title: Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3).
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.
authors.affiliation: From the ‡Center for Proteomics and Metabolomics.
authors.forename: Rosina
authors.initials: R
authors.lastname: Plomp
authors.affiliation: ¶Department of Experimental Immunohematology.
authors.forename: Gillian
authors.initials: G
authors.lastname: Dekkers
authors.affiliation: From the ‡Center for Proteomics and Metabolomics, §Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands;
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rombouts
authors.affiliation: ¶Department of Experimental Immunohematology.
authors.forename: Remco
authors.initials: R
authors.lastname: Visser
authors.affiliation: From the ‡Center for Proteomics and Metabolomics.
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.affiliation: From the ‡Center for Proteomics and Metabolomics.
authors.forename: Guinevere S M
authors.initials: GS
authors.lastname: Kammeijer
authors.affiliation: From the ‡Center for Proteomics and Metabolomics.
authors.forename: Bas C
authors.initials: BC
authors.lastname: Jansen
authors.affiliation: ¶¶Department of Immunopathology, Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.affiliation: From the ‡Center for Proteomics and Metabolomics.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.affiliation: ¶Department of Experimental Immunohematology.
authors.forename: Gestur
authors.initials: G
authors.lastname: Vidarsson
authors.affiliation: From the ‡Center for Proteomics and Metabolomics, **Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands m.wuhrer@lumc.nl.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000032:analysis*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins; D013912:Threonine; D014357:Trypsin