title: Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.
abstract: Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Faculty of Medicine and Dentistry, Department of Immunology, Palacky University in Olomouc, Olomouc, Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Raska
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Faculty of Medicine and Dentistry, Department of Immunology, Palacky University in Olomouc, Olomouc, Czech Republic.
authors.forename: Milada
authors.initials: M
authors.lastname: Stuchlova Horynova
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Stacy D
authors.initials: SD
authors.lastname: Hall
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Knud
authors.initials: K
authors.lastname: Poulsen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Mogens
authors.initials: M
authors.lastname: Kilian
authors.affiliation: Fujita Health University School of Health Sciences, Toyoake, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Hiki
authors.affiliation: Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yuzawa
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Zina
authors.initials: Z
authors.lastname: Moldoveanu
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Julian
authors.affiliation: UAB Biomedical FT-ICR MS Laboratory, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Renfrow
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Jan
authors.initials: J
authors.lastname: Novak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D005922:Glomerulonephritis, IGA / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism; D057809:HEK293 Cells; D006372:Helix, Snails / Q000378:metabolism; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis / Q000378:metabolism*
chemical_list: D007070:Immunoglobulin A; D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D016695:Glycosyltransferases