Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins.
The interaction between P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal 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. TheC-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 β1,6GnT-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 Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed.
Stefan
S
Gaunitz
Jining
J
Liu
Anki
A
Nilsson
Niclas
N
Karlsson
Jan
J
Holgersson
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D009077:Mucins; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008024:Ligands; D008562:Membrane Glycoproteins; D009077:Mucins; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; C109963:hemagglutinin, avian influenza A virus; D019158:N-Acetylneuraminic Acid