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Recombinant mucin-type proteins carrying LacdiNAc on differentO-glycan core chains fail to supportH. pyloribinding

Journal article
Authors Yolanda H. Mthembu
Chunsheng Jin
Médea Padra
Jining Liu
J. O. Edlund
H. Y. Ma
János T Padra
S. Oscarson
T. Boren
Niclas G. Karlsson
Sara K. Lindén
Jan Holgersson
Published in Molecular Omics
Volume 16
Issue 3
Pages 243-257
ISSN 1742-206X
Publication year 2020
Published at Institute of Biomedicine
Department of Laboratory Medicine
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 243-257
Language en
Links dx.doi.org/10.1039/c9mo00175a
Keywords helicobacter-pylori infection, chinese-hamster ovary, human gastric, mucin, molecular-cloning, dependent binding, adhesion, expression, baba, cancer, glcnac, Biochemistry & Molecular Biology
Subject categories Biochemistry and Molecular Biology

Abstract

The beta 4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a beta 1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment ofHelicobacter pylorito the gastric mucosaviabinding to the LDN determinant. TheO-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried byO-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positiveH. pyloriJ99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on differentO-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of(125)I-LDN-BSA to various clinicalH. pyloriisolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a beta 1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion ofH. pyloristrains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1O-glycans were detected in a microtiter well-based adhesion assay and no binding of a(125)I-labelled LDN-BSA neoglycoconjugate to clinicalH. pyloriisolates was identified.

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