviations used are:IgG
      immunoglobulin G
      ACN
      acetonitrile
      ConA
      concanavalin A
      Fab
      antigen binding fragment
      Fc
      crystallizable fragment
      GlcNAc
      N-acetylglucosamine
      HILIC
      hydrophilic interaction liquid chromatography
      SPE
      solid phase extraction.
      is the most abundant representative of this group, with serum concentrations of ∼10 mg/ml (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). It consists of two heavy chains (γ-chains) made up of three constant regions (CH1, CH2, and CH3) and one variable region (VH). Attached to each heavy chain is a light chain (λ or κ). Based on chemical and biological properties, different regions can be distinguished in the IgG molecule: two antigen binding fragments (obtained as F(ab′)2 by IdeS treatment; herein referred to as Fab) and a crystallizable fragment (Fc). The structure of IgG is schematically presented in Fig. 1.
      Figure thumbnail gr1
      Fig. 1Schematic representation of IgG with the heavy γ chains (dark blue), light chains (lighter blue), and N-glycans. In the top right-hand corner of the Fc and Fab areas, the percentages of galactosylation, sialylation, bisection, and fucosylation are depicted. The inset represents the stable heptasaccharide core with possible extensions.
      IgGs are glycoproteins, and N-glycans are present at Asn297 of the CH2 domain. These glycans consist of a constant heptasaccharide core that is often modified by a core fucose and is in part decorated with bisecting N-acetylglucosamine (GlcNAc), galactose(s), and sialic acid(s) (Fig. 1) (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ). The Fc glycans have been extensively studied, and glycosylation changes have been found to be associated with disease (e.g. rheumatoid arthritis) (
      • Parekh R.B.
      • Dwek R.A.
      • Sutton B.J.
      • Fernandes D.L.
      • Leung A.
      • Stanworth D.
      • Rademacher T.W.
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      • Taniguchi T.
      • Matsuta K.
      • Takeuchi F.
      • Nagano Y.
      • Miyamoto T.
      • Kobata A.
      Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
      ,
      • Bondt A.
      • Selman M.H.J.
      • Deelder A.M.
      • Hazes J.M.W.
      • Willemsen S.P.
      • Wuhrer M.
      • Dolhain R.J.E.M.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      ) and aging (
      • Parekh R.
      • Roitt I.
      • Isenberg D.
      • Dwek R.
      • Rademacher T.
      Age-related galactosylation of the N-linked oligosaccharides of human serum IgG.
      ,
      • Ruhaak L.R.
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      • Beekman M.
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      • Hokke C.H.
      • Westendorp R.G.J.
      • Wuhrer M.
      • Houwing-Duistermaat J.J.
      • Slagboom P.E.
      • Deelder A.M.
      Decreased levels of bisecting GlcNAc glycoforms of IgG are associated with human longevity.
      ,
      • Yamada E.
      • Tsukamoto Y.
      • Sasaki R.
      • Yagyu K.
      • Takahashi N.
      Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum.
      ). Several immune regulatory properties have been demonstrated for IgG Fc glycans (
      • Shields R.L.
      • Lai J.
      • Keck R.
      • O'Connell L.Y.
      • Hong K.
      • Meng Y.G.
      • Weikert S.H.A.
      • Presta L.G.
      Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity.
      ,
      • Ferrara C.
      • Grau S.
      • Jäger C.
      • Sondermann P.
      • Brünker P.
      • Waldhauer I.
      • Hennig M.
      • Ruf A.
      • Rufer A.C.
      • Stihle M.
      • Umaña P.
      • Benz J.
      Unique carbohydrate–carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose.
      ,
      • Malhotra R.
      • Wormald M.R.
      • Rudd P.M.
      • Fischer P.B.
      • Dwek R.A.
      • Sim R.B.
      Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.
      ,
      • Rademacher T.W.
      • Williams P.
      • Dwek R.A.
      Agalactosyl glycoforms of IgG autoantibodies are pathogenic.
      ,
      • Schwab I.
      • Seeling M.
      • Biburger M.
      • Aschermann S.
      • Nitschke L.
      • Nimmerjahn F.
      B cells and CD22 are dispensable for the immediate antiinflammatory activity of intravenous immunoglobulins in vivo.
      ,
      • Nesspor T.C.
      • Raju T.S.
      • Chin C.N.
      • Vafa O.
      • Brezski R.J.
      Avidity confers FcgammaR binding and immune effector function to aglycosylated immunoglobulin G1.
      ,
      • Jung S.T.
      • Reddy S.T.
      • Kang T.H.
      • Borrok M.J.
      • Sandlie I.
      • Tucker P.W.
      • Georgiou G.
      Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.
      ). For example, Fc-linked glycans influence the IgG effector function by altering the three-dimensional structure of the protein, and thereby the binding to Fcγ-receptors (
      • Nesspor T.C.
      • Raju T.S.
      • Chin C.N.
      • Vafa O.
      • Brezski R.J.
      Avidity confers FcgammaR binding and immune effector function to aglycosylated immunoglobulin G1.
      ,
      • Jung S.T.
      • Reddy S.T.
      • Kang T.H.
      • Borrok M.J.
      • Sandlie I.
      • Tucker P.W.
      • Georgiou G.
      Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.
      ). Additionally, glycan–glycan interactions occur between IgG and Fcγ-receptor-IIIa (
      • Ferrara C.
      • Grau S.
      • Jäger C.
      • Sondermann P.
      • Brünker P.
      • Waldhauer I.
      • Hennig M.
      • Ruf A.
      • Rufer A.C.
      • Stihle M.
      • Umaña P.
      • Benz J.
      Unique carbohydrate–carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose.
      ), with the presence of a core fucose decreasing this affinity by ∼2 orders of magnitude (
      • Shields R.L.
      • Lai J.
      • Keck R.
      • O'Connell L.Y.
      • Hong K.
      • Meng Y.G.
      • Weikert S.H.A.
      • Presta L.G.
      Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity.
      ).
      The Fab portion consists of the heavy chain CH1 and VH regions combined with a light chain and exhibits the antigen binding sites formed by the variable and hypervariable regions of those two chains. N-glycans are known to occur on 15% to 25% of the IgG Fab portions (
      • Arnold J.N.
      • Wormald M.R.
      • Sim R.B.
      • Rudd P.M.
      • Dwek R.A.
      The impact of glycosylation on the biological function and structure of human immunoglobulins.
      ,
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Stadlmann J.
      • Pabst M.
      • Altmann F.
      Analytical and functional aspects of antibody sialylation.
      ). The Fab N-glycans can be involved in immunomodulation, because they influence the affinity and avidity of antibodies for antigens (
      • Xu P.-C.
      • Gou S.-J.
      • Yang X.-W.
      • Cui Z.
      • Jia X.-Y.
      • Chen M.
      • Zhao M.-H.
      Influence of variable domain glycosylation on anti-neutrophil cytoplasmic autoantibodies and anti-glomerular basement membrane autoantibodies.
      ,
      • Coloma M.J.
      • Trinh R.K.
      • Martinez A.R.
      • Morrison S.L.
      Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1→6) dextran antibody.
      ,
      • Man Sung C.
      • Scheinberg D.A.
      • Avdalovic N.M.
      • McGraw K.
      • Vasquez M.
      • Caron P.C.
      • Queen C.
      Genetically engineered deglycosylation of the variable domain increases the affinity of an anti-CD33 monoclonal antibody.
      ,
      • Wright A.
      • Tao M.H.
      • Kabat E.A.
      • Morrison S.L.
      Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.
      ), as well as antibody half-life (
      • Coloma M.J.
      • Trinh R.K.
      • Martinez A.R.
      • Morrison S.L.
      Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1→6) dextran antibody.
      ,
      • Huang L.
      • Biolsi S.
      • Bales K.R.
      • Kuchibhotla U.
      Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
      ). The glycans of the Fab have been described as biantennary complex-type structures that are, in contrast to Fc glycans, highly sialylated (
      • Youings A.
      • Chang S.C.
      • Dwek R.A.
      • Scragg I.G.
      Site-specific glycosylation of human immunoglobulin G is altered in four rheumatoid arthritis patients.
      ,
      • Mimura Y.
      • Ashton P.R.
      • Takahashi N.
      • Harvey D.J.
      • Jefferis R.
      Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
      ,
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). Additionally, high-mannose-type structures have been said to be located on the Fab portion (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ).
      Pregnancy is known to be associated with overall changes in IgG glycosylation. Indeed, a marked increase of galactosylation and sialylation has been observed in IgG Fc glycosylation during pregnancy (
      • Bondt A.
      • Selman M.H.J.
      • Deelder A.M.
      • Hazes J.M.W.
      • Willemsen S.P.
      • Wuhrer M.
      • Dolhain R.J.E.M.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      ,
      • Selman M.H.J.
      • Derks R.J.E.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.M.
      • van de Geijn F.E.
      • Dolhain R.J.E.M.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ,
      • van de Geijn F.E.
      • Wuhrer M.
      • Selman M.H.
      • Willemsen S.P.
      • de Man Y.A.
      • Deelder A.M.
      • Hazes J.M.
      • Dolhain R.J.
      Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.
      ). In addition, lectin binding studies suggest changes in Fab glycosylation of IgG during pregnancy (
      • Zenclussen A.C.
      • Gentile T.
      • Kortebani G.
      • Mazzolli A.
      • Margni R.
      Asymmetric antibodies and pregnancy.
      ), which may be caused by increased levels of progesterone (
      • Prados M.B.
      • La Blunda J.
      • Szekeres-Bartho J.
      • Caramelo J.
      • Miranda S.
      Progesterone induces a switch in oligosaccharyltransferase isoform expression: consequences on IgG N-glycosylation.
      ). Changes in glycosylation during pregnancy could be one of the mechanisms that contribute to acceptance of the fetal allograft by the maternal immune system (
      • Zenclussen A.C.
      • Gentile T.
      • Kortebani G.
      • Mazzolli A.
      • Margni R.
      Asymmetric antibodies and pregnancy.
      ).
      Our knowledge on the Fab glycosylation of IgGs from peripheral blood is scarce, which is in part due to difficulty detecting the glycans in a Fab-region-specific manner. Because of the polyclonal nature of serum IgG, one may expect Fab glycans to be attached to a large variety of sequence motifs arising from somatic rearrangements and mutations (
      • Tonegawa S.
      Somatic generation of antibody diversity.
      ), making the analysis of Fab glycopeptides from polyclonal serum IgG very demanding, if feasible at all. Therefore, study of the Fab glycosylation of polyclonal serum IgG has mainly been pursued at the level of released glycans (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). Difficulties lie in the purification of IgG and the separation of Fc and Fab glycosylation, which is essential for the assignment of the glycans to either part of the IgG molecule.
      Here we present a high-throughput method for studying Fab glycosylation at the level of released glycans obtained from serum-derived polyclonal IgG. Using state-of-the-art affinity capturing beads and enzymes, we were able to obtain Fab and Fc separately, which, after glycan release, resulted in Fc- and Fab-specific glycan pools. The released glycans were subjected to a novel derivatization protocol resulting in linkage-specific modification of sialic acids, followed by HILIC sample purification and MALDI-TOF-MS. Finally, because marked changes in glycosylation during pregnancy have been described, the technique was applied to consecutive serum samples from a cohort of pregnant women. This approach was chosen to determine the usefulness of this technique in a clinical setting. The method proved to be able to demonstrate pregnancy-related changes in glycosylation of the Fab portion, in addition to the already known changes in Fc glycosylation (
      • Bondt A.
      • Selman M.H.J.
      • Deelder A.M.
      • Hazes J.M.W.
      • Willemsen S.P.
      • Wuhrer M.
      • Dolhain R.J.E.M.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      ,
      • Selman M.H.J.
      • Derks R.J.E.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.M.
      • van de Geijn F.E.
      • Dolhain R.J.E.M.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ,
      • van de Geijn F.E.
      • Wuhrer M.
      • Selman M.H.
      • Willemsen S.P.
      • de Man Y.A.
      • Deelder A.M.
      • Hazes J.M.
      • Dolhain R.J.
      Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.
      ).

      DISCUSSION

      Here we present a high-throughput profiling method for analyzing the glycosylation of both the constant and the variable region of polyclonal serum immunoglobulin G in a straightforward workflow. We applied the method to compare Fab and Fc glycosylation profiles of young women and to study pregnancy-associated changes in IgG Fab and Fc glycosylation in the same group. The analysis of Fc released glycans using the method described in this manuscript yielded results that were highly comparable to those previously obtained for the same sample set via LC-MS (
      • Bondt A.
      • Selman M.H.J.
      • Deelder A.M.
      • Hazes J.M.W.
      • Willemsen S.P.
      • Wuhrer M.
      • Dolhain R.J.E.M.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      ,
      • Selman M.H.J.
      • Derks R.J.E.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.M.
      • van de Geijn F.E.
      • Dolhain R.J.E.M.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ). In addition, this approach for the first time provided high-quality Fab glycosylation data for a clinical cohort.
      Glycosylation analysis of the Fab domain of IgG has been performed before, but mainly on monoclonal antibodies (
      • Huang L.
      • Biolsi S.
      • Bales K.R.
      • Kuchibhotla U.
      Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
      ,
      • Mimura Y.
      • Ashton P.R.
      • Takahashi N.
      • Harvey D.J.
      • Jefferis R.
      Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
      ,
      • Nallet S.
      • Fornelli L.
      • Schmitt S.
      • Parra J.
      • Baldi L.
      • Tsybin Y.O.
      • Wurm F.M.
      Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells.
      ,
      • Stadlmann J.
      • Pabst M.
      • Kolarich D.
      • Kunert R.
      • Altmann F.
      Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.
      ,
      • Chevreux G.
      • Tilly N.
      • Bihoreau N.
      Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.
      ,
      • Qian J.
      • Liu T.
      • Yang L.
      • Daus A.
      • Crowley R.
      • Zhou Q.
      Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
      ). To our knowledge only a few papers describe analysis of the Fab glycosylation of polyclonal IgG derived from peripheral blood, and only very limited numbers of sera were studied (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ,
      • Youings A.
      • Chang S.C.
      • Dwek R.A.
      • Scragg I.G.
      Site-specific glycosylation of human immunoglobulin G is altered in four rheumatoid arthritis patients.
      ,
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ), leaving the nature of human polyclonal IgG Fab glycans and their biological variation largely obscure. Additionally, Sambucus nigra agglutinin and concanavalin A (ConA) assays provided inconclusive insights into the molecular nature of human Fab glycosylation (
      • Xu P.-C.
      • Gou S.-J.
      • Yang X.-W.
      • Cui Z.
      • Jia X.-Y.
      • Chen M.
      • Zhao M.-H.
      Influence of variable domain glycosylation on anti-neutrophil cytoplasmic autoantibodies and anti-glomerular basement membrane autoantibodies.
      ,
      • Zenclussen A.C.
      • Gentile T.
      • Kortebani G.
      • Mazzolli A.
      • Margni R.
      Asymmetric antibodies and pregnancy.
      ,
      • Kelemen K.
      • Bognar I.
      • Paal M.
      • Szekeres-Bartho J.
      A progesterone-induced protein increases the synthesis of asymmetric antibodies.
      ,
      • Barrientos G.
      • Fuchs D.
      • Schröcksnadel K.
      • Ruecke M.
      • Garcia M.G.
      • Klapp B.F.
      • Raghupathy R.
      • Miranda S.
      • Arck P.C.
      • Blois S.M.
      Low levels of serum asymmetric antibodies as a marker of threatened pregnancy.
      ,
      • Stadlmann J.
      • Weber A.
      • Pabst M.
      • Anderle H.
      • Kunert R.J.
      • Ehrlich H.
      • Peter Schwarz H.
      • Altmann F.
      A close look at human IgG sialylation and subclass distribution after lectin fractionation.
      ,
      • Taniguchi T.
      • Mizuochi T.
      • Beale M.
      • Dwek R.A.
      • Rademacher T.W.
      • Kobata A.
      Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment.
      ). In contrast to these lectin-based assays, the method described in this paper uses mass spectrometric detection of IgG-derived N-glycans and, in combination with an optimized method for the preparation of Fc and Fab portions, provides detailed information on relative abundances, compositions, and major structural features of IgG Fab and Fc N-glycans. Obviously, the mass spectrometric approach for the detection of released glycans is relative by nature, and no absolute quantitation results can be obtained. Furthermore, due to the normalization on total intensity, relative abundances are reported throughout.
      We used an innovative on-bead digestion preventing the need for additional purification procedures to obtain separate Fc and Fab samples. The IdeS enzyme has been used before for the generation of Fc and Fab portions and their glycosylation analysis, but mainly on monoclonal antibodies (
      • Chevreux G.
      • Tilly N.
      • Bihoreau N.
      Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.
      ,
      • Janin-Bussat M.-C.
      • Tonini L.
      • Huillet C.
      • Colas O.
      • Klinguer-Hamour C.
      • Corvaïa N.
      • Beck A.
      Cetuximab Fab and Fc N-glycan fast characterization using IdeS digestion and liquid chromatography coupled to electrospray ionization mass spectrometry.
      ,
      • Ayoub D.
      • Jabs W.
      • Resemann A.
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      • Evans C.
      • Main L.
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      • Wagner-Rousset E.
      • Suckau D.
      • Beck A.
      Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.
      ). IdeS is known to cleave only IgG at a specific cleavage site (
      • von Pawel-Rammingen U.
      • Johansson B.P.
      • Bjorck L.
      IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
      ), leaving both Fab and Fc intact. Alternative enzymes come with some disadvantages, like a varying digestion site (
      • Bennett K.L.
      • Smith S.V.
      • Truscott R.J.W.
      • Sheil M.M.
      Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry.
      ) or multiple digestion sites in the Fc portion (
      • Holland M.
      • Yagi H.
      • Takahashi N.
      • Kato K.
      • Savage C.O.
      • Goodall D.M.
      • Jefferis R.
      Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.
      ).
      The ethyl esterification of sialic acids used in this study allowed for sensitive detection of glycans using MALDI-TOF-MS in reflectron positive mode (
      • Selman M.H.
      • Hemayatkar M.
      • Deelder A.M.
      • Wuhrer M.
      Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides.
      ). This resulted in high-resolution spectra, without the loss of sialic acids that is common in reflectron positive MALDI measurements (
      • Powell A.K.
      • Harvey D.J.
      Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry.
      ). We registered levels of Fc sialylation similar to those determined via LC-MS (
      • Bondt A.
      • Selman M.H.J.
      • Deelder A.M.
      • Hazes J.M.W.
      • Willemsen S.P.
      • Wuhrer M.
      • Dolhain R.J.E.M.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      ,
      • Selman M.H.J.
      • Derks R.J.E.
      • Bondt A.
      • Palmblad M.
      • Schoenmaker B.
      • Koeleman C.A.M.
      • van de Geijn F.E.
      • Dolhain R.J.E.M.
      • Deelder A.M.
      • Wuhrer M.
      Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.
      ); without stabilization, sialic acids exhibit low relative abundances in MALDI-TOF-MS experiments (
      • van de Geijn F.E.
      • Wuhrer M.
      • Selman M.H.
      • Willemsen S.P.
      • de Man Y.A.
      • Deelder A.M.
      • Hazes J.M.
      • Dolhain R.J.
      Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.
      ). In addition, sialic acid ethyl esterification results in linkage-specific mass differences (
      • Reiding K.R.
      • Blank D.
      • Kuijper D.M.
      • Deelder A.M.
      • Wuhrer M.
      High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.
      ). This allowed the facile differentiation of α2,3- and α2,6-linked sialic acids in a high-throughput fashion.
      Analysis of our data confirmed previous reports of the presence of high-mannose structures on the Fab portion of IgG (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). The data indicate higher levels of these glycans than observed by Anumula (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). Additionally, we observed drastically lower levels of H3N4F1 and H4N4F1 structures on Fab than reported previously. As these glycans may be largely regarded as Fc markers, this might indicate greater purity of the Fab preparation in our study than in the previous one (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ). One possible explanation is that the sequential release of glycans by different enzymes features a less stringent specificity for Fab and Fc than indicated (
      • Anumula K.R.
      Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.
      ).
      We analyzed 174 serum samples of 29 individuals covering six time points during and after pregnancy. In this way we were able to observe several changes over time (Fig. 4). Biological variation seemed greater on the Fab portion than on the Fc, as reflected by increased standard error that could not be attributed to technical variation. By applying this method, we demonstrated various changes in Fab glycosylation with pregnancy.
      The calculated glycosylation traits show different behaviors for Fc and Fab. For example, although different levels were observed for Fc galactosylation during pregnancy and 6 weeks after pregnancy, this did not occur for Fab galactosylation. Similarly, although to a lesser extent, Fab fucosylation did change after delivery, whereas Fc fucosylation remained at very similar levels throughout. Furthermore, for monosialylation, opposite changes were observed. Monosialylated structures were increased on the Fc portion during pregnancy and decreased after delivery. In contrast, Fab monosialylation was decreased during pregnancy relative to the time points after delivery, which might reflect increased turnover of Fab mono- into disialylated species. However, for both Fab and Fc glycosylation, the level of bisection decreased during pregnancy, and total sialylation increased.
      In contrast to the Fc glycosylation, limited information is available with regard to Fab glycosylation function. In fact, most of the data have been obtained recently. Initially it was shown that the anti-inflammatory properties of intravenous immunoglobulins were due to sialylation of the Fc portion (
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      ). However, an increasing body of evidence suggests that the Fab portion is involved (
      • Wiedeman A.E.
      • Santer D.M.
      • Yan W.
      • Miescher S.
      • Käsermann F.
      • Elkon K.B.
      Contrasting mechanisms of interferon-α inhibition by intravenous immunoglobulin after induction by immune complexes versus Toll-like receptor agonists.
      ,
      • Trinath J.
      • Hegde P.
      • Sharma M.
      • Maddur M.S.
      • Rabin M.
      • Vallat J.-M.
      • Magy L.
      • Balaji K.N.
      • Kaveri S.V.
      • Bayry J.
      Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2–dependent prostaglandin E2 in human dendritic cells.
      ). Lectins such as Siglecs (sialic acid–binding immunoglobulin-type lectins) and DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), recognizing one of the sugar moieties on either one of the IgG portions, may (
      • Trinath J.
      • Hegde P.
      • Sharma M.
      • Maddur M.S.
      • Rabin M.
      • Vallat J.-M.
      • Magy L.
      • Balaji K.N.
      • Kaveri S.V.
      • Bayry J.
      Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2–dependent prostaglandin E2 in human dendritic cells.
      ,
      • Séité J.-F.
      • Guerrier T.
      • Cornec D.
      • Jamin C.
      • Youinou P.
      • Hillion S.
      TLR9 responses of B cells are repressed by intravenous immunoglobulin through the recruitment of phosphatase.
      ,
      • Anthony R.M.
      • Wermeling F.
      • Karlsson M.C.
      • Ravetch J.V.
      Identification of a receptor required for the anti-inflammatory activity of IVIG.
      ) or may not (
      • Schwab I.
      • Seeling M.
      • Biburger M.
      • Aschermann S.
      • Nitschke L.
      • Nimmerjahn F.
      B cells and CD22 are dispensable for the immediate antiinflammatory activity of intravenous immunoglobulins in vivo.
      ,
      • Wiedeman A.E.
      • Santer D.M.
      • Yan W.
      • Miescher S.
      • Käsermann F.
      • Elkon K.B.
      Contrasting mechanisms of interferon-α inhibition by intravenous immunoglobulin after induction by immune complexes versus Toll-like receptor agonists.
      ) be involved in these processes. In addition to the influence of Fab glycans on cellular receptors, the glycans may also be involved in modulating antigen binding and antibody half-life (
      • Coloma M.J.
      • Trinh R.K.
      • Martinez A.R.
      • Morrison S.L.
      Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1→6) dextran antibody.
      ,
      • Huang L.
      • Biolsi S.
      • Bales K.R.
      • Kuchibhotla U.
      Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization.
      ).
      Increased ConA reactivity during pregnancy has been described in the literature (
      • Zenclussen A.C.
      • Gentile T.
      • Kortebani G.
      • Mazzolli A.
      • Margni R.
      Asymmetric antibodies and pregnancy.
      ,
      • Kelemen K.
      • Bognar I.
      • Paal M.
      • Szekeres-Bartho J.
      A progesterone-induced protein increases the synthesis of asymmetric antibodies.
      ,
      • Barrientos G.
      • Fuchs D.
      • Schröcksnadel K.
      • Ruecke M.
      • Garcia M.G.
      • Klapp B.F.
      • Raghupathy R.
      • Miranda S.
      • Arck P.C.
      • Blois S.M.
      Low levels of serum asymmetric antibodies as a marker of threatened pregnancy.
      ). This is generally interpreted as an increase in Fab glycosylated (also called asymmetrical) antibodies, which occurs under the influence of progesterone (
      • Kelemen K.
      • Bognar I.
      • Paal M.
      • Szekeres-Bartho J.
      A progesterone-induced protein increases the synthesis of asymmetric antibodies.
      ), among other factors. An increase in ConA reactivity in vitro has been confirmed by some (
      • Prados M.B.
      • La Blunda J.
      • Szekeres-Bartho J.
      • Caramelo J.
      • Miranda S.
      Progesterone induces a switch in oligosaccharyltransferase isoform expression: consequences on IgG N-glycosylation.
      ); others observed similar increases in ConA reactivity with low progesterone concentrations, whereas high concentrations resulted in a decrease (
      • Canellada A.
      • Blois S.
      • Gentile T.
      • Margni Idehu R.A.
      In vitro modulation of protective antibody responses by estrogen, progesterone and interleukin-6.
      ). The increase in ConA reactivity is believed to reflect increased levels of high-mannose glycans only present on the Fab portion, excluding interference from the Fc portion (Asn297), which is known to bear hardly any oligomannosidic glycans (
      • Parekh R.B.
      • Dwek R.A.
      • Sutton B.J.
      • Fernandes D.L.
      • Leung A.
      • Stanworth D.
      • Rademacher T.W.
      • Mizuochi T.
      • Taniguchi T.
      • Matsuta K.
      • Takeuchi F.
      • Nagano Y.
      • Miyamoto T.
      • Kobata A.
      Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.
      ,
      • Yamada E.
      • Tsukamoto Y.
      • Sasaki R.
      • Yagyu K.
      • Takahashi N.
      Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum.
      ,
      • Stadlmann J.
      • Pabst M.
      • Kolarich D.
      • Kunert R.
      • Altmann F.
      Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.
      ,
      • Neue K.
      • Mormann M.
      • Peter-Katalinic J.
      • Pohlentz G.
      Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides.
      ). However, ConA has also been reported to have affinity for non-bisected glycan structures (
      • Taniguchi T.
      • Mizuochi T.
      • Beale M.
      • Dwek R.A.
      • Rademacher T.W.
      • Kobata A.
      Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment.
      ), thereby exhibiting increased binding with decreased levels of bisecting GlcNAc with pregnancy on both Fc and Fab. One may speculate that the glycosylation changes observed in our study were likewise caused by hormonal changes, but more studies are needed to reveal how the cellular glycosylation machinery is regulated by hormones.
      In conclusion, we developed a high-throughput method enabling the separate detection of glycans derived from human polyclonal IgG, Fab, and Fc. When this technique was applied to consecutive serum samples from a cohort of pregnant women, it revealed clear differences between Fc and Fab glycosylation of immunoglobulin G. In addition, this technique proved to be suitable for demonstrating pregnancy-associated changes in glycosylation not only for Fc, but also for Fab.

      Acknowledgments

      We thank Irina Dragan for helping with the sample purity controls, and Hae-Won Uh for advice on the statistical approach.

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