Glycosylation Characterization of an Influenza H5N7 Hemagglutinin Series with Engineered Glycosylation Patterns: Implications for Structure-Function Relationships

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 ManGlcNAc glycoform, 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-MS analysis of glycosylation site microheterogeneity. Glycosylation site occupancy was also determined by LC-MS. Our major findings include (1) decreasing complexity of glycosylation from the stem to the globular head, (2) absence of glycosylation at N and N, (3) complex glycans at N in 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.