title: N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy.
abstract: BACKGROUND
  Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a β1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing.
  
  
  METHODS
  We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry.
  
  
  RESULTS
  ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide.
  
  
  CONCLUSIONS
  Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.
authors.affiliation: Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Milada
authors.initials: M
authors.lastname: Stuchlova Horynova
authors.affiliation: Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Alena
authors.initials: A
authors.lastname: Vrablikova
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Stewart
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Lydie
authors.initials: L
authors.lastname: Czernekova
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo 113-8421, Japan.
authors.forename: Koshi
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo 113-8421, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Julian
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Renfrow
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Novak
authors.affiliation: Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Milan
authors.initials: M
authors.lastname: Raska
mesh_terms: D001324:Autoantigens / Q000276:immunology*; D002478:Cells, Cultured; D005690:Galactose / Q000172:deficiency*; D005922:Glomerulonephritis, IGA / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism*; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*
chemical_list: D001324:Autoantigens; D007070:Immunoglobulin A; D011994:Recombinant Proteins; D012799:Sialyltransferases; C088391:galactosyl-1-3-N-acetylgalactosaminyl-specific 2,6-sialyltransferase; D019158:N-Acetylneuraminic Acid; D005690:Galactose
keywords: IgA nephropathy; aberrant O-glycosylation; galactose-deficient IgA1; immunoglobulin A1; α2,6 sialyltransferase ST6GalNAc-II