Format

Send to

Choose Destination
Mol Cell Proteomics. 2015 May;14(5):1373-84. doi: 10.1074/mcp.M114.047381. Epub 2015 Mar 10.

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

Author information

1
From the ‡Center for Proteomics and Metabolomics.
2
¶Department of Experimental Immunohematology.
3
From the ‡Center for Proteomics and Metabolomics, §Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands;
4
¶¶Department of Immunopathology, Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
5
From the ‡Center for Proteomics and Metabolomics, **Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands m.wuhrer@lumc.nl.

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.

PMID:
25759508
PMCID:
PMC4424406
DOI:
10.1074/mcp.M114.047381
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center