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

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 <i>N</i>-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 <i>N</i>-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 <i>N</i>-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

<i>Caenorhabditis elegans</i> Bacterial Pathogen Resistant <i>bus-4</i> Mutants Produce Altered Mucins

<div><p><i>Caenorabditis elegans bus-4</i> glycosyltransferase mutants are resistant to infection by <i>Microbacterium nematophilum</i>, <i>Yersinia pestis</i> and <i>Yersinia pseudotuberculosis</i> and have altered susceptibility to two <i>Leucobacter</i> species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the <i>bus-4 N</i>- and <i>O</i>-glycan pools. We observed dramatic changes in <i>O</i>-glycans and moderate ones in <i>N</i>-glycan pools compared to the parent strain. <i>Ce</i> core-I glycans, the nematode's mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl <i>O</i>-glycans, <i>Ce</i> core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl <i>N</i>-glycans. Quantitative expression analysis revealed that two mucins, <i>let-653</i> and <i>osm-8</i>, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.</p></div

Substitution ratios of <i>bus-4 Ce</i> core-I glycans differ from wild type.

a<p>Total molar ratios differ slightly from those reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107250#pone-0107250-g002" target="_blank">Figure 2</a> as these are calculated only on Ce core-I glycans examined through exhaustive MSⁿ and not the total <i>O</i>-glycan pool as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107250#pone-0107250-g002" target="_blank">Figure 2</a>.</p>b<p>Figures present in parentheses represent 1SD.</p>c<p>Figures in parentheses represent 1SD expressed in %.</p><p>Substitution ratios of <i>bus-4 Ce</i> core-I glycans differ from wild type.</p

The structural configurations of the <i>Ce</i> core-I <i>O</i>-glycans defined in this study are shown.

<p>The structural configurations of the <i>Ce</i> core-I <i>O</i>-glycans defined in this study are shown.</p

Composition of <i>C. elegans</i> permethylated <i>O</i>-glycans released from N2 wild type and the mutant strain <i>bus</i>-<i>4</i> determined by MALDI-TOF MS analysis.

a<p>All ion compositions detected as sodium adducts</p><p>Composition of <i>C. elegans</i> permethylated <i>O</i>-glycans released from N2 wild type and the mutant strain <i>bus</i>-<i>4</i> determined by MALDI-TOF MS analysis.</p

MALDI-TOF MS permethylation profiling of N2 and <i>bus-4</i> O-glycans.

<p>Histograms represent the average of three independent analyses. Light gray histograns are N2 and black are <i>bus-4</i>. Sample spectra are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107250#pone.0107250.s003" target="_blank">Figure S3</a>. Error bars represent one standard deviation. A: Relative quantitation of Ce core-I neutral, Ce core-I charged and fucosyl glycoforms, B; Relative quantitation of Ce core-II glycoforms, C; Relative abundances of <i>O</i>-glycan subclasses, D; GC/MS monosaccharide abundances detected in this study</p

Lectin staining patterns of fixed <i>bus-4</i> nematodes are altered.

<p>Texas Red-conjugated <i>Agaricus bisporus</i> (ABA) Gal (β1,3) GalNAc specific lectin staining of whole mounted delipidated and live <i>C. elegans</i> strains are shown. A: the left columns shows differential interference contrast (DIC) micrographs and right columns show fluorescence micrographs of ABA stained fixed nematodes with and without prior incubation with inhibitory sugar (β-D-galactose). Top panels are N2 Bristol. Bottom panels are <i>bus-4</i>. B; Shorter exposures of ABA lectin are shown along with DIC micrographs to the left. Cuticle staining seen in the ventral tail region leading up to the anus in the N2 parent is absent in the <i>bus-4</i> strain. C; Live ABA stained nematodes are shown. The surface coat of the <i>bus-4</i> nematodes stains more intensely than in the N2 parent. The staining is most concentrated in the head and tail regions. Staining in the intestine is also increased abundances of soluble mucin-like proteins are indicated.</p

The CID MS² analysis of matched N2 and <i>bus-4</i> Hex₃HexNAc₁HexA₁-ol at <i>m/z</i> 1160.6 [M+Na]⁺.

<p>Data were collected under identical conditions using a Thermo LTQ-XL ion trap equipped with an Advion Nanomate sample infusion system. Five predicted isomeric structures (IX – XIII) are represented. The origin of key fragment ions are shown. Structural pairs IX and X and XI and XII differ only by the position of the substituent Glc at either C4 or C6 of the partner Gal residue as indicated by the dashed line. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107250#pone-0107250-g002" target="_blank">Figure 2</a> for inferred monosaccharide identities.</p

Quantitative RT-PCR analysis <i>osm-8</i>, <i>let-653</i> and <i>gmd-2</i> gene expression: Larval stage L4 of C. elegans larval stage 4 wt (N2) and bus-4 nematodes were analyzed as biological triplicates.

<p>Data were presented as relative ΔCts values and normalized to the N2 samples. Error bars represent ± SEMs.</p

The CID MS² analysis of matched N2 and <i>bus-4</i> permethylated Hex₃HexNAc₁-ol at <i>m/z</i> 942.6 [M+Na]⁺.

<p>Data were collected under identical conditions using a Thermo LTQ-XL ion trap equipped with an Advion Nanomate sample infusion system. Three predicted isomeric structures (IV, V and VI) are represented. The origin of key fragment ions are shown. Structures IV and V differ only by the position of the substituent Glc at either C4 or C6 of the partner Gal residue as indicated by the dashed line. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107250#pone-0107250-g002" target="_blank">Figure 2</a> for inferred monosaccharide identities.</p