Glycosylation Analysis of Engineered H3N2 ... - ACS Publications

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Glycosylation Analysis of Engineered H3N2 Influenza A Virus Hemagglutinins with Sequentially Added Historically Relevant Glycosylation Sites Yanming An,† Jonathan A. McCullers,‡,§ Irina Alymova,‡,∥ Lisa M. Parsons,† and John F. Cipollo*,† †

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 ‡

S Supporting Information *

ABSTRACT: 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−MSE for glycopeptide composition, sequence and site occupancy analysis, and MALDITOF 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. KEYWORDS: influenza, hemagglutinin, glycoprotein, glycan, mass spectrometry, glycopeptide, LC−MS, SP-D, antigenic site, H3N2

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INTRODUCTION

translational modification occurs at specific Asn residues within the sequon Asn-Xaa-Ser/Thr (where Xaa is any amino acid except for Pro). The N-linked oligosaccharide moieties at the globular head region can modulate virus receptor binding,4−7 HA antigenicity, and affect host immune response.8−11 The glycosylation sites on the stem region are conserved and are required for the proper folding of HA molecules.12 A/H3N2 and A/H1N1 viruses have gradually increased Nglycosylation sequons in the globular head region during virus adaptation in humans.13−15 The 1968 pandemic, represented by the H3N2 strain A/Hong Kong/1/1968 (H3N2: HK68), resulted in up to 750,000 deaths.16 Attenuated variants of this virus continue to circulate in human populations, causing seasonal epidemics. Evolutionary studies with H3N2 IAV show that, while five N-linked glycosylation sites on the stem region (8, 22, 38, 285, and 483; numbering is based on the mature HA molecule) are highly conserved, the N-linked glycosylation sequons in the virus globular head have gradually increased in number [12, 14]. The HAs of pandemic HK68 and the majority of epidemic H3N2 viruses circulating between 1968 and 1974

Influenza virus, a member of the Orthomyxoviridae family, causes disease in avian and mammalian species. The virus spreads in human populations in seasonal epidemics, resulting in ∼36,000 deaths and more than 200,000 hospitalizations each year in the United States.1 Influenza A and B viruses are the major types circulating in human populations. Influenza A virus (IAV) exhibits great amino acid sequence variability2 and a high pandemic potential. Surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) of IAV are the major antigenic components. On the basis of the antigenic nature and sequence of their HA and NA, IAVs are further divided into subtypes (H1−H18 and N1−N11). Two IAV subtypes, A/H3N2 and A/ H1N1, are currently circulating among humans.3 HA plays a key role during IAV infection. It binds to cell surface receptors, facilitating the penetration of the virus into the cell cytoplasm through a viral envelope and host vesicle fusion. HA is a noncovalently linked homotrimer. Each HA polypeptide chain consists of an ectodomain, composed of a globular head and a stem region, a carboxyl-terminal proximal transmembrane domain, and a cytoplasmic tail. The HA polypeptide undergoes N-linked glycosylation in the host cell secretory system on its ectodomain globular head and stem regions. This co- and post© XXXX American Chemical Society

Received: May 15, 2015

A

DOI: 10.1021/acs.jproteome.5b00416 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

Here, we have used variously glycosylated HK68 viruses and a mass spectrometry-based approach to investigate the composition of glycans at each site, the overall range of glycans on these viruses (including HA), and the site occupancy of each site. Predominant glycans at each site were modeled onto the HK68 HA three-dimensional surface. Our study provides structural insights as to how these chemical entities may modulate influenza interactions with the host immune system and may have implications for future vaccine design.

had only two globular head domain glycosylation sites, which were present at Asn 81 and Asn 165. By 1975, H3N2 IAV gradually added N-linked glycosylation sites at Asn positions 63 and 126, and by 1995−97, at residues 122, 133, and 246.17 Some human isolates have also lost globular head glycans over this time (most notably at residue 81). The recent seasonal H3N2 IAVs have six to seven glycosylation sites on the globular head, following this pattern. The trend for increased HA glycosylation is not limited to H3N2. A similar trend, in which glycosylation sites are added to the globular head over time, has been observed in H1N1 viruses.18 By comparison, the past pandemic strains, including the 2009 H1N1 pandemic strain, have HAs with little glycosylation on the globular head. The accumulation of glycosylation sites on the HA globular head has been shown to influence H3N2 IAV pathogenicity and immunogenicity.17,19 On the basis of the pandemic HK68 strain HA, we constructed virus mutants bearing 1, 2, and 4 additional glycosylation sites in the globular head region (denoted as HK68 + 1, HK68 + 2 and HK68 + 4 hereafter). These constructs were based on the natural additions that occurred between 1968 and 2002.15 Other than differences in HA glycosylation, and the required AA change to introduce an N-glycosylation sequon, the HK68 (WT), HK68 + 1, HK68 + 2, and HK68 + 4 viruses are isogenic. In immunologic studies of these mutants, we observed progressive reduction in morbidity and mortality and in viral lung titers in infected mice as the level of HA glycosylation increased.11 That attenuation was at least in part mediated by mouse lung surfactant protein D (SP-D), which induced virus neutralization and expedited clearance from the lungs. Similar H3N2 IAV neutralization effects were reported by others for mouse serum mannose-binding lectin and rat lung SP-D.19 These findings suggested that collectins play a key role in the innate defense against H3N2 IAV, and the degree of virus glycosylation can be an important factor in its virulence.19 These studies also showed that prior infection of mice with higher glycosylated variants of HK68 afford lower protection than when prior infection is by less glycosylated virus. Thus, mice infected with the HK68 + 4 virus and then challenged with HK68 were not protected from infection and experienced significant T-cellmediated immunopathology. The higher glycosylated HA virus variant elicited significantly lower antibody responses than the wild-type or moderately glycosylated viruses. These data were consistent with observations from another research group, where H3N2 IAV HAs with 2 to 4 more glycosylation sites were less efficiently recovered by immunoprecipitation with human antisera than wild-type HA.17 It has been reported that the glycans can mask antibody epitopes, which is a mechanism for viral evasion [4, 7]. Because the intracellular transport and cell fusion activity of the higher glycosylated mutants were not affected by additional glycosylation, it was concluded that the addition of new oligosaccharides to the globular head of H3N2 HA may provide the virus an ability to evade antibody pressure by changing antigenicity without an unacceptable defect in biological activity.17 The studies with lower or higher glycosylated variants of H3N2 viruses strongly suggest that the addition of glycosylation sites to influenza HA is part of a survival strategy that is likely delicately balanced to provide a selective advantage whereby the replicative cycle is not excessively disadvantaged while antigenicity is altered by the presence of glycans at key sites. However, it should be noted that in all these studies glycosylation was not examined beyond N-glycosylation sequon prediction.

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EXPERIMENTAL SECTION

Viruses

Engineered H3N2 IAV containing six internal gene segments of H1N1 A/Puerto Rico/8/34, the HA and NA of HK68, and modification of the HA to generate two mutant variants with additional N-linked glycosylation sites at positions 63 (HK68 + 1), 63 and 126 (HK68 + 2), or at positions 63, 126, 133, and 246 (HK68 + 4) were previously described.11 Numbering of glycosylation is based on the crystal structure of the mature HK68 HA molecule.20 For the current study, viruses were grown in Madin−Darby canine kidney epithelium cells, concentrated, and purified through a gradient of 30−50% sucrose in PBS and fully sequenced to ensure no inadvertent mutations occurred during passaging. Chemicals and Reagents

Sep-Pak C18 cartridges were purchased from Waters Corporation (Milford, MA). TSKgel Amide-80 particles were purchased from Tosoh Bioscience LLC (Montgomeryville, PA). Porous graphitic carbon (PGC) cartridges were purchased from Thermo Fisher Scientific Incorporated (Waltham, MA). Sequencing-grade modified trypsin was purchased from Promega Corporation (Madison, WI). N-Glycosidase A and endoproteinase Asp-N were purchased from Roche Diagnostics Corporation (Indianapolis, IN). Iodomethane, dimethyl sulfoxide (DMSO), sodium hydroxide beads, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO); solvents were of HPLC grade or higher. All other reagents were ACS grade or higher. Microwave-Assisted Protein Trypsin Digestion

The HA proteins of HK68 viruses were dissolved in 50 mM ammonium bicarbonate at a concentration of ∼1 μg/μL and denatured at 95 °C for 5 min. Dithiothreitol (DTT) was added to reduce the disulfide bonds at a final concentration of 5 mM for 45 min at 60 °C. The samples were chilled to room temperature, and iodoacetamide (IAA) was added to alkylate the reduced cysteine residues at a concentration of 15 mM for 45 min in the dark at room temperature. Trypsin was added (enzyme/protein, 1:50, w/w), and the samples were put into a Discover microwave apparatus (CEM Corporation; Matthews, NC) for digestion. The incubation time was 15 min with a fixed microwave irradiation power of 50 W and a temperature of 45 °C. After digestion, the samples were boiled for 10 min to inactivate trypsin, and the solutions were vacuum-dried for downstream analysis. Endoproteinase Asp-N Digestion

The dried tryptic peptides were suspended in 50 mM sodium phosphate buffer, pH 8.0, at a concentration of ∼1 μg/μL. Asp-N was added (enzyme/protein, 1:100, w/w), and the incubation time was 18 h at 37 °C. B


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Figure 1. Work flow diagram of the glycoprotein analysis approach used in this study, intact glycopeptide analysis by nanoLC−MSE with HILIC enrichment, and glycan permethylation profiling.

Enrichment of Glycopeptides with Hydrophilic Interaction Chromatography (HILIC)

glycans were eluted with 1 mL of 0.1% TFA/30% ACN and 1 mL of 0.1% TFA/60% ACN and vacuum-dried. Solid-phase permethylation was conducted according to the published protocol of Mechref and colleagues22 with modifications. Sodium hydroxide beads were packed in an empty spin column (Harvard Apparatus, MA) with acetonitrile. The column was washed with 200 μL of DMSO twice by centrifugation. The N-glycans were dissolved in 90 μL of DMSO and 35 μL of iodomethane. The sample was loaded onto the column and then centrifuged at low speed for 30 s. The sample was recycled through the spin column eight times. The spin column was centrifuged to dryness and washed with 50 μL acetonitrile twice, and the eluents were mixed together. Two milliliters of 0.1% TFA/water was added to the permethylated N-glycans. The sample was applied to a preconditioned C18 Sep-Pak cartridge. The cartridge was preconditioned with 2 mL of ethanol and 2 mL of water. After applying the permethylated N-glycans, the C18 cartridge was washed with 2 mL of 0.1% TFA/water, and the derivatized Nglycans were eluted with 1.5 mL of 90% ACN/5% isopropanol/ 5% water. The eluent was vacuum-dried.

Intact glycopeptides were enriched via solid phase extraction with TSKgel Amide 80 HILIC resin according to our previous report.21 Approximately 200 mg (400 μL of wet resin) of Amide80 resin was placed into a Supelco fritted 1 mL column and washed with 1 mL of 0.1% TFA/water. The column was conditioned with 1 mL of 0.1% TFA/80% ACN. The peptides were suspended in 0.1% TFA/80% ACN and applied to the column. The hydrophobic species were washed away with 3 mL of 0.1% TFA/80% ACN. The glycopeptides were eluted with 1 mL of 0.1%TFA/60% ACN and 1 mL of 0.1% TFA/40% ACN. The eluents were mixed and vacuum-dried prior to reverse phase LC−MS analysis. Permethylation of Released N-Glycans and Preparation of Glycan-Denuded Peptides

The glycopeptides were suspended in 50 mM ammonium acetate (pH 5.0), and 2 μL of N-glycosidase A (PNGase A, 5 mU/100 μL) was added to release glycans from the peptides at 37 °C for 16 h. The deglycosylated peptides mixture was used in site occupancy experiments where nanoLC−MS E was used. Conditions used for site occupancy experiments was identical to those described in the Reverse Phase NanoLC−MSE Analysis of Glycopeptides section. A C18 Sep-Pak cartridge was preconditioned with 2 mL of ethanol and 2 mL of water. The mixture after glycan release was applied to the C18 cartridge, and the N-glycans were eluted with 5 mL of water. Then, the N-glycans were cleaned with a porous graphitic carbon cartridge (PGC, Thermo Fisher Scientific, Incorporated). The PGC cartridge was wetted with 1 mL of 0.1% TFA/ACN and sequentially equilibrated with 1 mL of 0.1% TFA/60% ACN, 1 mL of 0.1% TFA/30% ACN, and 1 mL of 0.1% TFA/water. The N-glycans in water were applied to the cartridge and washed with 3 mL of 0.1% TFA/water. The N-

Reverse Phase NanoLC−MSE Analysis of Glycopeptides

NanoLC−MSE was used to analyze intact glycopeptides as described previously23,24 with modifications. NanoLC−MSE is a peptide mapping approach, which collects MS data in an alternating scan mode between low and elevated collision energies. No precursor ions are selected in the method, and therefore, scanning is data independent. All precursor ions are subjected to collision conditions, and MS/MS data are reconstructed according to the physiochemical properties of the parent ions and their fragments in association with their chromatographic positions during the LC−MS experiment. The precursor ion data are collected during the low energy MS scan, and then the collision energy is ramped increasing over a range of voltages in the dissociation stage to fragment the precursor ions. C


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score of 0.5 or higher indicates a high probability of occupancy. In cross-validated performance testing, the software could identify 86% of the glycosylated sequons and 61% of the nonglycosylated sequons with an overall accuracy of 76%.25 We next used nanoLC−MSE to assess site occupancy. The HA proteins were dissolved in 50 mM ammonium bicarbonate at a concentration of ∼1 μg/μL and denatured at 95 °C for 5 min. Dithiothreitol (DTT) was added to reduce the disulfide bonds at a final concentration of 5 mM for 45 min at 60 °C. The samples were chilled to room temperature, and iodoacetamide (IAA) was added to alkylate the reduced cysteine residues at a concentration of 15 mM for 45 min in the dark at room temperature. Trypsin was added (enzyme/protein, 1:50, w/w), and the samples were placed into a Discover microwave apparatus (CEM Corporation, Matthews, NC) for digestion. The incubation time was 15 min with a fixed microwave irradiation power of 50 W and a temperature of 45 °C. After digestion, the samples were boiled for 10 min to inactive trypsin and cooled to room temperature. Glacial acetic acid was added to adjust the peptide solution pH to 5.0. PNGase A (0.1 mU) was added, and the mixture was incubated for 16 h at 37 °C. The digestion mixture was analyzed by LC−MSE. Some possible sources of variability should be noted. Assignment of deglycosylated peptides was by accurate mass measurement (90% occupancy of 9 of the 11 sites, including 38, 63, 81, 126, 133, 165, 246, 285, and 483. However, S/N ratios for sites 8 and 22 were too low to assign % abundance with high confidence, although these sites were confirmed to be occupied as well by MSE decompositional analysis.

been missing in the user-defined library provided to BiopharmaLynx. In addition, GLYMPS assignments weigh heavily upon fragmentation profiles rather than total mass, as in BiopharmaLynx, and thus can be used to increase confidence of assignments made by BiopharmaLynx. GLYMPS greatly increases the speed at which MSE fragments in each spectrum can be identified. It can also filter assignments based on key spectral components and the number of files in which the assignment is found. Three-Dimensional (3D) Modeling of Glycosylated Hemagglutinins

The protein sequence of HK68 HA, with the four added mutations, was modeled by Iterative Threading ASSEmbly Refinement (i-tasser).26,27 Protein sequence and modifications are as previously described.11,28 The glycans were mapped onto the surface of the HA 3D structure (PDB ID: 4FNK). The predominant glycan at each site, as detected by MSE extracted ion chromatogram analysis, was attached using the Glycoprotein builder feature of Glycam.29 No molecular dynamics simulations were attempted beyond the defaults used at dev.glycam.org. Figure 6 was created using the molecular visualization system PyMol (www.pymol.org) and edited using GIMP (GNU Image Manipulation Program) (www.gimp.org)

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RESULTS

Analysis of Glycosylation Site Occupancy

Four HK68 viruses were analyzed for the presence of N-linked glycans on the HA stem region and globular head. Table 1 lists Table 1. Sequential Modification to the HA A/Hong Kong/1/ 1968 and NetNGlyc Prediction amino acid position

HK68 consensus sequence

81 165 63 (+1) 126 (+2) 246 (+4) 133 (+4)

NETW NVTM DCTL TWTG NSNG NGGS

8 22 38 285 483

NSTA NGTL NATE NGSI NGTY

sequence modification

Globular Head none none NCTL NWTG NSTG NGTS Stalk none none none none none

potential

N-Glyc result

0.5962 0.7471 0.6844 0.5469 0.5079 0.6791

+ ++ ++ + + ++

0.8122 0.7044 0.5083 0.6789 0.5656

+++ ++ + ++ ++

Characterization of Influenza H3N2 Glycoforms

The N-glycans released from whole virus were permethylated and analyzed by MALDI-TOF MS. Permethylated glycan profiles are highly reproducible and can be used to provide semiquantitative oligosaccharide comparisons.31,32 Figure 2 shows a histogram of the relative abundances of the N-glycans from the HK68 WT, HK68 + 1, HK68 + 2, and HK68 + 4 variants in this study. The relative abundance of each glycan subclass is displayed as a fraction of the total abundance with standard deviations listed in the figure legend. See Figure S2 for representative MALDI-TOF MS spectra for each HA. The high mannose glycans, Man5GlcNAc2 to Man9GlcNAc2, were between 70 and 89% of the total glycan ion abundance across the four virus samples. Complex glycans were between 9 and 21%, and hybrid glycans were between 2 and 10% across whole virus samples. The compositions detected are listed in Table S1. Those that were summed to represent each subclass are shown in Table S1. Also shown in the table are the glycan compositions detected by both permethylation analysis and on the peptide by LC−MSE. The compositions detected by both methods are consistent with the ladder method being more sensitive and detecting more forms of related compounds. The majority of complex glycans were biantennary. Fucose was detected on both hybrid and complex glycans with compositions suggesting that it was present in both core and antennae substitutions. Glycopeptide data showing the sequence of the same and related glycan compositions confirmed the presence of both substitution types.The HK68 + 1 whole virus sample has the highest abundance of high mannose glycan (89%), whereas HK68 + 2 and HK68 + 4 contained the highest abundances of complex

the protein sequence changes to the globular head of each HA in our series and the glycosylation potential of each site as predicted by NetNGlyc1.0. All of these HAs contain the five conserved glycosylation sites of the stem region at Asn 8, 22, 38, 285, and 483 and the two sites on the globular head, Asn 81 and 165. The mutant HAs have more glycosylation sites on the globular head: HK68 + 1 mutant HA has the addition of a site at Asn 63; HK68 + 2 mutant HA has the addition of sites at Asn 63 and 126; and HK68 + 4 mutant has the addition of sites at Asn 63, 126, 133, and 246. We chose to prescan each of the predicted protein glycosylation sites using NetNGlyc 1.0 to evaluate predictive modeling versus the experimental results in this study. The software predicted that all seven glycosylation sites of the HK68 WT HA are glycosylated, and this is consistent with a previous E


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Figure 2. N-Glycan profile of the HA mutants. Glycans from each virus were released, permethylated, and analyzed by MALDI-TOF MS. Measurements were performed in triplicate, and subclass individual isobars were summed. Standard deviation (%) for each strain’s permethylated glycans were as follows for high mannose and complex and hybrid glycans, respectively: (WT) 0.7, 0.2, 0.4; (+1) 0.9, 0.2, 0.2; (+2) 1.2, 0.5, 0.4; and (+4) 3.2, 0.2, 0.3.

Figure 3. ( a) M S/MS spectrum o f tryptic glyc opeptide c ontaining A sn81 from WT HA, (Fuc 1 He x 6 HexNAc 5 ) ILDGIDCTLIDALLGDPHCDVFQNETWDLFVER; (b) MS/MS spectrum of tryptic-AspN glycopeptide containing Asn63 from +1 HA, (Hex6HexNAc5)DGINCTLI. The colors in the figure indicate the following: red, y-ions; blue, b-ions; green, y/b-ions after neutral loss; pink, glycopeptides after neutral loss assigned by Biopharmalynx; gray, unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: blue square, GlcNAc; green circle, Man; red triangle, Fuc; yellow circle, Gal. Peptide fragments with glycosyl neutral loss are indicated. F


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Journal of Proteome Research Table 2. N-Glycans Detected at the Globular Head Region of Influenza HA Mutants glycosylation site 81

165

63 (+1)

wt

+1

+2

+4

Hex5HexNAc6 Hex6HexNAc5 Hex6HexNAc6 dHex1Hex6HexNAc4 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex1Hex7HexNAc5 dHex1Hex7HexNAc6 dHex2Hex5HexNAc5 dHex2Hex6HexNAc5 dHex2Hex6HexNAc6 Hex6HexNAc2 Hex7HexNAc2

dHex1Hex5HexNAc5 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex1Hex7HexNAc6 dHex2Hex6HexNAc6

dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex1Hex7HexNAc6 dHex2Hex6HexNAc6

dHex1Hex5HexNAc4 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex1Hex7HexNAc6 dHex2Hex6HexNAc6

Hex5HexNAc2 Hex6HexNAc2 Hex7HexNAc2 Hex8HexNAc2 Hex9HexNAc2 Hex5HexNAc4 Hex5HexNAc5 Hex6HexNAc4 Hex6HexNAc5 Hex6HexNAc6 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6

Hex7HexNAc2 Hex8HexNAc2 Hex9HexNAc2

Hex7HexNAc2 Hex8HexNAc2 Hex9HexNAc2

Hex6HexNAc4 Hex6HexNAc5 Hex6HexNAc6 Hex7HexNAc6 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex1Hex7HexNAc6 Hex6HexNAc5 dHex1Hex5HexNAc4 dHex1Hex5HexNAc5 dHex1Hex6HexNAc4 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6 dHex2Hex5HexNAc5 dHex2Hex6HexNAc5

Hex6HexNAc5 Hex6HexNAc6 dHex1Hex5HexNAc5 dHex1Hex6HexNAc5 dHex1Hex6HexNAc6

126 (+2, + 4)/133 (+4)

246 (+4)

Hex4HexNAc5 Hex5HexNAc2 Hex5HexNAc4 Hex6HexNAc2 Hex6HexNAc3 Hex6HexNAc4 Hex7HexNAc2 Hex8HexNAc2 dHex1Hex5HexNAc5 dHex1Hex3HexNAc4 dHex1Hex6HexNAc4 dHex2Hex5HexNAc5 Hex5HexNAc2 Hex6HexNAc2 Hex7HexNAc2 Hex8HexNAc2 Hex9HexNAc2

diagnostic of a Fuc linked to the aglycone most GlcNAc of the core. The parent ion is 6023.62, which is colored green in the figure. The majority of glycan compositions detected at this site across all HAs were complex type containing two or more antennae. The first additional site to the globular head analyzed was Asn 63. The position of this site presented a challenge as it is very close in sequence to Asn 81 and is predicted to coincide with Asn 81 in the tryptic peptide. The tryptic peptide containing the two sites is large, containing 33 amino acid residues and two oligosaccharide chains. The peptide with Asn 81 only, derived from WT HA, ionized and fragmented well, as discussed above, and the spectrum is shown in Figure 3a. However, early LC−MSE analyses using only trypsin produced poor spectra for the glycopeptides containing both Asn 63 and 81. We used AspN protease in addition to trypsin to further digest the peptide, which resulted in higher quality data with the detection of two shorter peptides with one site on each: DGIN* CTLI and DVFQN*ETW. This strategy was applied for all HAs with Asn

glycans at 20 and 21%, respectively. Hybrid glycans were most abundant in WT HK68 (12%). NanoLC−MSE Analysis of Globular Head Glycopeptides

Sites Asn 81 and Asn 165 were present on wild type HA A/Hong Kong/1/68 and all three engineered HAs (Table 1). Glycans present at Asn 81 were complex-type for all 4 HAs. Although WT Asn 81 contained glycans with and without Fuc, HK68 + 1, HK68 + 2, and HK68 + 4 Asn 81 contained complex glycans exclusively with Fuc. All of these contained core Fuc with some bearing Fuc at antennae. Figure 3a shows the processed deconvoluted MS/MS spectrum of a glycopeptide containing Asn 81 produced from the HK68 HA. Six y-ions were detected as indicated in the figure with red lines, identifying the peptide moiety. The ion at m/z 3889.85 is the peptide after loss of the entire saccharide moiety. The ion at m/z 4092.92 is the peptide + GlcNAc. Also annotated in the figure are a series of the glycosidic fragments, exhibiting partial loss of the glycan moiety with retention of the peptide, which were used to derive the glycan composition and some sequence. The ion at m/z 4238.97 was G


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Figure 4. MS/MS spectrum of glycopeptide with site Asn165, (Man9GlcNAc2) SGSTYPVLNVTMPNN. The colors in the figure indicate the following: red, y-ions; blue, b -ions; green, y/b-ions after neutral loss; pink, glycopeptides after neutral loss assigned by Biopharmalynx; gray, unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: blue square, GlcNAc; green circle, Man. Peptide fragments with glycosyl neutral loss are indicated.

Figure 5. (a) MS/MS spectrum of a glycopeptide containing Asn 126 from +2 HA, (Fuc1Hex5HexNAc5)SLVASSGTLEFITEGFNWTGVTQNGGSIACK; (b) MS/MS spectrum of a glycopeptide containing Asn 126 and Asn 133 from +4 HA (Hex7HexNAc2) (Hex6HexNAc2)SLVASSGTLEFITEGFNWTGVTQNGTSIACK. The colors in the figure indicate the following: red, y-ions; blue, bions; green, y/b-ions after neutral loss; gray, unassigned ions by Biopharmalynx (some have been manually assigned as indicated). Monosaccharide symbols: blue square, GlcNAc; green circle, Man; red triangle, Fuc; yellow circle, Gal. Peptide fragments with glycosyl neutral loss are indicated.

63 and Asn 81 (HK68 + 1, HK68 + 2, and HK68 + 4), resulting in successful detection of a range of glycans at both sites in the HK68 + 1, HK68 + 2, and HK68 + 4 HAs. The glycan linked to Asn 63 in all cases is complex type. An example is shown in Figure 3b generated from the HK68 + 1 HA. The range of complex glycans detected in the HAs is shown in Table 2. Glycan compositions are similar to those at site 81, containing up to 4

antennae with and without Fuc. The example shown in Figure 3b shows ion abundances for peptide fragments (i.e., m/z 905.44), glycosidic fragments, and peptide + GlcNAc (m/z 1108.52). The oligosaccharides linked to conserved Asn 165 were exclusively high-mannose type in all HAs and ranged in composition from Man5GlcNAc2 to Man9GlcNAc2 (Table 2). Figure 4 is a processed MS/MS spectrum of a glycopeptide H


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able to confirm that both Asn’s are glycosylated via conversion to Asp* by the enzyme.

containing Asn165 with Man9GlcNAc2 produced from the WT HA. The spectrum contains abundant b- and y-ion intensities, as indicated in the figure, and peptide + GlcNAc ion abundance at m/z 1796.83, confirming the amino acid sequence and glycopeptide status. Key glycosidic fragments and oxonium ions are indicated in the figure as well, providing composition and sequence information for the glycan moiety. The second site added to the HK68 HA polypeptide backbone was located at Asn 126 and was present in the HK68 + 2 and HK68 + 4 HAs. Figure 5a shows an MS/MS spectrum of the glycopeptide from HK68 + 2 HA with a glycan composition of dHex1Hex5HexNAc5. The b- and y-ion abundances are indicated in the figure as well as the peptide + GlcNAc at m/z 3434.64, peptide + dHexGlcNAc at m/z 3581.69, and a series of glycosidic fragments along the spectrum. Additionally, there are oxonium ions, such as those at m/z 204.09, 528.19, and 731.27. The HK68 + 2 HA glycans attached to this site are complex type and highly branched with most containing Fuc at the core and some at the antennae. The range of glycoforms detected was especially diverse in the HK68 + 4 HA-derived glycopeptide. The third site added to the HK68 HA polypeptide backbone was located at Asn133 and was present in only the HK68 + 4 HA. This site is only seven amino acid residues separated from the site at Asn126. An example spectrum of the HK68 + 4 glycopeptide is shown in Figure 5b. Because of the sequence and close proximity of the two glycosylation sites, no proteolytic strategy was available to cleave the dual site-containing peptide. Although the composition for the two glycans could be determined, which one resided at each site was not resolved. In the example in Figure 5b, the glycans attached to Asn 126 and Asn 133 are both high mannose. Again, peptide fragment ion, glycosidic, peptide + GlcNAc, and oxonium ions were detected, allowing sequencing of both peptide and glycan moieties (see figure for annotations). We identified 13 glycopeptides with two glycans and 9 glycopeptides with one glycan. The glycan types included high mannose and complex. On the basis of the observation that the HK68 + 2 HA peptide with only Asn126 contained exclusively complex glycans, and that all peptides with both Asn 126 and Asn 133 glycosylations contained at least one high mannose-type glycan, it is likely that Asn 133 contains mostly high mannosetype glycans. The final glycosylation site analyzed was Asn 246, which was present only in HK68 + 4 HA. As shown in Table 2, we have identified 5 glycopeptides containing this site, and the glycans are exclusively high mannose type, ranging in composition from Man5GlcNAc2 to Man9GlcNAc2. Key fragment ion abundances were detected for peptide and glycan moiety assignments as discussed for other peptides. An example spectrum is shown in Figure S2.

Mapping of Glycosylation to the Three-Dimensional Surface

Figure 6 shows the 3D structure of the HK68 HA monomer with the glycans modeled on its surface. The X-ray crystal structure of the H3N2 HK68 HA was reported previously20 (PDB ID: 4FNK). The four added glycosylation sites representative of the HK68 + 1, HK68 + 2, and HK68 + 4 recombinant HAs were modeled onto the HA sequence using i-tasser. The i-tasser software models protein structures by iterative threading onto known structures in the Protein Data Bank and has been ranked as the number one protein structure prediction server in several Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments.26,27 There are five antigenic sites defined on the HA surface, denoted as sites A, B, C, D, and E. All are at the globular head region except for site C, as previously described.15,33−35 These antigenic sites were located on the protein surface with various colors. Site A, shown in red, is a protruding loop from amino acids 140−146 and surrounding residues 122, 126, and 133−139; site B, shown in blue, comprises the external residues 187−196 of an α helix and adjacent residues 155−160 and 163−165, which are along the upper edge of a pocket of conserved residues of the host receptor binding site; site D, shown in cyan, is in the interface region between subunits in the HA trimer and includes residues 174, 182, 201−220, 226, and 242−248; and site E, shown in yellow, is from 63 to 83.33−35 The glycosylation sites at the globular head all locate in or around one of the antigenic sites: Asn 133 is within site A, Asn 126 is at the boundary of sites A and B, Asn 165 is at the edge of site B, Asn 246 is within site D, and Asn 63 and Asn 81 are at site E.15,34,35 To visualize the glycan shielding of the antigenic sites, we mapped glycans representative of those found to be highly abundant on each glycosylation site based on trace ion chromatogram intensity to the protein surface using the Glycoprotein builder feather of Glycam.29 The glycan types detected on each glycosylation site are high mannose glycans at Asn 133, 165, 246, and 285 and complex glycans at Asn 38, 63, 81, 126 and 483. The three highest abundance glycans detected at each site, as determined by extracted ion chromatogram peak areas, are shown in Table S2. The glycan structures are inferred through a combination of our data and a preponderance of biological data reported in the literature.

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DISCUSSION While circulating in human populations during the past 40 years, the influenza H3N2 variants HA have gradually gained glycosylation sites in the globular head region. In 1968, the pandemic strain A/Hong Kong/1968 had only two glycosylation sites on the globular head, located at Asn 81 and 165, and five on the stem, located at Asn 8, 22, 38, 285, and 483. Recent H3N2 strains are highly glycosylated with up to 7 sites in the globular head region. The virus HAs studied here have been constructed to mimic sites added over time to this pandemic 1968 H3N2 strain during adaptation in human populations between 1968 and 2002. The globular head region of the A/Hong Kong/1968 H3N2 HA has been modified to contain an additional 1, 2, or 4 N-glycosylation sequons, which are represented in this study (Table 1). Previously, we characterized the immunologic effect of these sequential additions.11,28 We have shown that additional Nlinked glycosylation on the globular head of H3N2 influenza ̈ mice. A major viruses attenuates the severity of infection in naive mediator of this affect is likely improved neutralization via host

NanoLC−MSE Analysis of Stem Region Glycopeptides

Glycopeptides of the HAs’ stem region were also characterized (Table S1). These include Asn 8, 22, 38, 285, and 483. These are present in all HAs studied here and conserved across the H3N2 variants over the past three decades. Asn 38 glycans were diverse, containing all three N-glycan subtypes, high mannose, complex, and hybrid. Asn 285 is occupied by high mannose and hybrid glycans. Asn 483 is occupied by complex glycans. Asn 8 and Asn 22 were on the same tryptic peptide, and the specific location of glycan subtypes was not determined. This glycopeptide was not well ionized. However, high mannose and complex glycans were detected. When the glycans were released by PNGase A, we were I


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Figure 6. HA monomer with predominant glycans attached. The bottom structure is rotated 120 degrees clockwise from the top one. Each figure displays the protein through two different representations of the same orientation. The left side of each figure depicts the proteins as cartoons and the glycans as sticks. On the right, the accessible surface area of the protein is displayed with the glycans as balls. Glycans are colored by atom: green is carbon, blue is nitrogen, and pink is oxygen. The protein is colored gray except for the antigenic sites on the head region as described in this paper. Site A is red, site B is blue, site D is cyan, and site E is yellow. The glycan composition attached to each glycosylation site is as follows: Hex5HexNAc4-Asn38, Hex5HexNAc5-Asn63, Fuc1Hex5HexNAc5-Asn81, Fuc1Hex5HexNAc5-Asn126, Hex7HexNAc2-Asn133, Hex6HexNAc2-Asn165, Hex9HexNAc2Asn246, Hex6HexNAc2-Asn285, and Fuc1Hex6HexNAc5-Asn483. The figure was generated as described in the Experimental Section.

lung collectin SP-D.28 Other mechanisms may be involved as the virus with HK68 + 4 HA elicited significantly lower antibody responses than the WT and HK68 + 2 HA virus and was unable to elicit neutralization antibodies against WT virus reinfection in mice.11 In this report, we have analyzed the glycosylation status of these HAs using a range of mass spectrometry-based techniques to reveal how glycosylation is involved in the above changes. Our data show that the high mannose glycans are the major glycoforms on viral proteins contributing 70−89% of the total amount of the glycans released, whereas complex and hybrid glycans account for considerably less, 9−21% and 2−12%,

respectively (Figure 2). However, when we analyzed the abundances of glycans on HA by nano-LC−MSE, the abundance of glycans at each site reveals that complex glycans account for at least 50% of total glycans of WT HA, and the percent increases as glycosylation sites are added (see Table S2). Therefore, HA contains more complex glycan than whole virus released glycans. Influenza neuraminidase is the second most abundant protein and is glycosylated; these data suggest that NA may be primarily substituted with high mannose glycans in these virions. Each glycosylation site on the globular head is occupied at 90% or greater. This suggests that these sites have been selected for glycosylation at high efficiency and supports the notion that they J


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Journal of Proteome Research play a role in viral fitness as it adapts to the human host immune response. Our site specific glycosylation study revealed the glycosylation pattern residing on the globular head and stem of each of the HAs. We mapped the glycans onto the 3D structure of the 1968 H3N2 HA (PDB ID: 4FNK) monomer (Figure 6) and determined the range and relative abundance (Table S2) of glycoforms detected at each site (Table 2, head region; Table S1, stem). Sites 63, 126, 133, and 246 are the glycosylation sites that appeared after 1968 in seasonal strains. We have determined that sites 165 and 246 are substituted exclusively with high mannose glycans, and site 133 is substituted primarily by high mannose glycans. These three glycosylation sites cluster in the lung collectin SP-D binding domain of HA and will be discussed later. The overall trend of glycosylation patterning is a clustering of high mannose glycans at sites 133, 165, and 246, all in the vicinity of the SP-D binding site, and more complex glycans at sites 63, 81, and 126. The majority of glycans on the stem, sites 8, 22, 38, 285, and 483, are primarily complex type glycans, many of which are highly branched, especially at Asn sites 38 and 483. That Asn 133, 165, and 246 are occupied only by high mannose glycans directly impacts SP-D binding. SP-D is an important component of the family of collagenous Ca2+dependent defense lectins present in respiratory secretions, and it preferentially binds to high mannose glycans. Asn 165 is the most conserved glycosylation site in H3N2 viruses.9 Decreased virulence of highly glycosylated virus is associated with the increased clearance of virus by the lung-resident surfactant protein D (SP-D).28 Previously, we reported that hemagglutination inhibition capacity of recombinant human SPD was maximized after the addition of Asn 246 and remained the same with the addition of Asn 133.28 The high mannose glycans detected on sites Asn 133 and 246 in the current study demonstrate conclusively that the increased affinity of the virus HA for SP-D is based on the interaction between SP-D and high mannose glycans at sites 133, 165, and 246 in the mutants. The high-mannose glycans on conserved Asn 165 are key in the SP-D interaction as H3 virus becomes resistant to β-inhibitors, including SP-D, after loss of glycosylation site Asn 165.36 Asn 165 and 246 are particularly important in determining HA sensitivity to SP-D as removal of one or both of these two sites decreases affinity to collectin.37 Tethering of SP-D to the viral HA may occur by initial binding to the glycan at Asn 165. Once bound, SP-D can interact with additional sites, leading to greater sensitivity to neutralization. It has been reported that binding of the carbohydrate recognition domains of the SP-D trimer and glycan at Asn 133 of the HA trimer are energetically favorable based on the crystal structures of SP-D and HA.38 Taken together, these studies strongly support the notion that the additional glycosylation on the globular head of influenza H3N2 HA is associated with the attenuation of the virulence mediated by improved collectin binding and subsequent viral neutralization. The reason that some influenza strains tolerate increased susceptibility to SP-D may reside in the balance between other changes affecting both the humoral and innate arms of the immune system. The orientation of the high mannose glycan at Asn 165 was determined in the 1968 X-31 HA crystal structure.8 The glycan was located near the tip of the molecule oriented in a position predicted to block access of antibodies to the molecular surface between residues 166 and 168. No antigenic variant has been observed in this region since 1968, consistent with shielding of this site. In further support of a shielding effect, the HA of H1N1

A/PR/8/34 has no glycosylation sites in this region and shows amino acid substitution in the 165−168 region in monoclonal antibody-selected antigenic variants.39 It has been proposed that the gain of oligosaccharide chains on HA can provide an advantage for the virus becauase the additional glycans can mask antigenic sites leading to evasion of antibody neutralization.8,17 In the early 1980s, Wiley et al. described the 3D structure of the 1968 H3N2 HA and identified its antigenic sites using the available primary sequence information on natural and laboratory-selected antigenic variants.35 There are five antigenic sites on the HA surface: A, B, C, D, and E, and all but C are at the globular head region. The glycosylation sites at the globular head all locate in one or more of the antigenic sites: Asn 133 is within site A; Asn 126 is at the boundary of sites A and B; Asn 165 is at the edge of site B; Asn 246 is within site D; and Asn 63 and 81 are at site E.15,34,35 Skehel et al. provided direct evidence that the glycan at Asn 63 of H3N2 HA blocked recognition by an inhibitory monoclonal antibody at the region.8 Asn 81 was present in the 1968 H3N2 HA and later replaced by Asn 63 around 1975. These two sites are very close in their 3D structures, and our data reveal that Asn 63 and 81 both bear large complex-type glycans with up to four antennae, which supports shielding of antigenic site E.35 HK68 HA in our study had glycans limited to antigenic site E; in HK68 + 2 HA, the additional glycans shield the overlap region of antigenic sites A and B, whereas in HK68 + 4 HA, the glycans shield all four antigenic sites (A, B, D, and E) on the globular head, consistent with our observations of weaker antibody production and virus neutralization produced by the high glycosylation HA viral strain. Similar observations have been reported using a series of increasing glycosylated mutants of A/ Aichi/2/68 (H3N2) virus HA. Intracellular transport, receptor binding, and cell fusion activities were studied.17 HAs with added glycans to the globular head had 20−75% reduced hemagglutination inhibition titer compared to the antisera against less glycosylated strains,17 similar to our studies. Additionally, they found that receptor binding affinity was reduced with increased glycosylation but that cell fusion and transport were unaffected, suggesting that glycosylation-dependent reduction in receptor binding does not significantly disadvantage the virus. Therefore, in terms of receptor binding affinity cost, antigenic masking by glycans can be affordable. The impact of glycosylation on viral fitness may have important implications for vaccine design. Genetic removal of glycosylation sites on the HA globular head to expose antigenic sites may improve vaccine immunogenicity.5 The H1N1 strain in use before 2009 was a highly glycosylated virus and showed poor ability to protect from infection during the 2009 H1N1 pandemic.10,11,18 Medina and co-workers generated a set of recombinant glycosylation-deleted viruses from a recent seasonal H1N1 strain (A/Texas/36/1991). They found that the mice infected with viruses containing glycosylation-deleted HA survived the lethal challenge with the pH1N1 2009 virus whereas the mice infected with the WT highly glycosylated seasonal virus, A/Texas/36/1991 H1N1, all succumbed to the pH1N1 2009 challenge.40 Glycosylation denuded HA virus was more protective. Recombinant HAs bearing a single GlcNAc at each glycosylation site have been reported to provide better protection against H5N1 viruses than the respective fully glycosylated HAs and that protection covers a broader range of influenza variants.5 Again, glycan-denuded HA virus was more protective. Glycosylation reduction strategies may offer an avenue for improvement in vaccine design. Glycosylation-driven K


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hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc. Natl. Acad. Sci. U. S. A. 1984, 81 (6), 1779−83. (9) Das, S. R.; Puigbo, P.; Hensley, S. E.; Hurt, D. E.; Bennink, J. R.; Yewdell, J. W. Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain. PLoS Pathog. 2010, 6 (11), e1001211. (10) Wei, C. J.; Boyington, J. C.; Dai, K.; Houser, K. V.; Pearce, M. B.; Kong, W. P.; Yang, Z. Y.; Tumpey, T. M.; Nabel, G. J. Crossneutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design. Sci. Transl. Med. 2010, 2 (24), 24ra21. (11) Wanzeck, K.; Boyd, K. L.; McCullers, J. A. Glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice. Am. J. Respir. Crit. Care Med. 2011, 183 (6), 767−73. (12) Daniels, R.; Kurowski, B.; Johnson, A. E.; Hebert, D. N. N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin. Mol. Cell 2003, 11 (1), 79−90. (13) Cherry, J. L.; Lipman, D. J.; Nikolskaya, A.; Wolf, Y. I. Evolutionary dynamics of N-glycosylation sites of influenza virus hemagglutinin. PLoS Curr. 2009, 1, RRN1001. (14) Sun, S.; Wang, Q.; Zhao, F.; Chen, W.; Li, Z. Glycosylation site alteration in the evolution of influenza A (H1N1) viruses. PLoS One 2011, 6 (7), e22844. (15) Kobayashi, Y.; Suzuki, Y. Evidence for N-glycan shielding of antigenic sites during evolution of human influenza A virus hemagglutinin. J. Virol 2012, 86 (7), 3446−51. (16) Paul, W. E. Fundamental Immunology, 6th ed.; Lippincott Williams and Wilkins, 2008. (17) Abe, Y.; Takashita, E.; Sugawara, K.; Matsuzaki, Y.; Muraki, Y.; Hongo, S. Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin. J. Virol 2004, 78 (18), 9605−11. (18) Reichert, T.; Chowell, G.; Nishiura, H.; Christensen, R. A.; McCullers, J. A. Does Glycosylation as a modifier of Original Antigenic Sin explain the case age distribution and unusual toxicity in pandemic novel H1N1 influenza? BMC Infect. Dis. 2010, 10, 5. (19) Reading, P. C.; Morey, L. S.; Crouch, E. C.; Anders, E. M. Collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice. J. Virol. 1997, 71 (11), 8204−8212. (20) Wilson, I. A.; Skehel, J. J.; Wiley, D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 1981, 289 (5796), 366−73. (21) An, Y.; Cipollo, J. F. An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin. Anal. Biochem. 2011, 415 (1), 67−80. (22) Mechref, Y.; Kang, P.; Novotny, M. V. Solid-phase permethylation for glycomic analysis. Methods Mol. Biol. 2009, 534, 53−64. (23) Xie, H.; Doneanu, C.; Chen, W.; Rininger, J.; Mazzeo, J. R. Characterization of a recombinant influenza vaccine candidate using complementary LC−MS methods. Curr. Pharm. Biotechnol. 2011, 12 (10), 1568−1579. (24) An, Y.; Rininger, J. A.; Jarvis, D. L.; Jing, X.; Ye, Z.; Aumiller, J. J.; Eichelberger, M.; Cipollo, J. F. Comparative glycomics analysis of influenza Hemagglutinin (H5N1) produced in vaccine relevant cell platforms. J. Proteome Res. 2013, 12 (8), 3707−20. (25) Gupta, R. J.; Jung, E.; Brunak Prediction of N-Glycosylation Sites in Human Proteins, 2004; unpublished manuscript (26) Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinf. 2008, 9, 40. (27) Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. The ITASSER Suite: protein structure and function prediction. Nat. Methods 2014, 12 (1), 7−8. (28) Vigerust, D. J.; Ulett, K. B.; Boyd, K. L.; Madsen, J.; Hawgood, S.; McCullers, J. A. N-linked glycosylation attenuates H3N2 influenza viruses. J. Virol 2007, 81 (16), 8593−600. (29) Kirschner, K. N.; Yongye, A. B.; Tschampel, S. M.; GonzalezOuteirino, J.; Daniels, C. R.; Foley, B. L.; Woods, R. J. GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J. Comput. Chem. 2008, 29 (4), 622−55.

strategies may not be limited to antigenic deshielding. HA glycans with terminal mannose may improve neutralization and uptake into dendritic cells by targeting lectins and surfactants, such as SP-D.41 Therefore, the design of HA antigen with high mannose glycans may provide an advantage for antigen uptake and T-cell recognition.42 Influenza uses HA glycosylation in fitness strategies. Our understanding of these structure−function relationships may provide new opportunities toward better influenza vaccines.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00416. MS/MS spectra of the deglycosylated peptides and glycopeptide at site Asn246, representative MALDI-MS spectra of permethylated N-glycan of each virus strain, and tables of N-glycans detected at stalk region of influenza HA mutants, estimated abundance of the top 3 glycoforms for each site, and summary of glycan compositions detected by MALDI−MS and ESI−MSE (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by ALSAC to J.A.M. and the CBER Pandemic Flu and Medical Counter Measures targeted internal funds to J.F.C. We would like to acknowledge Drs. Ian York and Terry Tumpey of the Influenza Division of the CDC for helpful discussions, and Shane Gansebom of the Infectious Disease Department of St. Jude Children’s Research Hospital for technical assistance.

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REFERENCES

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Journal of Proteome Research (30) Ward, C. W.; Gleeson, P. A.; Dopheide, T. A. Carbohydrate composition of the oligosaccharide units of the haemagglutinin from the Hong Kong influenza virus A/Memphis/102/72. Biochem. J. 1980, 189 (3), 649−652. (31) Costello, C. E.; Contado-Miller, J. M.; Cipollo, J. F. A glycomics platform for the analysis of permethylated oligosaccharide alditols. J. Am. Soc. Mass Spectrom. 2007, 18 (10), 1799−1812. (32) Cipollo, J. F.; Awad, A. M.; Costello, C. E.; Hirschberg, C. B. srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates. J. Biol. Chem. 2004, 279 (51), 52893−903. (33) Suzuki, Y. Three-dimensional window analysis for detecting positive selection at structural regions of proteins. Mol. Biol. Evol. 2004, 21 (12), 2352−9. (34) Underwood, P. A. Mapping of antigenic changes in the haemagglutinin of Hong Kong influenza (H3N2) strains using a large panel of monoclonal antibodies. J. Gen. Virol. 1982, 62 (1), 153−169. (35) Wiley, D. C.; Wilson, I. A.; Skehel, J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 1981, 289 (5796), 373− 8. (36) Anders, E. M.; Hartley, C. A.; Jackson, D. C. Bovine and mouse serum beta inhibitors of influenza A viruses are mannose-binding lectins. Proc. Natl. Acad. Sci. U. S. A. 1990, 87 (12), 4485−9. (37) Tate, M. D.; Job, E. R.; Brooks, A. G.; Reading, P. C. Glycosylation of the hemagglutinin modulates the sensitivity of H3N2 influenza viruses to innate proteins in airway secretions and virulence in mice. Virology 2011, 413 (1), 84−92. (38) Hartshorn, K. L.; Webby, R.; White, M. R.; Tecle, T.; Pan, C.; Boucher, S.; Moreland, R. J.; Crouch, E. C.; Scheule, R. K. Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D. Respir Res. 2008, 9, 65. (39) Caton, A. J.; Brownlee, G. G.; Yewdell, J. W.; Gerhard, W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 1982, 31 (2), 417−427. (40) Medina, R. A.; Stertz, S.; Manicassamy, B.; Zimmermann, P.; Sun, X.; Albrecht, R. A.; Uusi-Kerttula, H.; Zagordi, O.; Belshe, R. B.; Frey, S. E.; Tumpey, T. M.; Garcia-Sastre, A. Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses. Sci. Transl. Med. 2013, 5 (187), 187ra70. (41) Reading, P. C.; Tate, M. D.; Pickett, D. L.; Brooks, A. G. Glycosylation as a target for recognition of influenza viruses by the innate immune system. Adv. Exp. Med. Biol. 2007, 598, 279−292. (42) Engering, A. J.; Cella, M.; Fluitsma, D. M.; Hoefsmit, E. C.; Lanzavecchia, A.; Pieters, J. Mannose receptor mediated antigen uptake and presentation in human dendritic cells. Adv. Exp. Med. Biol. 1997, 417, 183−7.

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