Influenza A viruses can bind sialic acid–terminating glycan receptors, and species specificity is often correlated with sialic acid linkage with avian strains recognizing α2,3-linked sialylated glycans and mammalian strains preferring α2,6-linked sialylated glycans. These paradigms derive primarily from studies involving erythrocyte agglutination, binding to synthetic receptor analogs or binding to undefined surface markers on cells or tissues. Here, we present the first examination of the N-glycome of the human lung for identifying natural receptors for a range of avian and mammalian influenza viruses. We found that the human lung contains many α2,3- and α2,6-linked sialylated glycan determinants bound by virus, but all viruses also bound to phosphorylated, nonsialylated glycans.

Here, we report our characterization of the N-glycome from human lung tissue and our generation of the first human lung–shotgun N-glycan microarray (HL-SGM) to define the natural receptors recognized by a range of IAV. We identified a broad spectrum of both α2,3- and α2,6-linked sialylated N-glycans of various branch patterns and chain length, with and without core fucose modifications, demonstrating a multitude of potential sialylated glycan receptors for IAV. A range of avian, swine, and human IAV of various subtypes exhibited differing spectrums of binding profiles, from very broad binding to a variety of glycans to highly selective ligand specificity. We found that all IAV strains examined also bind well to phosphorylated, nonsialylated N-glycans. While Sia recognition by HA is an established feature of IAV, our findings suggest a possible role for alternative ligands, such as the phosphorylated glycans identified here, as cofactors or facilitators of virus entry, possibly opening new/additional routes to influenza treatment.

Influenza A viruses (IAVs) are a substantial annual burden on human health and the economy, and novel pandemic strains emerge from wild waterfowl hosts at unpredictable intervals. Sialic acid (Sia)–terminating cell surface glycans have been identified as receptors for IAV hemagglutinin (HA), and Sia linkage specificity is thought to provide a critical barrier for cross-species transmission, with avian viruses binding α2,3-linked sialylated glycans and human viruses preferring α2,6-linked sialylated glycans ( 1 – 7 ). Many studies have examined the binding characteristics and the structural interactions of avian and human IAV HAs with glycan receptors and the effects caused by mutations in the HA binding site [reviewed in ( 8 , 9 , 10 )]. However, a preponderance of the historical data on IAV receptor binding is based on agglutination of erythrocytes from different species (or α2,3- or α2,6-linked resialylated erythrocytes), binding to synthetic sugars or binding to poorly defined surface ligands on cells or tissues. There appears to be a difference in the abundance of sialylated glycan receptors at sites of infection in the natural hosts, i.e., α2,3-linked sialylated glycans are more prevalent in the intestinal tract of birds and α2,6-linked sialylated glycans are enriched on mucosal surfaces of the human upper respiratory tract, largely based on the presumption of specificity of Sia binding by the plant lectins Sambucus nigra agglutinin (SNA; α2,6-linked Sia) and Maackia amurensis lectin (MAL-I; α2,3-linked Sia) ( 11 – 13 ).

RESULTS

Human lung–shotgun N-glycan microarray To identify the endogenous glycans recognized by IAV, we generated an HL-SGM using N-glycans isolated directly from human lung tissue (Fig. 1A). N-glycans were fluorescently labeled, separated by two-dimensional (2D) high-performance liquid chromatography (HPLC), and 120 fractions were collected and concentrated to equal volume to reflect the natural abundance in the lung and then were covalently printed on N-hydroxysuccinimide (NHS)–activated slides. We first interrogated the HL-SGM with a variety of lectins to confirm the functional immobilization and recognition of glycans and to broadly survey some major structural features of lung N-glycans (Fig. 1B). SNA and MAL-I, which recognize α2,6- and α2,3-linked sialylated glycans, respectively, bound to divergent glycan fractions with higher chart IDs (50 to 138), indicating the presence of both α2,3- and α2,6-linked Sia in the human lung. The lectins concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin, which bind to glycan determinants with terminal mannose, N-acetylglucosamine/Sia, and galactose/Sia residues, respectively, bound a variety of glycans. The fucose-binding lectin Aleuria aurantia lectin bound many glycans, demonstrating that fucosylation is common in lung N-glycans. The terminal galactose-binding lectin Erythrina cristagalli lectin bound relatively poorly to the HL-SGM, but its binding was enhanced following exposure of galactosyl residues upon removal of Sia by neuraminidase (NA) treatment (fig. S1). Lycopersicon esculentum (tomato) lectin, which recognizes LacNAc repeats within poly-N-acetyllactosamine sequences (-3Galβ1-4GlcNAcβ1-) n (14), bound weakly to a number of fractions, suggesting that LacNAc repeats are not abundant. To further analyze the sialylated glycans and the specificities of SNA and MAL-I, we treated HL-SGM with Arthrobacter ureafaciens NA, which cleaves α2,3-, α2,6-, and α2,8-linked Sia. This treatment completely eliminated binding of SNA, indicating complete removal of α2,6-linked Sia, but MAL-I binding was only partially reduced (fig. S1). This is consistent with our recent findings, demonstrating that MAL-I can also detect certain nonsialylated, galactose-terminating, branched complex-type N-glycans (15) and, therefore, binding by MAL-I does not directly correspond to the presence of sialylated glycans (16). Fig. 1 An HL-SGM was generated and validated by lectin binding. (A) To identify endogenous receptors recognized by IAV, we generated a shotgun glycan microarray comprising the N-glycans from the human lung (7, 42, 43). Lungs (provided by LifeLink) were processed following the oxidative release of natural glycans (ORNG) method (17) and labeled with the bifunctional linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB) (40). The labeled N-glycans were printed on glass slides to generate the HL-SGM. RFU, relative fluorescence units; m/z, mass/charge ratio; MS, mass spectrometry; MSn, tandem mass spectrometry; MAGS, Metadata Assisted Glycan Sequencing. (B) Biotinylated lectins were bound to the HL-SGM at the noted concentrations and were detected with cyanine 5–conjugated streptavidin. ConA, concanavalin A; WGA, wheat germ agglutinin; RCA, R. communis agglutinin; AAL, A. aurantia lectin; ECL, E. cristagalli lectin; LEL, L. esculentum lectin.

Binding of influenza viruses to HL-SGM We analyzed binding to the HL-SGM of a panel of 11 different IAVs, including avian, swine, and human strains of differing subtype, geographic location, and date of isolation (table S1). Each IAV exhibited differential binding to the HL-SGM (Fig. 2A). The human H1N1 vaccine and seasonal strains, A/Brisbane/59/2007 and A/Pennsylvania/08/2008 (Penn), displayed very broad binding profiles to many glycans, while the H1N1 pandemic isolates A/California/04/2009, A/Texas/15/2009, and A/Mexico/InDRE4487/2009, as well as the H3N2 seasonal strain A/New York/55/2004, bound in a more restricted fashion, preferring glycans with lower number chart IDs (1 to 48), which generally correspond to less sialylated glycans. The swine isolates, A/sw/Minnesota/02719/2009 and A/sw/Illinois/02860/2009, exhibit broad binding, while the binding profile for A/sw/Minnesota/02749/2009 was also restricted to the lower numbered chart IDs (1 to 48). The avian isolates display wide-ranging binding profiles. Fig. 2 A range of IAVs all displays binding on the HL-SGM to chart IDs not bound by sialylated glycan binding lectins, SNA, and MAL-I. (A) Fluorescently labeled viruses, representative of different subtypes and host species, were bound to the array and display divergent binding profiles. (B) Penn binding to HL-SGM and comparison to SNA and MAL-I. IAV virus Penn was labeled with Alexa Fluor 488 and bound to the HL-SGM. The panels for MAL-I and SNA binding are included to demonstrate the nonoverlap between the Sia-binding lectins (light blue boxes) and the virus binding in the lower fraction numbers (green box). We selected Penn for more detailed studies, as this virus exhibits a robust binding signal and a broad receptor recognition profile. For each fraction collected by HPLC, a comparison of virus binding activity to fluorescence signal [due to 2-amino-N-(2-aminoethyl)-benzamide (AEAB) label on all glycans] showed no correlation between binding specificity and glycan abundance (fig. S2). The Penn strain bound to glycans of lower fraction numbers, e.g., chart IDs (1 to 48), which are relatively nonsialylated (Fig. 2B), and to those in higher chart IDs (53 to 138), which are sialylated and bound by both MAL-I and SNA. To examine the Sia requirement for virus binding in more detail, we treated the HL-SGM slides with A. ureafaciens NA (Fig. 3A). As a control, we treated a defined sialylated N-glycan microarray side by side to establish reaction conditions on a known array that ensures the complete removal of Sia (fig. S3A). We considered the treatment complete once the binding of SNA to the N-glycan microarray was reduced to background levels. Penn retained binding to the HL-SGM after removal of Sia, and binding was mainly to glycans with lower chart IDs (1 to 48). These glycans did not bind SNA and MAL-I, indicating the presence of additional glycan binding determinants for IAV independent of Sia. We also examined avian and swine influenza strains on a desialylated slide, and both retained binding to the lower numbered fractions as observed with Penn, indicating that binding to nonsialylated glycans is not limited to the Penn strain (fig. S3B). Fig. 3 The Penn strain binds to sialylated glycans and to glycan fractions containing phosphorylated structures. (A) NA treatment of HL-SGM and interrogation with SNA (25 μg/ml; detected with cyanine 5–conjugated streptavidin) and Penn reveal that IAV binds to array after removal of Sia as determined by loss of SNA binding. (B) Matrix-assisted laser desorption/ionization–time-of-flight MS (MALDI-TOF-MS) analysis of fractions, R10N13 (chart ID 118; high binding) indicating sialylated N-glycans and R04N23 (chart ID 47; high binding after NA treatment) indicating phosphorylated glycans, and tandem MS (MS/MS) of selected peaks. (C) Neuraminidase treatment of highest binding fraction R10N13 (predicted structures shown to the left; blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose; purple diamond, Sia) and binding of SNA (red bars) and MAL-I (blue bars) reveal the presence of both linkage types within the fraction.

Mass spectrometry of high binding fractions of HL-SGM We analyzed the HL-SGM fractions broadly recognized by a range of viruses. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) analyses of glycans revealed both sialylated glycans and those that were phosphorylated (Fig. 3B and fig. S4). The presence of sialylated glycans was exemplified by the MS analysis of the fraction R10N13 (chart ID 118). We detected major molecular ions [e.g., mass/charge ratio (m/z) 2094, 2240, 2605, and 2896] with compositions that represent mono- or disialylated complex-type N-glycans. The glycans in this fraction, which exhibited the highest relative fluorescence units (RFUs) for Penn binding, are predicted biantennary N-glycans, some with a core fucose, some presenting an additional N-acetyllactosamine unit (Galβ1-4GlcNAcβ1-). All the glycans are sialylated, although only some are sialylated on both branches (Fig. 3C). To determine the terminal Sia linkage, we treated the HL-SGM with A. ureafaciens NA and with Streptococcus pneumoniae NA, the latter of which cleaves primarily α2,3-linked Sia. We examined the SNA and MAL-I binding to fraction R10N13 (chart ID 118) before and after the NA treatments and found that MAL-I binding is decreased after both NA treatments, indicating that α2,3-linked Sia was present, and that SNA binding was decreased after A. ureafaciens NA treatment, but not after S. pneumoniae NA treatment, indicating that α2,6-linked Sia was also present (Fig. 3C). The MALDI-TOF-MS profile of the fraction R04N23 (chart ID 47), a fraction for which binding activity is not reduced by neuraminidase treatment, revealed the presence of a series of phosphorylated glycans with the compositions of Hex 5–12 HexNAc 0–3 PO 3. On the basis of the knowledge of biosynthetic pathways of N-glycans, structures detected in this fraction are likely high mannose–type N-glycans with a phosphate moiety. Loss of the reducing end HexNAc units of a few molecular ions (e.g., m/z 1070, 1395, 1719, and 2043) was due to processing by the oxidative release of natural glycans (ORNG) method, as previously described (17). We also identified molecular ions corresponding to N-glycans as Hex 9 , e.g., that could be Man 9 -containing glycans, or with further extension of hexoses that could contain additional glucose residues (m/z 1881, 2043, and 2205). Three molecular ions (m/z 2004, 2166, and 2328) were observed to display an additional HexNAc unit, which was proposed to be in phosphodiester linkage to the high-mannose structures via a phosphate group, as previously described (18). The fragment ion at m/z 79 could not be generated from a sulfate group (m/z 97, HSO 4 −) by losing a water molecule. Therefore, the detection of the fragment ions at m/z 79 (PO 3 −) and 97 (H 2 PO 4 −) indicated that the molecular ion at m/z 2125 contained a phosphate moiety. In confirmation of this interpretation, treatment of the fractions R05N15 (chart ID 58) and R02N23 (chart ID 21) with NA and/or phosphatase caused peak shifts in the HPLC profiles that correspond to the loss of Sia or the loss of phosphorylation (fig. S5).

Significance of phosphorylated glycans for IAV binding To define whether phosphorylation of glycans is required for binding by Penn, we treated the HL-SGM with bovine alkaline phosphatase, A. ureafaciens NA, or a combination of both (Fig. 4). We confirmed that alkaline phosphatase was efficient, using a synthetic glycan array containing a few phosphorylated glycans, in which alkaline phosphatase caused loss of binding of the single-chain variable domain antibody fragment (scFv) M6P-1, specific for selected mannose-6-phosphate (Man6P)–containing glycans (fig. S6) (19). Notably, the dephosphorylation of the HL-SGM by alkaline phosphatase treatment following NA treatment resulted in a total loss of virus binding (Fig. 4). Treating the arrays separately with NA or phosphatase individually leads to the retention of some binding, although the profiles are separate and distinct, with IAV binding on the NA-treated slide to glycans of chart IDs 1 to 48, whereas IAV binding on the phosphatase-treated slide occurred to glycans in chart IDs 50 to 120. We also treated the HL-SGM with NA and then completed the virus binding experiments in the presence of Man6P scFv (Fv M6P-1). The Fv M6P-1 competitively inhibited virus binding to the NA-treated slide (Fig. 4). The complete reduction of virus binding to background levels following phosphatase treatment, combined with the competitive inhibition exhibited by Fv M6P-1, confirms that glycan phosphorylation is a key contributor to the Sia-independent binding by Penn. Fig. 4 A combination of NA and phosphatase treatment of the HL-SGM results in no binding of Penn, while single enzymatic treatments allow for binding to certain glycan fractions. Binding inhibition with Fv M6P-1 (100 μg/ml) is also shown. To further confirm the importance of glycan phosphorylation to IAV binding to the HL-SGM, we performed hapten inhibition studies (Fig. 5). Incubation of Penn with 20 mM Sia inhibited the binding to those sialylated glycans recognized by MAL-I and/or SNA. The binding profile of Sia inhibition mimics that of an NA-treated slide. Binding competition with 6-sialyllactose produces the same effect (fig. S7A). These results suggest that IAV binds sialylated glycans through the canonical Sia receptor–binding site (RBS) on HA. In competition experiments with 20 mM Sia on an NA-treated slide, IAV retains binding, albeit at somewhat lower RFUs, to the glycans not recognized by either MAL-I or SNA, suggesting that the binding is occurring in a Sia-independent manner and not via the RBS. When we included Man6P on a slide with the sialylated glycans intact, the hapten had no effect on binding to sialylated glycans, indicating that it is not interfering with the canonical RBS on HA. However, following desialylation of glycans on the HL-SGM, the inclusion of Man6P caused a reduction in binding to most of the glycans. This effect is most pronounced for Man6P; other sugars, phosphorylated or sulfated in the 1 or 6 position, including mannose-6-sulfate (Man6S) and fructose-6-phosphate (Fruc6P), had more limited inhibition of IAV binding. Inclusion of glucose-1-phosphate (Glc1P) and Glc6P reduced IAV binding to certain fractions such as chart IDs 26 to 33. Overall, these results indicate that Sia is an inhibitor of IAV binding to sialylated glycans and that Man6P is an inhibitor of binding to phosphorylated glycans. Fig. 5 Different phosphorylated sugars produce no inhibition to Penn on the HL-SGM in hapten competition assays, indicating that charge alone is not mediating virus binding to nonsialylated glycans. The IAV-only RFUs are represented in gray, while the IAV and hapten RFUs are in green. In addition, the HL-SGM was treated with neuraminidase A (denoted as NA) to remove Sia before binding experiments, and the IAV-only RFUs are in gray, while the IAV and hapten RFUs are in purple.