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J Proteome Res. 2015 Sep 4;14(9):3957-69. doi: 10.1021/acs.jproteome.5b00416. Epub 2015 Aug 10.

Glycosylation Analysis of Engineered H3N2 Influenza A Virus Hemagglutinins with Sequentially Added Historically Relevant Glycosylation Sites.

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Center for Biologics Evaluation and Research, Food and Drug Administration , Silver Spring, Maryland 20993, United States.
Department of Infectious Diseases, St. Jude Children's Research Hospital , Memphis, Tennessee 38105, United States.
Department of Pediatrics, University of Tennessee Health Science Center , Memphis, Tennessee 38103, United States.
Influenza Division, National Center for Immunization & Respiratory Diseases, Centers for Disease Control & Prevention , Atlanta, Georgia 30333, United States.


The influenza virus surface glycoprotein hemagglutinin (HA) is the major target of host neutralizing antibodies. The oligosaccharides of HA can contribute to HA's antigenic characteristics. After a leap to humans from a zoonotic host, influenza can gain N-glycosylation sequons over time as part of its fitness strategy. This glycosylation expansion has not been studied at the structural level. Here we examine HA N-glycosylation of H3N2 virus strains that we have engineered to closely mimic glycosylation sites gained between 1968 through 2002 starting with pandemic A/Hong Kong/1/68 (H3N2: HK68). HAs studied include HK68 and engineered forms with 1, 2, and 4 added sites. We have used: nano-LC-MS(E) for glycopeptide composition, sequence and site occupancy analysis, and MALDI-TOF MS permethylation profiling for characterization of released glycans. Our study reveals that 1) the majority of N-sequons are occupied at ≥90%, 2) the class and complexity of the glycans varies by region over the landscape of the proteins, 3) Asn 165 and Asn 246, which are associated with interactions between HA and SP-D lung collectin, are exclusively high mannose type. Based on this study and previous reports we provide structural insight as to how the immune system responses may differ depending on HA glycosylation.


H3N2; LC−MS; SP-D; antigenic site; glycan; glycopeptide; glycoprotein; hemagglutinin; influenza; mass spectrometry

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