IgA displays site- and subclass-specific glycoform differences despite equal glycoenzyme expression
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Background Glycosylation is an important posttranslational modification of proteins and in most cases indispensable for proper protein function. Like most soluble proteins, IgA, the second most prevalent antibody in human serum, contains several N- and O-glycosylation sites. While for IgG the impact of Fc glycosylation on effector functions and inflammatory potential has been intensively studied, only little is known for IgA. In addition, only glimpses exist regarding the regulation of IgA glycosylation. We have previously shown that IgA1 and IgA2 differ functionally and also show differences in their glycosylation pattern. The more pro-inflammatory IgA2 which is linked to autoimmune diseases displays decreased sialylation, galactosylation, fucosylation and bisection as compared to IgA1. In the present study, we aimed to investigate these differences in glycosylation in detail and to explore the mechanisms underlying them. Methods IgA1 and IgA2 was isolated from serum of 12 healthy donors. Site specific glycosylation was analyzed by mass spectrometry. In addition, human bone marrow plasma cells were investigated using single cell mRNA sequencing, flow cytometry and ELISpot. Results We found that certain glycoforms greatly differ in their abundance between IgA1 and IgA2 while others are equally abundant. Overall, the IgA2 glycans displayed a more immature phenotype with a higher prevalence of oligomannose and fewer fully processed glycans. Of note, these differences can’t be explained by differences in the glycosylation enzyme machinery as mRNA sequencing and flow cytometry analysis showed equal enzyme expression in IgA1 and IgA2 producing plasma cells. ELISpot analysis suggested a slightly increased antibody production rate in IgA2 producing plasma cells which might contribute to its lower glycan processing rates. But this difference was only minor, suggesting that further factors such as steric accessibility determine glycan processing. This is supported by the fact that glycans at different positions on the same IgA chain differ dramatically in fucosylation, sialylation and bisection. Conclusion In summary, our detailed overview of IgA1 and IgA2 glycosylation shows a class, subclass, and site-specific glycosylation fingerprint, most likely due to structural differences of the protein backbones.
Authors 14
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Glycomics and Clinical Proteomics Group, Leiden, Netherlands
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen · University Hospital Schleswig-Holstein
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Department of Internal Medicine I, Subsection Rheumatology and Clinical Immunology, University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Carolien A. M. Koeleman Aachen Center for Proteomics and Metabolomics Glycomics and Clinical Proteomics Group
Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Glycomics and Clinical Proteomics Group, Leiden, Netherlands
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Division of Molecular Immunology, Internal Medicine 3, Nikolaus-Fiebiger Center, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 5-Haematology and Clinical Oncology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
FAU Profile Center Immunomedicine (FAU I-MED), Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany
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Manfred Wuhrer Aachen Center for Proteomics and Metabolomics Glycomics and Clinical Proteomics Group
Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Center for Proteomics and Metabolomics, Glycomics and Clinical Proteomics Group, Leiden, Netherlands
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Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
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Ulrike Steffen corresponding
Friedrich-Alexander-Universität Erlangen-Nürnberg · Universitätsklinikum Erlangen
Affiliation as printed
Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-Universität Erlangen-Nürnberg and Uniklinikum Erlangen, Erlangen, Germany
FAU Profile Center Immunomedicine (FAU I-MED), Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany
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