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Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins

Journal article
Authors Stefan Gaunitz
Jining Liu
Anki Nilsson
Niclas G. Karlsson
Jan Holgersson
Published in Glycoconjugate Journal
Volume 31
Issue 2
Pages 145-159
ISSN 0282-0080
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 145-159
Language en
Links dx.doi.org/10.1007/s10719-013-9503-...
Keywords Mucins, Avian influenza, Hemagglutinin, Mass spectrometry, Biacore, A VIRUSES, CHEMOENZYMATIC SYNTHESIS, RECEPTOR-BINDING, MEMBRANE-FUSION, IMMUNE-SYSTEM, HOST-RANGE, ANTIBODIES, GLYCOSYLATION, INHIBITORS, INFECTION
Subject categories Clinical immunology

Abstract

The interaction between P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Sia alpha 2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG(2b) fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 beta 1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG(2b) is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG(2b) carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG(2b) mucin-type fusion protein carrying Sia alpha 2-3Gal as a multivalent inhibitor of influenza virus is discussed.

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