Shotgun glycomics of pig lung identifies naturalendogenous receptors for influenza virusesLauren Byrd-Leotisa,1, Renpeng Liub,c,1,2, Konrad C. Bradleya,3, Yi Lasanajakb,c, Sandra F. Cummingsa,c,Xuezheng Songb,c, Jamie Heimburg-Molinarob,c, Summer E. Gallowaya, Marie R. Culhaned, David F. Smithb,c,David A. Steinhauera,4, and Richard D. Cummingsb,c,4

Departments of aMicrobiology and Immunology and bBiochemistry and cThe Glycomics Center, Emory University School of Medicine, Atlanta, GA 30322;and dMinnesota Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, MN 55108

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved April 22, 2014 (received for review December 19, 2013)

Influenza viruses bind to host cell surface glycans containingterminal sialic acids, but as studies on influenza binding becomemore sophisticated, it is becoming evident that although sialic acidmay be necessary, it is not sufficient for productive binding. Tobetter define endogenous glycans that serve as viral receptors, wehave explored glycan recognition in the pig lung, because in-fluenza is broadly disseminated in swine, and swine have beenpostulated as an intermediary host for the emergence of pandemicstrains. For these studies, we used the technology of “shotgunglycomics” to identify natural receptor glycans. The total releasedN- and O-glycans from pig lung glycoproteins and glycolipid-derivedglycans were fluorescently tagged and separated by multidimen-sional HPLC, and individual glycans were covalently printed to gener-ate pig lung shotgun glycan microarrays. All viruses tested interactedwith one or more sialylated N-glycans but not O-glycans or glyco-lipid-derived glycans, and each virus demonstrated novel and un-expected differences in endogenous N-glycan recognition. The resultsillustrate the repertoire of specific, endogenous N-glycans of pig lungglycoproteins for virus recognition and offer a new direction forstudying endogenous glycan functions in viral pathogenesis.

receptor binding | hemagglutinin | virus attachment

Influenza A viruses infect the human population on a yearlybasis, causing hundreds of thousands of excess deaths, enor-

mous stress on health care systems worldwide, and extensiveeconomic burden. At unpredictable intervals, viruses with anti-genically novel glycoproteins emerge to cause pandemics of evengreater concern, and the pig is often implicated as an in-termediate host when this occurs (1). The waterfowl that act asnatural reservoirs for influenza A viruses harbor 16 antigenicallydistinct subtypes of the hemagglutinin (HA) surface antigen, and9 subtypes of the neuraminidase (NA) envelope protein, but theonly subtypes to emerge and circulate broadly in humans overthe past century are H1N1 in 1918, H2N2 in 1957, H3N2 in 1968,and H1N1 again in 2009. However, the possible emergence ofa novel subtype in humans remains a concern, because highlypathogenic H5 and H7 subtype “bird flu” viruses continue tocirculate in avian species, and over the past 15 y have infectedhumans, with limited ability to transmit between humans. Thereasons for such host–species restriction and mechanisms un-derlying cross species transmission are thought to be multifac-torial, but the receptor binding properties of the HA surfaceantigen are of paramount significance.Since the identification of sialic acids as critical components of

influenza receptors (2, 3), a broadly observable phenomenon hasbeen that avian strains generally prefer binding to glycanreceptors with sialic acids containing α2,3 glycosidic linkages tothe penultimate galactose in the carbohydrate chain, whereashuman viruses favor binding to substrates with α2,6 linkages (4–7). The description of mutant viruses for which specific changesin the HA receptor-binding site result in a switch from onebinding preference to the other provides clues on potentialmechanisms for altered host range (8–14) but perhaps leads to

the simplified perception that influenza binds rather indiscrim-inately, as long as the minimal sialic acid linkage is appropriate.Interestingly, proton-NMR binding data for avian-like or human-like HAs to monosialosides of preferred, or nonpreferred link-age, show binding affinity to be generally very weak, with dis-sociation constants in the millimolar range and only a two- tothreefold difference between cognate pairs and reciprocal pairs(15, 16). This suggests that productive virus attachment resultsfrom the cumulative effects of multiple HA receptor interactionsin which the consequences of very small differences are ampli-fied in a phenotypically distinguishable fashion. However, littlemore is known with regard to binding affinity for individualglycans of defined structure or of their presence and distributionin tissues encountered during natural infections.Initial studies using linkage-specific lectins as an indirect

probe for the presence and distribution of potential influenzaglycan receptors suggested that the intestinal tract of ducks isrich in α2,3-linked sialic acid (α2,3-Sia) and the upper respiratorytract of humans contains an abundance of sialic acid in α2,6linkage (17). These observations have been extended consider-ably, and although the literature on studies applying lectin his-tochemistry data is complicated, a general consensus suggeststhat human upper airways contain primarily α2,6-Sia, and the

Significance

Studies using novel “shotgun glycan microarray” technologyidentify, for the first time to our knowledge, the endogenousreceptors for influenza viruses from a natural host, the pig.Libraries of total N-glycans from pig lung were probed forbinding properties using a panel of influenza viruses isolatedfrom humans, birds, and swine. Natural glycan receptors wereidentified for all viruses examined, and although some dis-played the rather broad α2,3 or α2,6 sialic acid linkage speci-ficity conventionally associated with avian or human viruses,other strains were highly specific, revealing a complexity that hasnot been demonstrated previously. Because pigs are often impli-cated as intermediate hosts for pandemic viruses, these resultsand the approaches describedwill transform our understanding ofinfluenza host range, transmission, and pathogenicity.

Author contributions: L.B.-L., R.L., D.F.S., D.A.S., and R.D.C. designed research; L.B.-L., R.L.,K.C.B., Y.L., S.F.C., X.S., and S.E.G. performed research; M.R.C. contributed new reagents/analytic tools; L.B.-L., X.S., J.H.-M., D.F.S., D.A.S., and R.D.C. analyzed data; and L.B.-L., R.L.,J.H.-M., D.F.S., D.A.S., and R.D.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1L.B.-L. and R.L. contributed equally to this work.2Present address: New England Biolabs, Ipswitch, MA 01938.3Present address: Imperial College London, London SW7 2AZ, United Kingdom.4To whom correspondence may be addressed. E-mail: dsteinh@emory.edu or rdcummi@emory.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1323162111/-/DCSupplemental.

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lower respiratory tract contains a mixture with considerablerepresentation of both α2,3-Sia and α2,6-Sia receptor types (18–21) The respiratory tract of swine also contain both α2,3-Sia andα2,6-Sia, which appear to be distributed similarly to humans (17,22, 23). These observations have been used in support of thehypothesis that the pig may provide an intermediate host for theadaptation of avian strains before emergence in humans or forthe genesis of reassortant pandemic viruses capable of humaninfection following coinfection with avian and human strains (24).Although lectin histochemistry may provide an indicator for

the presence of glycans of particular sialic acid linkage, it doesnot identify actual glycan receptors. The continued developmentand use of new tools in carbohydrate chemistry and an expandingrange of viruses under scrutiny have made clear that not onlysialic acid linkage but underlying glycan composition and struc-ture, chain length, and presentation of receptors also play a rolein the complex interactions that lead to productive attachment ofinfluenza to host cells. Defined glycan microarrays have beendeveloped and successfully applied to examine the bindingspecificity of influenza, as well as other viruses and micro-organisms (25–32). However, a limitation of most such arrays isthat they are based on chemically or enzymatically synthesizedglycan structures and represent only a subset of the endogenouscomplex glycomes of natural hosts. The restrictions imposed bythe synthetic nature of such arrays is emphasized by the obser-vation that mutant influenza viruses have been identified whichreplicate both in cell culture or in the mouse respiratory tractand yet exhibit no detectable binding to the glycans on theConsortium of Functional Glycomics (CFG) array (33). In ad-dition, recent glycan profiles of swine epithelial cells (34), ortissues from human respiratory tissues (21), demonstrate a di-versity of glycan structures that are not comprehensively repre-sented on available glycan arrays. Furthermore, the observationthat α2,6-linked, as well as α2,3-linked, sialic acids were identi-fied by mass spectrometry (MS) in human lung glycans indicatesthat alveolar tropism of influenza virus may not be dictated by itsbinding only to α2,3 glycans (21). The available data highlight theneed for more focused arrays that represent the distribution ofnatural endogenous receptors derived from tissues in which in-fluenza replicates in natural hosts.Our group recently developed a strategy of shotgun glycomics

to successfully identify naturally occurring glycoconjugate ligandsfor glycan-binding proteins (GBPs) and serum antibodies (35). Inthis strategy, summarized in Fig. 1, glycans released from natu-rally occurring glycoconjugates are labeled with a bifunctionalfluorescent tag, fractionated and purified as individual glycans,printed as glycan microarrays, and interrogated with biologicaltargets, such as viruses. The glycans that are strongly bound areconsidered biologically relevant and are structurally character-ized. This novel approach is a shortcut toward defining relevantprotein–glycan interactions, because it limits the difficult task ofstructural characterization of an entire glycome and the labori-ous chemical synthesis of complex and possibly irrelevant gly-cans. Using this strategy for the study of influenza virus bindingis ideal, because natural glycan microarrays can be preparedfrom biologically relevant cells and tissues and directly screenedwith different strains of viruses. In this report, we describe theisolation, fractionation, purification, and interrogation of theN-glycome of the pig lung, a potential site of infection for in-fluenza viruses of avian, swine, and human origin and determinethe binding profiles for a panel of influenza viruses that includeisolates from each of these host species and represent severalHA subtypes. Although some of the expected trends of bindingto α2,3- or α2,6-linked sialic acid receptors were observed, thebinding profiles did not correlate with those of specific lectins,and receptor recognition was quite selective for particularstrains. In several examples, modifications to the glycan back-bone were influential, and for several viruses preferential rec-

ognition of branched chain di- and triantennary structures suggesteda role for multivalent interactions. To our knowledge, this is thefirst identification of binding characteristics of influenza viruses tospecific natural N-glycan derived receptor molecules.

ResultsPig Lung N-Glycan Library. Pig lung tissue was obtained froma commercial vendor or from a germ-free experimental animal,and in each case, 15 g of pig lung tissue yielded ∼3.1 μmol oftotal N-glycans. Following separation by HPLC, Lampire lung-derived glycans were determined to be a mixture of neutral(∼31%), monosialylated (∼23%), disialylated (∼40%), and tri-sialylated (∼6%) species (SI Appendix, Fig. S1). To estimaterecovery of the total N-glycans released by N-glycanase from thestarting material of tryptic glycopeptides, we followed the distri-bution of mannose, which is a common constituent of N-glycans.N-glycanase digestion released ∼90% of the total mannose,whereas ∼5% remained associated with residual N-glycanase–resistant glycopeptides. We analyzed total permethylated N-gly-cans in preparations from the two sources of lung tissue usingMALDI-TOF (36, 37). The results shown in SI Appendix, Fig.S2A revealed that the N-glycans from the purchased pig lunghave compositions that indicate they are dominated by sialylatedand fucosylated biantennary complex-type structures (m/z 2,966.9,2,605.3, etc.). A remarkable signal of predicted triantennarycomplex-type structures (m/z 3,777.2, etc.) is also observed. Signalsof high mannose-type N-glycans, ranging from Man5GlcNAc2to Man9GlcNAc2, were also detected in minor abundance (m/z1,579.8, 1,783.9, 1,988.0, 2,192.1, and 2,396.2). N-glycolylneur-aminic acid (Neu5Gc) (m/z 2,635.3, 2,996.7, etc.) and the pre-dicted αGal epitope (Gal-Gal) (m/z 2,809.5, etc.) are also foundin theN-glycans. Interestingly, we did not detect significant amountsof glycans with poly-N-acetyllactosamine (LacNAc) repeatingstructures (-3Galβ1,4GlcNAcβ1-n). Repeating this analysis onthe permethylated N-glycans from the pathogen-free pig lunggave a similar pattern (SI Appendix, Fig. S2B), which indicatedthe reproducibility of our preparative techniques and that theN-glycome was independent of pathogen exposure.

Fig. 1. Principle of shotgun glycan microarrays from pig lung. Total glycansare isolated from lung tissue glycoproteins or glycosphingolipids, fluo-rescently tagged and separated by HPLC to generate a tagged glycan library(TGL) of purified fractions collected in 96-well plates. Each glycan fraction isthen printed in equal amounts (∼1 nmol) on NHS-activated glass slides andinterrogated with fluorescently labeled influenza A virus. The glycans cor-responding to bound positions on the slide are retrieved from the TGL andstructures determined by MS and other methods to identify the candidateligands for specific viruses.

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Pig Lung N-Glycan Shotgun Glycan Microarrays. The released N-glycans were derivatized with 2-amino-N-(2-aminoethyl)-benza-mide (AEAB) as described previously (38) and separated inthe first dimension using normal-phase HPLC. Each peak fromthe normal phase column was rechromatographed by HPLC ona porous graphitized carbon (PGC) column to obtain a pig lungtagged glycan library (PL-TGL) comprised of 96 N-glycan frac-tions (SI Appendix, Table S1). This library, along with fourstandards, including glycans with α2,3- and α2,6-linked sialicacid, was printed on N-hydroxysuccinamide (NHS)-derivatizedglass slides to produce v1.0 of the pig lung shotgun glycanmicroarrays (PL-SGMs). The N-glycans were quantified basedon fluorescence to estimate relative abundance (SI Appendix,Table S1) and prepare them at 100 μM for printing 0.33 nL ofeach in replicates of four. Before printing, each entry of the PL-TGL was analyzed by MALDI-TOF to identify the molecularions in each glycan target. In many cases, the unique mass pro-vided monosaccharide composition, and most fractions containedonly one or two glycans, thus indicating the relative purity ofthe isolated glycans.

Lectin Binding to Pig Lung N-Glycan Microarray. To confirm that theN-glycans were printed on the PL-SGM, we interrogated thearray using biotinylated lectins with defined glycan bindingspecificity and the results are shown in Dataset S1, which pro-vides the average relative fluorescence units (RFUs), coefficientof variation (%CV), and ranking of the glycans in each analysis,as displayed in the histograms in Fig. 2. The rank of individualglycans in a binding assay was calculated as a percentile of RFUof the glycan divided by the highest RFU values in that assay.Sambucus nigra agglutinin (SNA) binding to the PL-SGM (Fig.2A) indicates the glycans that possess terminal α2,6-linked sialicacid to galactose (26, 39, 40). Maackia amurensis agglutinin-I(MAL-I) binding, which indicates the presence of terminal sialicacid linked α2,3 to galactose (41), is shown in Fig. 2B. Inter-estingly, most of the glycans appear to be homogeneous withrespect to sialic acid linkage because there was very little overlapin the SNA- and MAL-I–binding profiles. Comparison of therankings of MAL-I– and SNA-binding glycans (Dataset S1)indicates significant potential overlap in only three glycan frac-tions, glycans 24, 62, and 63. Binding of these lectins was lostupon treatment of the PL-SGM with neuraminidase, indicating

that recognition was sialic acid-dependent and not attributableto sulfate; recent studies indicate that SNA and MAL-I can bothinteract with 6-O– or 3-O–sulfated terminal galactose residues,respectively (42, 43). The N-glycan PL-SGM was interrogatedwith other defined biotinylated lectins including Aleuria aurantialectin (AAL), which binds α-linked fucose in a variety of link-ages (44) (Fig. 2C); concanavalin A (ConA), which binds complex-type biantennary, hybrid-type, and high mannose-type N-glycansbut not tri- or tetraantennary complex N-glycan structures (45–47) (Fig. 2D); Erythrina crystagalli lectin (ECL), which bindsterminal β1,4–linked galactose (48) (Fig. 2E); and Ricinus com-munis agglutinin-I (RCA-I), which binds terminal β-linked ga-lactose or galactose modified with α2,6-linked sialic acids but notα2,3-linked sialic acids (49) (Fig. 2F). The ECL binding profileafter neuraminidase digestion (Fig. 2G) indicates that the terminalsialic acids were predominantly linked to galactose in the glycanspresented on the PL-SGM. The behavior of lectins binding tothe four control glycans is consistent with the known specificity ofthe lectins, and, taken together, the data indicate that we weresuccessful in printing N-glycans on the PL-SGM and that theseglycans possess both α2,6- and α2,3-linked sialic acid.

Influenza Virus Binding to N-Glycans of the PL-SGM. To explore therange and diversity of potential influenza virus natural glycanreceptors present in the pig lung, a panel of viruses isolated fromhuman, swine, and avian species was examined. The viruses se-lected represent a broad spectrum of viruses encompassing dif-fering species of origin, time, and geographic location of isolationand various HA subtypes including H1, H2, H3, and H6. (Table 1).Included in the panel were seasonal human isolates of both anH1N1 subtype (A/Pennsylvania/08/2008) and an H3N2 subtype(A/New York/55/2004), as well as 2009 human pandemic (pdm)H1N1 strains A/California/04/2009, A/Mexico/4487/2009, and A/Texas/15/2009, which represent viruses that displaced the pre-viously circulating H1N1 seasonal strains (i.e., A/Pennsylvania/08/2008). These 2009 viruses are direct human isolates (not pas-saged in eggs) from the H1N1 pandemic, which caused differentpathogenic symptoms in humans and have strict α2,6-linked sialicacid specificities (50). The contemporary swine viruses of thepanel were isolated from pigs at the time of the 2009 humanpandemic and are thought to be representative of viruses with

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Fig. 2. Lectins binding to pig lung N-glycan micro-array. Glycan microarray TGL fractions were incubatedwith lectins of different specificity to validate thearrays. The lectins used were: SNA (specific forSiaα2,6Gal-R) (A); MAL-I (specific for Siaα2,3Gal-R) (B);AAL (specific for fucose) (C); ConA (specific for bian-tennary but not triantennary complex N-glycan struc-tures) (D); ECL (specific for terminal galactose) (E );and RCA-I (specific for terminal galactose or α2,6-linked sialic acids but not α2,3-linked sialic acids) (F).The slides were also treated with neuraminidase, fol-lowed by binding of ECL (G) to identify glycans withnewly exposed galactose sugars to which sialic acidshad been linked. Fractions 1–96 are N-glycans isolatedfrom pig lung: 1–14, neutral; 15–47, monosialyl; 48–77,disialyl; and 78–96, trisialyl. Standards include: 97, NA2(nonsialylated biantennary N-glycan); 98, NA2-Sia2,3;99, NA2-Sia2,6; and 100, Man5.

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related gene constellations that may have given rise to the hu-man pandemic strains. The three swine strains, A/sw/Minnesota/02719/2009 (H3N2), A/sw/Minnesota/02749/2009 (H1N1), andA/sw/Illinois/02860/2009 (H1N2), have several internal genes incommon with one another and with human pandemic strains butdifferent surface glycoprotein combinations. The Ruddy Turn-stone viruses are low-pathogenicity avian H6N1 and H1N9 sub-types. A/Brisbane/59/2007 (H1N1) is a vaccine strain that has beenpassaged extensively in eggs, and A/New York/55/2004 (H3N2) isa seasonal virus, which was passaged one to two times in eggsbefore transfer to MDCK cells. None of the other human or swineviruses analyzed have been passaged in embryonated chicken eggs,a common procedure for growing field or clinical isolates that isknown to select for changes in the binding characteristics, includingadaptation to α2,3-sialylated receptors (51). We have shown thatA/Brisbane/59/2007 (H1N1) has broad specificity for both α2,3- andα2,6-sialylated receptors (50).The 11 viruses were labeled with a fluorescent tag and ana-

lyzed on the glycan microarray (Version 5.1) developed by theCFG (www.functionalglycomics.org), which contains 610 immo-bilized chemoenzymatically generated glycans, representing avariety of defined glycan epitopes or determinants found onN- and O-glycans and glycosphingolipid-derived mammalian-type glycans. The binding patterns of the viruses on the CFGarray demonstrated that all of the viruses bound strongest tosialylated N-glycans and common N-glycan epitopes (Fig. 3).Each preparation of labeled virus was divided and examined inparallel on the CFG array and on the N-glycans of the PL-SGM.All of the isolates were prepared in this way to maintain consis-tency between array experiments. None of the viruses bound toneutral glycans, but many mono, di-, and trisialylated N-glycanswere bound by the different virus strains (Fig. 3), and each of theviruses displayed a distinct binding pattern to the PL-SGM.Waterfowl isolates. The binding patterns of A/Ruddy Turnstone/650625/2002 (H6N1) and A/Ruddy Turnstone/DE/650645/2002(H1N9) were compared on the CFG glycan array with the helpof the Cross Analysis tool in the GlycoPattern website (https://glycopattern.emory.edu). This comparison indicated a clear re-lationship between these two strains. Both avian strains dem-onstrated a definite specificity for NeuAcα2,3Gal-terminatingglycans. Of the two strains, A/Ruddy Turnstone/DE/650645/2002(H1N9) demonstrated a much more restricted specificity, bind-ing primarily to linear fucose-containing glycans. Interestingly,all of the glycans bound by strain A/Ruddy Turnstone/DE/650645/2002 (H1N9) were also bound by strain A/Ruddy Turn-stone/650625/2002 (H6N1); however, A/Ruddy Turnstone/650625/

2002 (H6N1) bound many more glycans with lower ranking(14–40 percentile), many of which were branched glycans. Whena similar comparison was made between these two strains onthe PL-SGM (Fig. 3 and Dataset S2), no such overlap was ob-served. The A/Ruddy Turnstone/650625/2002 (H6N1) strainbound 34 glycan fractions ranked between 100 and 20 percentile,whereas the four glycans bound strongest (ranked 73–100) bystrain A/Ruddy Turnstone/DE/650645/2002 (H1N9) were amongthe weaker binding glycans for strain A/Ruddy Turnstone/650625/2002 (H6N1).Swine isolates. The three swine isolates A/swMinnesota/02749/2009(H1N1), A/sw/Minnesota/02719/2009 (H3N2), and A/sw/Illinois/02860/2009 (H1N2) were analyzed on the defined glycan micro-array from the CFG and the PL-SGM (Fig. 3 and Dataset S2) inparallel. These three isolates were all generally specific forα2,6-linked sialic acids and bound strongest to sialylated linearand branched lactosamine- or polylactosamine-containing gly-cans on the CFG array. When analyzed on the PL-SGM, bindingof the three swine isolates correlated well with SNA binding,which was consistent with the and swine isolates being generallyspecific for α2,6-Sia. The three strains analyzed bound a numberof common glycan fractions, including glycans identified with chartidentification nos. 64–67 and 85–89 (Fig. 3 and Dataset S2).Human isolates. We analyzed a vaccine strain, A/Brisbane/59/2007(H1N1), seasonal strains A/Pennsylvania/08/08 (H1N1) andA/New York/55/2004 (H3N2), and three pandemic strains ofH1N1 A/California/04/2009, A/Mexico/INDRE4487/2009, andA/Texas/15/2009 side-by-side on defined glycan microarrays ofthe CFG and the PL-SGM (Fig. 3 and Dataset S2). The vaccinestrain A/Brisbane/59/2007 (H1N1), which had undergone mul-tiple passages in eggs, had an extremely broad specificity, binding∼100 of the 158 sialylated glycans on the CFG array, including50 of the 88 glycans that have α2,3-linked sialic acid. Among theseasonal and pandemic strains analyzed, the seasonal strainA/Pennsylvania/08/08 (H1N1) had the broadest specificity, bind-ing 55 glycans in the top 80 percentile ranking, which includedonly 11 glycans containing only α2,3–linked sialic acid. The otherseasonal strain A/New York/55/2004 (H3N2) displayed the mostrestricted specificity on the CFG-defined array with binding toonly 10 glycans considered as significant; all of which containedα2,6Sia, and in most cases, these terminated linear lactosamineand polylactosamine structures. Among the pandemic strains from2009, A/Texas/15/2009 (H1N1) showed the broadest specificity,binding 38 sialylated glycans, all containing α2,6-linked sialicacid. Glycans bound by the other pandemic strains, A/California/04/2009 (H1N1) and A/Mexico/INDRE4487/2009 (H1N1), werealso bound by A/Texas/15/2009. When the human isolates wereanalyzed on the PL-SGM, the vaccine strain A/Brisbane/59/2007(H1N1) appeared to have the broadest specificity for the pig lungN-glycans, binding 28 of the 96 glycans printed on the PL-SGM(Fig. 3 and Dataset S2), which is consistent with the broadspecificity of this strain on the defined CFG array. Similar to itsbehavior on the CFG array, the seasonal strain A/New York/55/2004 (H3N2) showed the most restricted specificity on the PL-SGM (Fig. 3 and Dataset S2), with only four glycans bound tosignificant levels. Thus, the general behavior of the human isolateson the PL-SGM was very similar to their behavior on the definedglycan array.Some general observations from the CFG and the PL-SGM

data can be made when comparing the isolates, especially be-cause they are grouped by specificity. The avian isolates, whichdemonstrated a preference for terminal α2,3-Sia, preferentiallybound to glycans displaying core fucosylation of N-glycans, es-pecially A/Ruddy Turnstone/DE/650645/2002 (H1N9). Forboth avian viruses, residues of the same structure tend tobind with greater apparent affinity if a core fucose is present.However, the subset of viruses that bind terminal α2,6Sia do notshow a preference for core fucosylated glycans. For example,

Table 1. Virus strains tested on the microarrays

Virus Subtype Host

Avian isolatesA/RuddyTurnstone/DE/650625/2002 H6N1 AvianA/RuddyTurnstone/DE/650645/2002 H1N9 Avian

Contemporary swine isolatesA/sw/Minnesota/02749/2009 H1N1 SwineA/sw/Minnesota/02719/2009 H3N2 SwineA/sw/Illinois/02860/2009 H1N2 Swine

Vaccine strainA/Brisbane/59/2007 H1N1 Human

SeasonalA/Pennsylvania/08/2008 H1N1 HumanA/New York/55/2004 H3N2 Human

Pandemic H1N1A/California/04/2009 H1N1 HumanA/Mexico/InDRE4487/2009 H1N1 HumanA/Texas/15/2009 H1N1 Human

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the A/sw/Minnesota/02749/2009 (H1N1) virus binds two struc-tures in the highest percentiles (100 and 96) that differ only inthe addition of core fucose residues. For the mammalian viru-ses, the sialic acid linkage and composition of the terminalsugars seem to be more critical determinants of binding, be-

cause all glycans with these terminal characteristics are bound withsimilar affinity, regardless of chain length or whether they arebranched or unbranched. This finding suggests that for the recog-nition of terminal α2,6-Sia, it is the extreme reducing end of theglycan structure that is important, and this may be attributable to

A

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Fig. 3. Influenza virus binding to glycan microarrays. Eleven viruses have been tested including isolates from vaccine (A/Brisbane/59/2007), human (A/NewYork/55/2004, A/Pennsylvania/08/2008, A/California/04/2009, A/Mexico/INDRE4487/2009, and A/Texas/15/2009), avian (the Ruddy Turnstone viruses), and swine(A/sw/Minnesota/02719/2009, A/sw/Minnesota/02749/2009, and A/sw/Illinois/02860/2009). (A) PL-SGM array. Fractions 1–96 are N-glycans isolated from piglung: 1–14, neutral; 15–47, monosialyl; 48–77, disialyl; and 78–96, trisialyl. Standards include: 97, NA2; 98, NA2-Sia2,3; 99, NA2-Sia2,6; and 100, Man5. (B) CFGarray. This microarray contains 610 immobilized glycans, representing a variety of defined glycan epitopes or determinants. Only the epitopes containingterminal sialic acid are represented here. The glycans are grouped by terminal linkage with the α2,3 linkage type highlighted in green, the α2,6 linkagehighlighted in blue, and the α2,8 linkage highlighted in purple.

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the way the terminal sialic acid in the α2,6 conformation fits intothe binding pocket on the HA. The avian viruses seem to rely moreon core structure and modification for binding preference incomparison with the mammalian strains, which could be a conse-quence of the HA receptor interactions beyond the sialic acidbinding pocket of HA.

Characterization of Glycans That May Function as Influenza VirusReceptors. The N-glycans on the PL-SGM that were consis-tently bound at high levels by labeled viruses were consideredcandidates as natural receptors for influenza and chosen forfurther structural analysis. Glycans 34, 64, 67, and 88 showedrelatively strong binding by human, swine, and avian influenzavirus strains, whereas glycans 56, 81, and 83 were recognizedby the avian virus strains and the human and swine strains,A/Brisbane/59/2007 (H1N1) and A/sw/Illinois/02860/2009 (H1N2),that showed a broader specificity of binding (Fig. 3 and DatasetS2). Therefore, the structures of these glycans were analyzed byMALDI-TOF, tandem mass (MS/MS) spectrometry, and thepattern of binding by lectins with defined specificities (SI Ap-pendix, Figs. S3–S8, Datasets S1 and S3, SI Appendix, Table S5),to allow a prediction of glycan structure using the general ap-proach of metadata-assisted glycan sequencing (MAGS) (52).The glycans were analyzed in both positive and negative mode

MALDI-TOF to check for sulfate groups in the glycans. A dif-ference of m/z 78 (or 56 because of sodium adduct) indicateda sulfo group. The glycan structures were also analyzed byMS/MS to determine the backbone structures. In addition, theglycans were further treated with neuraminidase to demonstrate thesialic acid moiety and linkage, which is also easier for MS/MS

characterization of glycan backbones. For example, when glycan67 was treated with neuraminidase, its HPLC profile was shifted,indicating sialic acid removal (Fig. 4A). MS revealed glycan 67has a molecular mass of 2,553.2 [M+Na]+, very close to calculatedmolecular mass of 2554.9 [M+Na]+, indicating a composition offive hexoses (Hex), four acetyl hexosamines (HexNAc), two sialicacids (Neu5Ac), and one fucose (Fuc) (Fuc1NeuAc2Hex5HexNAc4)(Fig. 4B). Together with the molecular weight change determinedby MS (Fig. 4C), we propose two sialic acids in the glycan moiety.The MS/MS further confirmed the backbone structures (Fig. 4C).Consistent with the MS data, the glycan was bound by SNA andRCA-I, which is consistent with Siaα2,6Galβ1,4GlcNAc, whereasConA binding is consistent with a biantennary N-glycan (not atriantennary complex N-glycan structure) and AAL binding withthe presence of fucose. Taken together, these results indicate thatglycan 67 is a biantennary complex-type N-glycan terminatedwith Siaα2,6Galβ1,4GlcNAc and containing a core fucoselinked to the reducing end GlcNAc, as shown in Fig. 5. Thisstructure is consistent with no binding by ECL (free terminalgalactose) or MAL-I (α2,3-linked sialic acids). The MS analysisof glycan 64 predicts a biantennary complex-type N-glycan withno core fucose (Fig. 5) and a lectin binding pattern identical toglycan 67 with the exception of AAL, which is consistent withthe absence of Fucose in this glycan. In addition, glycan 64demonstrated a lectin-binding profile identical to the standardglycan 99; glycans 64, 67, and 99 all showed similar virusbinding profiles.Based on a similar approach, the other glycans were identified

as bi- and triantennary complex-type N-glycans (Fig. 5), as wellas a sulfated biantennary structure (glycan 83). The sialic acid

2000 2250 2500 2750 3000 3250 3500 3750

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Fraction 87

Galactose (Gal) Glucose (Glc) N-Acetylglucosamine (GlcNAc)

Mannose (Man) Fucose (Fuc) N-Acetylneuraminic acid (Neu5Ac)

Fig. 4. Determination of the structure of fraction 67.(A) HPLC profile of fraction 67 before and after treat-ment of neuraminidase. (B) The MS spectra of fraction67, in both positive (Upper) and negative (Lower)mode. (C) The MS (Upper) analysis of fraction 67 aftertreatment of neuraminidase and MS/MS (Lower) anal-ysis of parent ion 1972.

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linkages were identified from the lectin-binding profiles (SNAand MAL-I). All of the candidate virus-binding receptors ter-minated in α2,6-Sia, except glycans 56, 81, and 83, which termi-nated in α2,3-Sia based on lectin binding. Based on thesepredicted structures, comparisons with the structures present onthe CFG array can be made. Glycans 64 and 67 have cognatestructures on the CFG array; glycans 55, 318, 325, and 366 cor-respond to glycan 64 on the PL-SGM; and glycans 482 and 483correspond to glycan 67 on the PL-SGM. The remaining struc-tures from the PL-SGM that we have characterized are not rep-resented on the CFG array. This finding confirms the noveltyand significance of the shotgun array technique. To our knowl-edge, these data demonstrate for the first time the direct asso-ciation of naturally occurring N-glycans from pig lung with bindingof influenza viruses, supporting their function as potential recep-tors for these influenza viruses in vivo.

Studies on Virus Binding to O-Glycan and Glycolipids. Although theabove results define the ability of influenza viruses to recognizeendogenous N-glycans, we also explored the potential of virusesto bind other classes of glycans, including O-glycans and glyco-lipids. Using the shotgun glycomics approach described pre-viously (35), we prepared total O-glycans and glycolipids. Thesewere derivatized with AEAB, separated by multidimensionalHPLC, and printed on glycan microarrays. Although a wide va-riety of glycans were detected by specific lectin binding (SI Ap-pendix, Figs. S9 and S11), as might be expected from previousstudies of lung tissue glycomics of other species, no significantbinding of influenza viruses was observed on shotgun micro-arrays of the O-glycans or glycolipid-derived glycans (SI Appen-dix, Figs. S10 and S12). Therefore, based on our available data,pig lung-derived N-glycans are recognized by all of the influenzaviruses studied, but we observed no significant binding to piglung-derived O-glycans or glycolipids.

DiscussionThe data presented here provide fundamental contributions tothe understanding of several separate but interrelated areas in-volving host–pathogen interactions. From a broad perspective, itrepresents, to our knowledge, the first interrogation of the piglung glycome and opens up the field for investigating numerouseconomically important respiratory diseases of swine. Further-more, the extension of the technology, reported here, to human

tissue should pave the way for the understanding of varioushuman respiratory diseases and promote the development ofnovel therapeutic intervention strategies. Our results also revealnovel insights into the recognition of natural glycan receptors inthe pig lung by a diverse array of influenza viruses and show thatbinding does not necessarily follow basic assumptions that havebeen made regarding α2,3-Sia and α2,6-Sia linkage specificityfor avian and human viruses, respectively.Because swine are susceptible to both avian and human in-

fluenza viruses and are considered a possible intermediate hostfor the generation of pandemic human viruses, there has beensignificant interest in characterizing the glycome using glycomicprofiling of cells and tissues by MS (34, 53). These studies revealthe presence of a diverse range of glycans terminating in Galα1-3Gal or sialic acid in both α2,3- and α2,6- linkages, where NeuAcwas more prevalent than NeuGc. Glycan microarray analysesof the same viruses that were used to infect primary swinerespiratory epithelial cells demonstrated the viruses preferredα2,6-linked sialic acid on the array, and infectivity studiesshowed a clear dependence of surface α2,6–linked sialic acidfor influenza infection (34). These studies indicated that Neu-Acα2,6-containing N-glycans are important for virus infection, butstructural definition of the natural glycan receptors was predictedonly by comparison of the similarity of the predicted glycanstructures determined by MS analysis with the defined glycans onthe CFG glycan microarray, and therefore these studies arecorrelative. In addition, glycans expressed by cultured cells iso-lated from organs may be misleading and not necessarily re-flective of glycans in the native organ, because cell cultureconditions have been known to select for changes in glycanstructures (54, 55). Our approaches involve direct analysis ofglycan structures in the relevant tissue. We analyzed two in-dependent samples of pig lung tissue, observing glycans in therange of 1,500–5,000 Da, and the data are similar to the profilesreported previously for trachea, lung, and primary swine re-spiratory epithelial cells. Whereas the MS profile of primaryswine respiratory epithelial cells observed glycans over an ex-tended mass range of 1,500–8,000 Da (34), the large, multi-antennary polylactosamine-containing glycans that constitutethe high end of the mass range were present in vanishingly lowamounts. We were unable to detect these glycans on the lungsample we analyzed, and they were not reported as present intrachea or lung by Sriwilaijaroen et al. (53); however, no testingof influenza binding to these glycans was performed in this study.The defined glycan microarray currently available from the CFGcontains a variety of multiantennary glycans possessing longstretches of polylactosamine terminated in sialic acid, and nu-merous analyses of influenza virus binding to this array of de-fined glycans demonstrate strong virus binding to such structures.Based on these data, it has been suggested that such long sialy-lated polylactosamine structures may be important in productivevirus infection (56, 57). Such conclusions, however, may be opento question, because the amounts of such structures actuallyidentifiable in the tissues appears to be extremely low or un-detectable. As we have demonstrated, if sufficient material isavailable to undergo detailed structural analysis, the shotgunglycan microarray approach makes it possible to identify po-tential receptors based on their presence in natural tissue andtheir ability to participate in functional binding.A significant body of work has accumulated from many in-

vestigators on analyses of influenza A virus binding to definedsialylated glycans on glycan microarrays and despite this ap-proach, we still have not defined the exact structure of a glycanreceptor that functions in productive infection. These data wererecently documented in a multidisciplinary study of influenza Avirus–glycan interactions using a glycomics approach (21). In thisextensive study, the N-glycan and O-glycan composition of hu-man lung and bronchus were analyzed by MS. A wide spectrum

α2,6

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Fig. 5. The structures of selected N-glycans. The bound glycans wereidentified as bi- and triantennary complex-type N-glycans, as well as a sul-fated biantennary structure with α2,6-linked sialic acids (Upper) or α2,3-linked (Lower) sialic acids. See Fig. 4 for glycan cartoon key.

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of α2,3-Sia– and α2,6-Sia–terminated glycans were found in boththe lung and the bronchus. These results indicate that alveolartropism by influenza viruses appears to involve both α2,3-Sia–and α2,6-Sia–terminated glycans and emphasizes the complexityof this system where the cell surface glycans, as well as glyco-conjugates of the respiratory tract fluid probably, play importantbut undefined roles. The MS data allowed for structural pre-dictions that were used in combination with influenza A virus-binding data from four published glycan arrays comprised ofdefined glycans to determine whether they could predict repli-cation of human, avian, and swine viruses in human ex vivo re-spiratory tract tissues. The CFG defined glycan microarray wasthe most comprehensive of the four evaluated. Although theCFG array possessed the greatest diversity of sialylated glycans,the influenza A binding data from this array was not predictive ofproductive virus replication in human respiratory tract tissue. Thestudy concluded that more comprehensive and focused arraysneed to be developed to evaluate emerging influenza A viruses. Itis tempting to conclude that the solution to this dilemma is simplyto synthesize the glycans corresponding to the sialylated glycanspredicted by glycomics analyses. However, the glycan structuresbased on profiling are predicted structures, and details of all ofthe structures are not available. Furthermore, synthesis of largeN-glycans is an extremely difficult task, and synthesis of all pos-sible glycans predicted by MS analysis of the human respiratorytract tissues is currently not possible. Thus, the shotgun glycomicsapproach, which will obtain and identify the glycans from thenatural source of tissue or glycoconjugate, has a high probabilityof presenting cellular glycans that function as viral receptors.With regard to the specific binding properties of influenza

viruses, our data are generally consistent with the well-docu-mented observations that some strains bind preferentially to ei-ther α2,3-Sia or α2,6-Sia and that these specificities can provideuseful indicators for potential involvement in host species spec-ificity. However, our results also indicate that influenza virusreceptor recognition is much more complicated than might beascertained from such basic assumptions. One important obser-vation is that virus binding profiles do not directly correlate withthe recognition profiles determined for the lectins SNA andMAL-I, which are often used to assay for the abundance ofspecific “receptors” of α2,6-Sia or α2,3-Sia linkages, respectively(17, 19, 20, 22, 58). Whereas some viruses, such as the vaccinestrain A/Brisbane/59/2007 (H1N1) and the low-pathogenicityavian isolates A/Ruddy Turnstone/650625/2002 (H6N1) andA/Ruddy Turnstone/650645/2002 (H1N9), bound to glycans thatbroadly reflected the profile observed with the specific lectins,other viruses, such as the pdmH1N1 strains A/California/04/2009, A/Mexico/INDRE4487/2009, and A/Texas/15/2009 and theseasonal strains A/Pennsylvania/08/2008 (H1N1) and A/NewYork/55/2004 (H3N2), bound to highly specific subsets of gly-cans. Interestingly, for a number of virus strains, significantbinding was observed to glycans recognized by neither SNA orMAL-I. In other examples, viruses bound efficiently to bothα2,3-Sia– and α2,6-Sia–linked glycans. A/swine/Minnesota/02719/2009 (H3N2) and A/swine/Illinois/02860/2009 (H1N2) each bindthe same three glycans that are recognized as α2,3-Sia and α2,6-Sia. Similarly, three glycans of both specificities are bound by A/Brisbane/59/2007 (H1N1), A/California/04/2009 (H1N1), and A/New York/55/2004 (H3N2). This dual specificity may have beenexpected for the vaccine strain A/Brisbane/59/2007 (H1N1),because it represents a human strain that had been subsequentlypassaged in embryonated chicken eggs, where recognition ofα2,3-Sia linkages may have been selected but was unexpected forother viruses that bound the same glycans or for the two avianstrains A/Ruddy Turnstone/DE/650645/2002 (H1N9) and A/RuddyTurnstone/650625/2002 (H6N1). Although these avian strainsshowed the expected binding to α2,3-Sia glycans that were notrecognized efficiently by human or swine strains, they also bound

to several of the α2,6-Sia–containing glycans. The swine virusesA/sw/Minnesota/02749/2009 (H1N1), A/sw/Minnesota/02719/2009(H3N2), and A/sw/Illinois/02860/2009 (H1N2) were all isolatedin 2009 but displayed broad differences with respect to the numberof glycans recognized, with the A/sw/Minnesota/02749/2009 (H1N1)virus showing a much more restricted binding profile. Althoughthese swine viruses were isolated in the US Midwest in the sameyear, they are different HA and NA subtypes. Another illumi-nating observation relates to the valency of receptors recognized,because some of the more highly specific strains were more se-lective for di- and triantennary glycans. Interestingly, the 2009human pandemic strains known generally to have low bindingactivity (50, 59), were more selective for the multivalent receptors.The glycans that bound virus and generated a ranking of

greater than 40% for at least one of the viruses interrogated areshown in Dataset S3. The glycans selected for further analysiswere glycans 34, 56, 64, 67, 81, 83, and 88, and their structuresare shown in Fig. 5. The characterized glycans do have somesimilarities, which suggests that these features might be relevantdeterminants of binding. Each structure is branched, eitherbiantennary or triantennary, and of the eight structures charac-terized, seven contain a core fucose modification. The motif ofterminal Sia-Gal-GlcNac is represented in all of the structuresthat have been characterized which is in accordance with generalobservations of influenza binding. These structures were chosenbecause of their abundance, purity, and relative ease of charac-terization. Each is a principal binder for at least one and oftenmany of the viruses tested, but they are not necessarily thehighest binding structure. Further structural characterization of thecomponents of the PL-SGM will reveal the true relevance of thesecomplex N-glycan structures with respect to influenza binding.The observation that swine and human isolates recognize

a number of the same glycans is consistent with our knowledge ofthe swine as a suitable host for a range of influenza viruses, andthis is supported by the observation that avian isolates examinedhere also recognized a range of both α2,3-Sia– or α2,6-Sia–linkedreceptors. The extension of the pig lung glycan array technologyreported here to human respiratory tract tissues, and to the tis-sues of the intestinal tract of avian species, will be required toidentify whether specific receptors exist that are critical deter-minants of host range.Our current understanding of influenza binding specificity has

been based largely on studies of binding to artificial receptoranalogs of defined α2,3-Sia or α2,6-Sia linkage or to heteroge-neous collections of glycans present on cell surfaces character-ized only by reactivity with specific lectins. Such studies haveproven useful for broad generalizations of species specificity, byrelating them to the amino acid composition, and structuralaspects of the HA-binding site using viruses isolated from dif-ferent hosts. However, a striking feature of these results pre-sented here relates to distinct differences in the binding profilesdetected, even among strains that would be broadly classified inthe same category based on overall α2,3-Sia or α2,6-Sia preference.These distinctions relate to glycan backbone, modifications, andvalency of the receptor species and highlight a level of com-plexity that is not easily explained based on our current knowledgeof HA structures. When considering productive virus attachmentin the context of a natural host, the situation becomes even morecomplicated, because factors relating to NA activity and speci-ficity, relative ratios and distribution of HA and NA glyco-proteins on viral surfaces, and virion morphology may all playa role. The mechanisms by which individual virus strains mod-ulate their detailed selectivity for receptors, and the implicationsfor host range and pathogenicity, are not currently appreciated.However, our current studies on swine tissue suggest that it willrequire a much broader understanding of the identity, density,and distribution of natural endogenous receptors at sites of in-fection in many of the natural hosts for influenza viruses.

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Experimental MethodsMaterials. Pig lung was purchased from Lampire Biological Products orobtained from germ-free animals from the College of Veterinary Medicine,University of Minnesota, and stored at −80 °C until use. Euthanasia andtissue harvest was approved by the University of Minnesota InstitutionalAnimal Care and Use Committee, was conducted in compliance with theAnimal Welfare Act, and adhered to principles stated in the Guide for theCare and Use of Laboratory Animals (60) (protocol 1011A92934). All chem-icals were purchased from Sigma-Aldrich and were used without furtherpurification. HPLC solvents were purchased from Fisher Scientific. An Ultra-flex-II TOF/TOF system (Bruker Daltonics) was used for MALDI-TOF MSanalysis. Both reflective positive and reflective negative modes were usedas indicated in the figures. The 2,5-dihydroxybenzoic acid [5 mg·mL−1 in 50%(vol/vol) acetonitrile, 0.1% TFA] was freshly prepared as the matrix and 0.5μL of matrix solution was spotted on an Anchorchip target plate (200 μm or400 μm) and air-dried. Then 0.5 μL of sample solution was applied and air-dried. An ozone generator (model OZV-8; Ozone Solutions) was used togenerate ozone from high purity oxygen.

Isolation of N-Linked Glycans. Frozen pig lung (free of trachea) was homog-enized at 4 °C and adjusted to a chloroform/methanol/water ratio of 4:8:3(vol/vol/vol). The soluble fraction was reserved as a source of glycolipids andthe insoluble material, collected by centrifugation, was resuspended in in8 M GuHCl in 0.2 M Tris (pH 8.2) and subsequently reduced by DTT andalkylated by iodoacetamide. Denatured material was dialyzed against waterovernight and lyophilized. The lyophilized proteins were digested withtrypsin in 50 mM phosphate buffer (pH 8.2) to generate glycopeptides thatare susceptible to digestion with Peptide N-Glycosidase F (PNGase F; NewEngland Biolabs). After PNGase F digestion, the released N-glycans wereobtained in the run-through of C-18 cartridges (Waters) and collected bysubsequent application to and elution from a Carbograph cartridge (All-tech), and subsequently the free glycans were derivatized with AEAB aspreviously described (38). The bifunctional fluorescent AEAB tag permitsquantification of total glycans and provides a primary amino group forcoupling to NHS-activated glass surfaces when printed as microarrays.

Isolation of O-Glycans and Glycolipids. O-glycans were isolated following thenonreductive elimination procedure using ammonium hydroxide/ammoniumcarbonate developed by Huang et al. (61). The free reducing glycans wereconjugated with AEAB as described for N-glycans. Free glycans from glyco-sphingolipids were isolated following the ozone-mediated nonreductive deli-pidation (62). The free reducing glycans were conjugated with AEAB asdescribed for N-glycans. One-dimensional HPLC using a normal phase columnwas carried out for bothO-glycans and glycans from glycolipids. Fractions werecollected, lyophilized, quantified based on fluorescence, and printed without2D HPLC separation. No apparent binding was observed for these microarrays.

Permethylation of Glycans. For structural profiling by MALDI-TOF analysis, theN-glycans were permethylated using sodium hydroxide and iodomethane inDMSO (37) before derivatization with AEAB. The permethylation reactionwas quenched with water after 1 h, and the samples were extracted withchloroform. The organic phase was washed three times by ddH2O and gentlydried with nitrogen gas under a hood and redissolved in 1:1 methanol:water.The samples were loaded onto C-18 cartridge and eluted stepwise with waterand 15%, 35%, 50%, and 75% aqueous acetonitrile. Permethylated glycanswere usually present in the 35%, 50%, and 75% acetonitrile fractions. Theglycans were annotated with the help of the GlycoWorkbench tool.

HPLC Separation of AEAB.An HPLC CBM-20A system (Shimadzu), coupled witha UV-light detector (SPD-20A) and a fluorescence detector (RF-10AXL), wasused for HPLC analysis and separation of AEAB-conjugated glycans; 2D HPLCseparation, using normal-phase and reverse-phase columns as previouslydescribed (38) provided multiple fractions that were largely homogeneous.The quantified fractions obtained after 2D HPLC were then lyophilized andadjusted to 200 μM with deionized water and stored frozen as the PL-TGL.

Viruses and Cells. Virus stocks representing swine, human, and avian isolateswere selected based on their availability and the relevance of the strain withrespect to circulation in a population. The virus stocks were grown in Madin–Darby canine kidney (MDCK) cells that were maintained using DMEMsupplemented with 5% FBS and penicillin–streptomycin. Briefly, 85–90%

confluent MDCK cells grown in T175 flasks were washed twice with PBS,overlaid with 10 mL of serum-free DMEM supplemented with 1 μg/mLtosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin, and infec-ted with a 1:1,000 or 1:10,000 dilution of the original virus stock. Cells wereincubated with rocking for 1 h at ambient temperature, the inoculum wasremoved, and 30 mL of serum-free DMEM supplemented with 1 μg/mLTPCK trypsin was added. The flasks were incubated at 37 °C for 2–3 d untilmonolayers were 80–90% destroyed. Virus was then harvested in the infectedcell supernatant and frozen at −80 °C for subsequent purification. The stocksof all viruses used in these studies were resequenced after the limited ampli-fication in MDCK cells before labeling for glycan array analysis. We detectedno sequence changes and no evidence for observable heterogeneity.

Purification of Virus Strains. Preparations of each virus were done so that eachwould yield enough volume for multiple experiments so that essentially thesame preparation could be used on each array at the same time. For viruspreparations, infected cell supernatants were clarified by low speed centri-fugation, and then pelleted through a 25% sucrose cushion in NTE buffer (100mM NaCl, 10 mM Tris, 1 mM EDTA). Purified viruses were pelleted by cen-trifugation in a Beckman Coulter SW32 rotor at 28,000 rpm for 2 h, resus-pended in 2 mL of PBS buffer, aliquoted, and frozen at −80 °C. Frozen, purifiedvirus strains were later thawed, and the hemagglutination units and plaqueforming units (plaque forming units per milliliter) titers were determined usingstandard techniques.

Agglutination of Erythrocytes. Chicken erythrocytes were acquired fromLampire Biologicals as whole-blood preparations. Whole-blood preparationswere washed two times with 1× PBS and diluted to 0.5% for hemaggluti-nation experiments, which were performed using standard techniques.Briefly, 0.5% erythrocyte preparations were added to twofold serial dilu-tions of 50 μL of virus stock for 1 h to determine the HA titer. This procedurewas done before and after labeling the virus with Alexa Fluor 488 to mon-itor the HA titer during the labeling process. Infectious titers of virus stockswere also determined by plaque assay on MDCK cell monolayers.

Fluorescently Labeled Viruses. Binding of fluorescently labeled influenza viruswas performed as previously described (50, 63, 64). Briefly, 200 μL of viruswas incubated with 25 μg of Alexa Fluor 488 (Invitrogen) in 1 M NaHCO3 (pH9.0) for 1 h at room temperature. Labeled viruses were dialyzed against PBSusing an Mr 7,000 molecular weight cutoff Slide-A-Lyzer minidialysis unit(Thermo Scientific) overnight at 4 °C. In all cases, labeled viruses were used inexperiments the following day.

Microarray Preparation and Analysis. NHS-activated slides (Nexterion H) werepurchased from SCHOTTNorth America. Corning Epoxy slides were purchasedfrom Corning Incorporated Life Sciences. Noncontact printing was performedusing a Piezoarray printer (Perkin-Elmer) as previously described (65). Allsamples were printed within 10% variation of 0.33 nL at a concentration of100 μM in phosphate buffer (300 mM sodium phosphates, pH 8.5). Beforeassay, the slides were rehydrated for 5 min in TSM buffer [20 mM Tris·HCl,150 mM sodium chloride (NaCl), 0.2 mM calcium chloride (CaCl2), and 0.2mM magnesium chloride (MgCl2)], and the analyses were carried out forlectins and viruses as previously described for 1 h at 4 °C to prevent neur-aminidase activity (63, 65). Biotinylated lectins (Vector Labs) were used ascontrols in the binding assay and the bound lectins were detected by a sec-ondary incubation with cyanine 5–streptavidin (5 μg·mL−1; Invitrogen). Formultipanel experiments on a single slide, the array was constructed accordingto the dimension of a standard 16-chamber adaptor. The adaptor was appliedto the slide to separate a single slide into 16 chambers sealed from each otherduring the assay. The slides were scanned with a ProScanArray microarrayscanner (Perkin-Elmer) equipped with four lasers covering an excitation rangefrom 488 to 637 nm, and the scanned images were processed to Excelspreadsheets as previously described (63, 65). For cyanine 5 fluorescence,649 nm (excitation) and 670 nm (emission) were used. For Alexa Fluor 488fluorescence, 495 nm (excitation) and 519 nm (emission) were used.

ACKNOWLEDGMENTS. This work was supported by the US Depart-ment of Health and Human Services Grants HHSN266200700006C andHHSN272201400006C (National Institute of Allergy and Infectious DiseasesCenters of Excellence for Influenza Research and Surveillance) and NationalInstitutes of Health Grants GM098791 (to R.D.C.) and GM085448 (to D.F.S.).

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