Milada Stuchlova Horynova1, Alena Vrablikova1, Tyler J Stewart2, Kazuo Takahashi3, Lydie Czernekova1, Koshi Yamada4, Hitoshi Suzuki4, Bruce A Julian5, Matthew B Renfrow6, Jan Novak2, Milan Raska1. 1. 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. 2. Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 3. 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. 4. 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. 5. Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 6. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
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.
BACKGROUND:Galactose-deficientO-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-deficientO-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-deficientIgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant humanST6GalNAc-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-deficientIgA1. As serum levels of galactose-deficientIgA1 with sialylated glycoforms are increased in IgANpatients, our data explain the corresponding part of the biosynthetic pathway.
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