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Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins

Journal article
Authors Reeja Maria Cherian
S. Gaunitz
A. Nilsson
Jining Liu
Niclas G. Karlsson
Jan Holgersson
Published in Glycobiology
Volume 24
Issue 1
Pages 26-38
ISSN 0959-6658
Publication year 2014
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 26-38
Language en
Links dx.doi.org/10.1093/glycob/cwt086
Keywords blood group P1, mass spectrometry, mucin, Shiga-like toxin, SPR, HEMOLYTIC-UREMIC SYNDROME, PIGEON EGG-WHITE, HELICOBACTER-PYLORI, COLONIZATION, UROPATHOGENIC ESCHERICHIA-COLI, N-GLYCAN STRUCTURES, SHIGELLA-DYSENTERIAE, MAJOR GLYCOPROTEINS, STREPTOCOCCUS-SUIS, CRYSTAL-STRUCTURE, GROUP-A
Subject categories Clinical Medicine

Abstract

The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG(2b) fusion protein, the pigeon alpha 1,4-galactosyltransferase (alpha 4Gal-T) and the core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG(2b) and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG(2b). In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG(2b) and a P-k-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG(2b) and P-k-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon alpha 4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG(2b) bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.

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