title: Glycosylation Characterization of an Influenza H5N7 Hemagglutinin Series with Engineered Glycosylation Patterns: Implications for Structure-Function Relationships.
abstract: The glycosylation patterns of four recombinant H5 hemagglutinins (HAs) derived from A/Mallard/Denmark/64650/03 (H5N7) have been characterized. The proteins were expressed in (i) HEK293T cells to produce complex glycoforms, (ii) HEK293T cells treated with Vibrio cholera neuraminidase to provide asialo-complex glycoforms, (iii) HEK293S GnTI(-) cells with predominantly the canonical Man5GlcNAc2glycoform, and (iv) Drosophila S2 insect cells producing primarily paucimannose glycoforms. Previously, these HAs were used to investigate the effect of different glycosylation states on the immune responses in chicken and mouse systems. Evidence was found that high-mannose glycans diminished antibody response via DC-SIGN interactions. We performed two semiquantitative analyses including MALDI-TOF MS permethylation analysis of released glycans and LC-MSEanalysis of glycosylation site microheterogeneity. Glycosylation site occupancy was also determined by LC-MSE. Our major findings include (1) decreasing complexity of glycosylation from the stem to the globular head, (2) absence of glycosylation at N10and N193, (3) complex glycans at N165in HEK293T cell HA but high mannose glycans at this site in HEK293S and S2 cells, and (4) differences between the three-dimensional structures of H3 and H5 HAs that may explain glycan type preferences at selected sites. Biological implications of the findings are discussed.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Parsons
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
authors.forename: Yanming
authors.initials: Y
authors.lastname: An
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University , 3584CG Utrecht, The Netherlands.
authors.forename: Robert P
authors.initials: RP
authors.lastname: de Vries
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584CL Utrecht, The Netherlands.
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
authors.forename: John F
authors.initials: JF
authors.lastname: Cipollo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D004331:Drosophila melanogaster; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009439:Neuraminidase / Q000737:chemistry; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D000072417:Protein Domains; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014734:Vibrio cholerae / Q000737:chemistry
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; C109963:hemagglutinin, avian influenza A virus; D009439:Neuraminidase; D008358:Mannose
keywords: GLYMPS; HEK293; S2; flu; glycosylation; hemagglutinin; influenza; mass spectrometry