Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)

Rosina Plomp, Gillian Dekkers, Yoann Rombouts, Remco Visser, Carolien A M Koeleman, Guinevere S M Kammeijer, Bas C Jansen, Theo Rispens, Paul J Hensbergen, Gestur Vidarsson, Manfred Wuhrer, Rosina Plomp, Gillian Dekkers, Yoann Rombouts, Remco Visser, Carolien A M Koeleman, Guinevere S M Kammeijer, Bas C Jansen, Theo Rispens, Paul J Hensbergen, Gestur Vidarsson, Manfred Wuhrer

Abstract

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

Fig. 1.
Fig. 1.
NanoLC-ESI-IT-CID spectra showing fragmentation of various trypsin-generated recombinant IgG3m(g) O-glycopeptides. Non-, mono- and disialylated core 1 type O-glycans (A–C) were seen attached to the peptide SCDTPPPCPR, as well as a disialylated O-glycan with an N-acetyllactosamine (N-acetylglucosamine + galactose) (D). These structures are partially based on literature since it is difficult to distinguish between different types of hexoses and N-acetylhexosamines with mass spectrometry. The triply charged peak at m/z 895.48 in panel D has a higher mass than the precursor mass, and thus likely originates from a contaminant. The MS1 precursor peak is shown for each fragmentation spectrum. Pep = peptide; yellow square = N-acetylgalactosamine, yellow circle = galactose; blue square = N-acetylglucosamine; purple diamond = N-acetylneuraminic acid.
Fig. 2.
Fig. 2.
A schematic overview of IgG3, which consists of two heavy chains (shown in dark gray, one variable and three conserved domains) and two light chains (light gray, one variable and one conserved domain). Black bars represent interchain disulfide bonds. An N-glycosylation site is present in domain CH2. Each IgG3 heavy chain contains three hinge repeat sequences with two or three partially occupied O-glycosylation sites. The peptide sequences of the major trypsin- and proteinase K-generated O-glycopeptides are shown.
Fig. 3.
Fig. 3.
(A) NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation of a sialidase- and galactosidase-treated tryptic glycopeptide of recombinant IgG3m(s). Peptide backbone fragmentation (c- and z-ions) and loss of the N-acetylhexosamine (HexNAc) or cysteine side chain (C) were observed. (B–C) CE-ESI-beam-type CID analysis was done on sialidase- and galactosidase-treated tryptic peptides of IgG3 derived from pooled plasma, and fragmentation spectra are shown for (B) the unoccupied tryptic hinge peptide SCDTPPPCPR and (C) the O-glycosylated version of the same peptide with a single N-acetylgalactosamine attached. The b- and y-ions are annotated. The MS1 precursor peak is shown on the right. The doubly charged peak at m/z 694.327 in panel C is likely a coeluting compound that appears in the spectrum because it was present within the precursor m/z window, and the peak at m/z 694.321 is the same compound after loss of water. The cysteine residues have been underlined to denote carbomidomethylation.
Fig. 4.
Fig. 4.
(A) Relative quantification of IgG3 O-glycosylation based on nanoLC-ESI-IT-MS analysis of tryptic glycopeptides from various IgG3 samples (IgG3 derived from pooled plasma, two monoclonal IgG3 allotypes and IgG3 purified from six donors (D1–6)). The signal intensities were normalized on the sum of all hinge-derived O-glycopeptides. The relative abundance and technical variation are based on LC-MS analyses of four distinct tryptic digests, each of them measured twice. The values given for glycopeptide NHS are expected to be significantly lower than the actual values because the triply charged compound overlapped with the doubly charged hinge peptide with a putative acetylation modification in all samples, leaving only the doubly charged peak for relative quantification. (B) An estimate of the total percentage of O-glycosylation was derived from relative quantification of tryptic (glyco)peptides that had been treated with exoglycosidases, trimming all O-glycans down to a single HexNAc. The averages and standard deviation are based on two LC-MS analyses of the same sample. (C) The number of N-acetylneuraminic acids per O-glycan was calculated from the same O-glycopeptide data listed under (A). A comprehensive list of values is available in supplemental Table S5.

References

    1. Stoop J. W., Zegers B. J., Sander P. C., Ballieux R. E. (1969) Serum immunoglobulin levels in healthy children and adults. Clin. Experiment. Immunol. 4, 101–112
    1. Morell A., Terry W. D., Waldmann T. A. (1970) Metabolic properties of IgG subclasses in man. J. Clin. Invest. 49, 673–680
    1. Vidarsson G., Dekkers G., Rispens T. (2014) IgG subclasses and allotypes: From structure to effector functions. Frontiers Immunol. 5, 520
    1. Roux K. H., Strelets L., Michaelsen T. E. (1997) Flexibility of human IgG subclasses. J. Immunol. 159, 3372–3382
    1. Dangl J. L., Wensel T. G., Morrison S. L., Stryer L., Herzenberg L. A., Oi V. T. (1988) Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies. EMBO J. 7, 1989–1994
    1. Scharf O., Golding H., King L. R., Eller N., Frazier D., Golding B., Scott D. E. (2001) Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1. J. Virol. 75, 6558–6565
    1. Cavacini L. A., Emes C. L., Power J., Desharnais F. D., Duval M., Montefiori D., Posner M. R. (1995) Influence of heavy chain constant regions on antigen binding and HIV-1 neutralization by a human monoclonal antibody. J. Immunol. 155, 3638–3644
    1. Redpath S., Michaelsen T., Sandlie I., Clark M. R. (1998) Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52. Immunology 93, 595–600
    1. Norderhaug L., Brekke O. H., Bremnes B., Sandin R., Aase A., Michaelsen T. E., Sandlie I. (1991) Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge. European J. Immunol. 21, 2379–2384
    1. Natsume A., In M., Takamura H., Nakagawa T., Shimizu Y., Kitajima K., Wakitani M., Ohta S., Satoh M., Shitara K., Niwa R. (2008) Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. Cancer Res. 68, 3863–3872
    1. Hogarth P. M., Pietersz G. A. (2012) Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nature Rev. Drug Discovery 11, 311–331
    1. Canfield S. M., Morrison S. L. (1991) The binding-affinity of human-IgG for its high-affinity Fc receptor is determined by multiple amino-acids in the Ch2 domain and is modulated by the hinge region. J. Exper. Med. 173, 1483–1491
    1. Stapleton N. M., Andersen J. T., Stemerding A. M., Bjarnarson S. P., Verheul R. C., Gerritsen J., Zhao Y., Kleijer M., Sandlie I., de Haas M., Jonsdottir I., van der Schoot C. E., Vidarsson G. (2011) Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential. Nature Commun. 2, 599.
    1. Einarsdottir H., Ji Y., Visser R., Mo C., Luo G., Scherjon S., van der Schoot C. E., Vidarsson G. (2014) H435-containing immunoglobulin G3 allotypes are transported efficiently across the human placenta: Implications for alloantibody-mediated diseases of the newborn. Transfusion 54, 665–671
    1. Dard P., Lefranc M. P., Osipova L., Sanchez-Mazas A. (2001) DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations. Eur. J. Human Genetics 9, 765–772
    1. Jefferis R., Lefranc M. P. (2009) Human immunoglobulin allotypes: Possible implications for immunogenicity. mAbs 1, 332–338
    1. Dard P., Huck S., Frippiat J. P., Lefranc G., Langaney A., Lefranc M. P., Sanchez-Mazas A. (1997) The IGHG3 gene shows a structural polymorphism characterized by different hinge lengths: Sequence of a new 2-exon hinge gene. Human Genetics 99, 138–141
    1. Anthony R. M., Nimmerjahn F. (2011) The role of differential IgG glycosylation in the interaction of antibodies with FcγRs in vivo. Curr. Op. Organ Transplant. 16, 7–14
    1. Shields R. L., Lai J., Keck R., O'Connell L. Y., Hong K., Meng Y. G., Weikert S. H., Presta L. G. (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biolog. Chem. 277, 26733–26740
    1. Kapur R., Kustiawan I., Vestrheim A., Koeleman C. A., Visser R., Einarsdottir H. K., Porcelijn L., Jackson D., Kumpel B., Deelder A. M., Blank D., Skogen B., Killie M. K., Michaelsen T. E., de Haas M., Rispens T., van der Schoot C. E., Wuhrer M., Vidarsson G. (2014) A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy. Blood 123, 471–480
    1. Kaneko Y., Nimmerjahn F., Ravetch J. V. (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673
    1. Karsten C. M., Pandey M. K., Figge J., Kilchenstein R., Taylor P. R., Rosas M., McDonald J. U., Orr S. J., Berger M., Petzold D., Blanchard V., Winkler A., Hess C., Reid D. M., Majoul I. V., Strait R. T., Harris N. L., Köhl G., Wex E., Ludwig R., Zillikens D., Nimmerjahn F., Finkelman F. D., Brown G. D., Ehlers M., Köhl J. (2012) Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of Fc[gamma[RIIB and dectin-1. Nature Med. 18, 1401–1406
    1. Kim H., Yamaguchi Y., Masuda K., Matsunaga C., Yamamoto K., Irimura T., Takahashi N., Kato K., Arata Y. (1994) O-glycosylation in hinge region of mouse immunoglobulin G2b. J. Biolog. Chem. 269, 12345–12350
    1. Mattu T. S., Pleass R. J., Willis A. C., Kilian M., Wormald M. R., Lellouch A. C., Rudd P. M., Woof J. M., Dwek R. A. (1998) The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions. J. Biolog. Chem. 273, 2260–2272
    1. Takahashi N., Tetaert D., Debuire B., Lin L. C., Putnam F. W. (1982) Complete amino acid sequence of the delta heavy chain of human immunoglobulin D. Proc. Natl. Acad. Sci. U.S.A. 79, 2850–2854
    1. Takayasu T., Suzuki S., Kametani F., Takahashi N., Shinoda T., Okuyama T., Munekata E. (1982) Amino acid sequence of galactosamine-containing glycopeptides in the hinge region of a human immunoglobulin D. Biochem. Biophys. Res. Commun.105, 1066–1071
    1. Reinholdt J., Tomana M., Mortensen S. B., Kilian M. (1990) Molecular aspects of immunoglobulin A1 degradation by oral streptococci. Infection Immun. 58, 1186–1194
    1. Kruijsen D., Einarsdottir H. K., Schijf M. A., Coenjaerts F. E., van der Schoot E. C., Vidarsson G., van Bleek G. M. (2013) Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice. J. Virol. 87, 7550–7557
    1. Vink T., Oudshoorn-Dickmann M., Roza M., Reitsma J. J., de Jong R. N. (2014) A simple, robust and highly efficient transient expression system for producing antibodies. Methods 65, 5–10
    1. Rispens T., Davies A. M., Ooijevaar-de Heer P., Absalah S., Bende O., Sutton B. J., Vidarsson G., Aalberse R. C. (2014) Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange. J. Biolog. Chem. 289, 6098–6109
    1. Selman M. H., Derks R. J., Bondt A., Palmblad M., Schoenmaker B., Koeleman C. A., van de Geijn F. E., Dolhain R. J., Deelder A. M., Wuhrer M. (2012) Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. J. Proteomics 75, 1318–1329
    1. Tarp M. A., Clausen H. (2008) Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim. Biophys. Acta 1780, 546–563
    1. Domon B., Costello C. E. (1988) A systematic nomenclature for carbohydrate fragmentations in Fab-Ms Ms spectra of glycoconjugates. Glycoconjugate J. 5, 397–409
    1. Edelman G. M., Cunningham B. A., Gall W. E., Gottlieb P. D., Rutishauser U., Waxdal M. J. (1969) The covalent structure of an entire gamma G immunoglobulin molecule. Proc. Natl. Acad. Sci. U.S.A. 63, 78–85
    1. Stavenhagen K., Hinneburg H., Thaysen-Andersen M., Hartmann L., Varón Silva D., Fuchser J., Kaspar S., Rapp E., Seeberger P. H., Kolarich D. (2013) Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J. Mass Spec. 48, 627–639
    1. Baković M. P., Selman M. H., Hoffmann M., Rudan I., Campbell H., Deelder A. M., Lauc G., Wuhrer M. (2013) High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides. J. Proteome Res. 12, 821–831
    1. Croset A., Delafosse L., Gaudry J. P., Arod C., Glez L., Losberger C., Begue D., Krstanovic A., Robert F., Vilbois F., Chevalet L., Antonsson B. (2012) Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells. J. Biotech. 161, 336–348
    1. Chen F. T., Dobashi T. S., Evangelista R. A. (1998) Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis. Glycobiology 8, 1045–1052
    1. Pedroso M. M., Pesquero N. C., Thomaz S. M., Roque-Barreira M. C., Faria R. C., Bueno P. R. (2012) Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring. Glycobiology 22, 326–331
    1. Hortin G. L., Trimpe B. L. (1990) Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose. Anal. Biochem. 188, 271–277
    1. Wilson I. B., Gavel Y., von Heijne G. (1991) Amino acid distributions around O-linked glycosylation sites. Biochem. J. 275, 529–534
    1. Johnson P. M., Michaelsen T. E., Scopes P. M. (1975) Conformation of the hinge region and various fragments of human IgG3. Scandinavian J. Immunol. 4, 113–119
    1. Julenius K., Mølgaard A., Gupta R., Brunak S. (2005) Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15, 153–164
    1. Ahmed A. A., Giddens J., Pincetic A., Lomino J. V., Ravetch J. V., Wang L. X., Bjorkman P. J. (2014) Structural characterization of anti-inflammatory immunoglobulin g fc proteins. J. Molecular Biol. 426, 3166–3179
    1. Brezski R. J., Jordan R. E. (2010) Cleavage of IgGs by proteases associated with invasive diseases: An evasion tactic against host immunity? mAbs 2, 212–220
    1. Brezski R. J., Vafa O., Petrone D., Tam S. H., Powers G., Ryan M. H., Luongo J. L., Oberholtzer A., Knight D. M., Jordan R. E. (2009) Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge. Proc. Natl. Acad. Sci. U.S.A. 106, 17864–17869
    1. Baici A., Knöpfel M., Fehr K., Skvaril F., Böni A. (1980) Kinetics of the different susceptibilities of the four human immunoglobulin G subclasses to proteolysis by human lysosomal elastase. Scandinavian J. Immunol. 12, 41–50
    1. Virella G., Parkhouse R. M. (1971) Papain sensitivity of heavy chain sub-classes in normal human IgG and localizaton of antigenic determinants for the sub-classes. Immunochemistry 8, 243–250
    1. Turner M. W., Bennich H. H., Natvig J. B. (1970) Pepsin digestion of human G-myeloma proteins of different subclasses. I. The characteristic features of pepsin cleavage as a function of time. Clin. Experiment. Immunol. 7, 603–625
    1. Tomana M., Novak J., Julian B. A., Matousovic K., Konecny K., Mestecky J. (1999) Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J. Clin. Invest. 104, 73–81
    1. Lehoux S., Mi R., Aryal R. P., Wang Y., Schjoldager K. T., Clausen H., van Die I., Han Y., Chapman A. B., Cummings R. D., Ju T. (2014) Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin a (IgA) nephropathy and healthy individuals. Mol. Cell. Proteomics 13, 3097–3113
    1. Imai H., Hamai K., Komatsuda A., Ohtani H., Miura A. B. (1997) IgG subclasses in patients with membranoproliferative glomerulonephritis, membranous nephropathy, and lupus nephritis. Kidney Int. 51, 270–276

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