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Shotgun glycomics of pig lung identifies natural ... · PDF file Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved April 22, 2014 (received

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  • Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses Lauren 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 containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because in- fluenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of “shotgun glycomics” to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans 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 interacted with 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 results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying 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 extensive economic burden. At unpredictable intervals, viruses with anti- genically novel glycoproteins emerge to cause pandemics of even greater concern, and the pig is often implicated as an in- termediate host when this occurs (1). The waterfowl that act as natural reservoirs for influenza A viruses harbor 16 antigenically distinct subtypes of the hemagglutinin (HA) surface antigen, and 9 subtypes of the neuraminidase (NA) envelope protein, but the only subtypes to emerge and circulate broadly in humans over the past century are H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, and H1N1 again in 2009. However, the possible emergence of a novel subtype in humans remains a concern, because highly pathogenic H5 and H7 subtype “bird flu” viruses continue to circulate in avian species, and over the past 15 y have infected humans, with limited ability to transmit between humans. The reasons 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 surface antigen are of paramount significance. Since the identification of sialic acids as critical components of

    influenza receptors (2, 3), a broadly observable phenomenon has been that avian strains generally prefer binding to glycan receptors with sialic acids containing α2,3 glycosidic linkages to the penultimate galactose in the carbohydrate chain, whereas human viruses favor binding to substrates with α2,6 linkages (4– 7). The description of mutant viruses for which specific changes in the HA receptor-binding site result in a switch from one binding preference to the other provides clues on potential mechanisms 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- to threefold difference between cognate pairs and reciprocal pairs (15, 16). This suggests that productive virus attachment results from the cumulative effects of multiple HA receptor interactions in which the consequences of very small differences are ampli- fied in a phenotypically distinguishable fashion. However, little more is known with regard to binding affinity for individual glycans of defined structure or of their presence and distribution in tissues encountered during natural infections. Initial studies using linkage-specific lectins as an indirect

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


    Studies using novel “shotgun glycan microarray” technology identify, for the first time to our knowledge, the endogenous receptors for influenza viruses from a natural host, the pig. Libraries of total N-glycans from pig lung were probed for binding properties using a panel of influenza viruses isolated from humans, birds, and swine. Natural glycan receptors were identified 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 has not been demonstrated previously. Because pigs are often impli- cated as intermediate hosts for pandemic viruses, these results and the approaches describedwill transform our understanding of influenza 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: [email protected] or [email protected]

    This article contains supporting information online at 1073/pnas.1323162111/-/DCSupplemental. PNAS | Published online May 19, 2014 | E2241–E2250

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  • lower respiratory tract contains a mixture with considerable representation of both α2,3-Sia and α2,6-Sia receptor types (18– 21) The respiratory tract of swine also contain both α2,3-Sia and α2,6-Sia, which appear to be distributed similarly to humans (17, 22, 23). These observations have been used in support of the hypothesis that the pig may provide an intermediate host for the adaptation of avian strains before emergence in humans or for the genesis of reassortant pandemic viruses capable of human infection 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 does not identify actual glycan receptors. The continued development and use of new tools in carbohydrate chemistry and an expanding range of viruses under scrutiny have made clear that not only sialic acid linkage but underlying glycan composition and struc- ture, chain length, and presentation of receptors also play a role in the complex interactions that lead to productive attachment of influenza to host cells. Defined glycan microarrays have been developed and successfully applied to examine the binding specificity of influenza, as well as other viruses and micro- organisms (25–32). However, a limitation of most such arrays is that they are based on chemically or enzymatically synthesized glycan structures and represent only a subset of the endogenous complex glycomes of natural hosts. The restrictions imposed by the synthetic nature of such arrays is emphasized by the obser- vation that mutant influenza viruses have been identified which replicate both in cell culture or in the mouse respiratory tract and yet exhibit no detectable binding to the glycans on the Consortium of Functional Glycomics (CFG) array (33). In ad- dition, recent glycan profiles of swine epithelial

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