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Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding

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Abstract

The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-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-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of 125I-LDN–BSA to various clinical H. pylori isolates 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 β1,4-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 of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a 125I-labelled LDN–BSA neoglycoconjugate to clinical H. pylori isolates was identified.

Graphical abstract: Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding

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Supplementary files

Article information


Submitted
30 Nov 2019
Accepted
20 Mar 2020
First published
30 Mar 2020

Mol. Omics, 2020, Advance Article
Article type
Research Article

Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding

Y. H. Mthembu, C. Jin, M. Padra, J. Liu, J. O. Edlund, H. Ma, J. Padra, S. Oscarson, T. Borén, N. G. Karlsson, S. K. Lindén and J. Holgersson, Mol. Omics, 2020, Advance Article , DOI: 10.1039/C9MO00175A

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