Preferential Recognition of Avian-Like Receptors in Human Influenza A H7N9 Viruses Rui Xu et al. Science 342, 1230 (2013); DOI: 10.1126/science.1243761

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REPORTS To determine whether IFESCs functionally require the Wnt that they produce, we treated human epidermal keratinocytes with IWP-2, a validated small-molecule inhibitor of Wnt secretion, and cultured them at clonal density in a defined medium. IWP-2–treated keratinocytes were sparsely distributed and became large and flattened with arrested growth, unlike the densely packed, cuboidally shaped, control keratinocytes (Fig. 4, C and E). Many more IWP-2–treated keratinocytes also expressed high levels of involucrin, a marker of advanced keratinocyte differentiation (Fig. 4, D and F). These data are consistent with our in vivo observations that IFESCs undergo premature differentiation upon loss-of-function mutations in Wnt signaling (Fig. 3, E and F). If IFESCs are both the source and the target of Wnt signals, how might they escape from this autocrine loop and enter a differentiation process? Several genes for secreted Wnt inhibitors, including Dickkopf-1 (Dkk1), Dkk3, and Wnt Inhibitory Factor-1 (WIF1) are expressed in the skin (17–19). With double-labeling RNA in situ hybridization, we saw overlapping expression of Dkks and Axin2 expression in basal cells (Fig. 4G and fig. S6C). This is similar to the situation in human skin, in which primary human basal cells, either isolated from skin tissue or cultured in vitro, express Dkks (Fig. 4H and fig. S7). Although the Dkk (Fig. 4, G and H, and fig. S6C) and WIF1 (19) mRNAs are mostly located in basal cells, the secreted WIF1 and Dkk3 proteins accumulate at high levels in the suprabasal layers (18, 19). By antibody staining for the Dkk3 protein, we confirmed that Dkk3 is localized to the suprabasal layers, directly adjacent to the Axin2-expressing basal progenitors (Fig. 4I and figs. S8, A and B, and S9) (18). We tested whether Dkk influences stem cells in the skin by adenoviral overexpression of Dkk, finding that this caused a thinned and hypoproliferative epidermis (fig. S10) resembling b-catenin mutant skin (Fig. 3A). These data suggest that the differential diffusion of Wnts and Dkk from the basal epidermal stem cells may restrict autocrine Wnt/b-catenin signaling to the basal layer of the epidermis (fig. S8C). IFESCs leaving the basal layer would encounter increased Wnt inhibitors and differentiate. Functional redundancy between the various Wnt inhibitors and Wnts expressed in the skin (Fig. 4, A and G, and fig. S6, B and C) may explain the absence of overt phenotypes in mice mutant for these genes (20). However, there is genetic evidence supporting an essential role for Wnt signals in the epidermis. Porcn-knockout mice display a thinned epidermis, similar to that seen in human patients bearing Porcn mutations who develop focal dermal hypoplasia (21–23). Mutations in both Wnt effectors Tcf3 and Tcf4 result in a thinner epidermis (24), whereas deleting b-catenin using the basal epidermal specific driver Keratin-5-rtTA/tet-O-Cre also results in a thinner and hypoproliferative plantar epidermis (25).

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Signals emerging from a distinct niche cell compartment are thought to be the main drivers of stem cell self-renewal. We find that epidermal stem cells themselves can be the source of their own self-renewing signals and differentiating signals for their progeny. We postulate that the multiplicity of Wnts and Wnt inhibitors produced by epidermal stem cells allows for fine-tuning of epidermal thickness and wound repair. References and Notes 1. J. Huelsken, R. Vogel, B. Erdmann, G. Cotsarelis, W. Birchmeier, Cell 105, 533–545 (2001). 2. S. Beronja et al., Nature 501, 185–190 (2013). 3. H. J. Snippert et al., Science 327, 1385–1389 (2010). 4. V. P. Losick, L. X. Morris, D. T. Fox, A. Spradling, Dev. Cell 21, 159–171 (2011). 5. H. Ueno, I. L. Weissman, Dev. Cell 11, 519–533 (2006). 6. I. C. Mackenzie, Nature 226, 653–655 (1970). 7. C. S. Potten, Cell Tissue Kinet. 7, 77–88 (1974). 8. E. Clayton et al., Nature 446, 185–189 (2007). 9. D. P. Doupé, A. M. Klein, B. D. Simons, P. H. Jones, Dev. Cell 18, 317–323 (2010). 10. G. Mascre et al., Nature 489, 257–262 (2012). 11. A. M. Klein, B. D. Simons, Development 138, 3103–3111 (2011). 12. J. R. Bickenbach, J. McCutecheon, I. C. Mackenzie, Cell Prolif. 19, 325–333 (1986). 13. K. M. Braun et al., Development 130, 5241–5255 (2003). 14. S. Reddy et al., Mech. Dev. 107, 69–82 (2001). 15. F. Witte, J. Dokas, F. Neuendorf, S. Mundlos, S. Stricker, Gene Expr. Patterns 9, 215–223 (2009). 16. N. Radoja, A. Gazel, T. Banno, S. Yano, M. Blumenberg, Physiol. Genomics 27, 65–78 (2006). 17. Y. Yamaguchi et al., J. Cell Biol. 165, 275–285 (2004).

18. G. Du et al., Exp. Dermatol. 20, 273–277 (2011). 19. H. Schlüter, H.-J. Stark, D. Sinha, P. Boukamp, P. Kaur, J. Invest. Dermatol. 133, 1669–1673 (2013). 20. I. del Barco Barrantes et al., Mol. Cell. Biol. 26, 2317–2326 (2006). 21. J. J. Barrott, G. M. Cash, A. P. Smith, J. R. Barrow, L. C. Murtaugh, Proc. Natl. Acad. Sci. U.S.A. 108, 12752–12757 (2011). 22. W. Liu et al., PLOS ONE 7, e32331 (2012). 23. J. L. Bolognia, J. L. Jorizzo, J. V. Schaffer, in Dermatology (Mosby-Saunders, London, 2012), pp. 869–885. 24. H. Nguyen et al., Nat. Genet. 41, 1068–1075 (2009). 25. Y. S. Choi et al., Cell Stem Cell 10.1016/j.stem.2013.10.00 (2013). Acknowledgments: These studies were supported by the HHMI, California Institute of Regenerative Medicine grant TR1-01249, and NIH grants NIH 1U01DK085527, 1R01DK085720, and 5K08DK096048. We thank L. De Simone, A. E. Marcy, and P. H. Chia for cell quantification assistance; C. Logan, S. J. Habib, and A. Oro for manuscript comments; and J. Akech and L.-C. Wang at Advanced Cell Diagnostics for assistance with RNA in situ hybridization. X.L., S.H.T., W.L.C.K., and R.M.W.C. are supported by National Science Scholarships from A*STAR, Singapore. A.M.K. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. K.S.Y. has a Burroughs Wellcome Fund Career Award for Medical Scientists. R.v.A. was supported by a European Molecular Biology Organization long-term fellowship (ALTF 122-2007) and a Dutch Cancer Society fellowship.

Supplementary Materials www.sciencemag.org/content/342/6163/1226/suppl/DC1 Materials and Methods Supplementary Theory and Data Analysis Figs. S1 to S10 References (26–40) 29 April 2013; accepted 28 October 2013 10.1126/science.1239730

Preferential Recognition of Avian-Like Receptors in Human Influenza A H7N9 Viruses Rui Xu,1 Robert P. de Vries,2 Xueyong Zhu,1 Corwin M. Nycholat,2 Ryan McBride,2 Wenli Yu,1 James C. Paulson,2* Ian A. Wilson1,3* The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu226, a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike a2-6–linked receptors and strong preference for a subset of avian-like a2-3–linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission. n the spring of 2013, an outbreak of human infections caused by avian-origin H7N9 subtype influenza A virus occurred in the eastern provinces of China (1). By the end of May 2013, 132 cases of laboratory-confirmed H7N9 influenza were reported, resulting in 37 deaths (2). These patients generally presented influenza-like illnesses that frequently progressed to acute respiratory distress syndrome and severe pneumonia

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(3, 4). However, natural infection by H7N9 viruses in avian hosts are asymptomatic, which allows the virus to spread among birds and not be readily detected by surveillance (2). The H7N9 outbreak has raised concerns about its potential for causing human pandemics or epidemics (5, 6). Compared with H5N1 viruses, H7N9 appears to transmit from birds to humans more readily, with reports of a relatively large

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REPORTS number of recent human infections in a short period of time. Fortunately, avian influenza viruses such as H7N9 must overcome a species barrier that prevents efficient transmission from human to human and thereby attain wide circulation in an antigenically naïve human population. Early reports have suggested that exposure to poultry was responsible for over 75% of the documented human cases of H7N9 influenza, but limited humanto-human transmission cannot be ruled out, especially in a few small clusters of human infections (4). Sequence analysis of the H7N9 viral proteins revealed that the virus has acquired several amino acid changes associated with adaptation to human receptor binding and transmission in prior human pandemics (7–9). The PB2 protein of the H7N9 virus contains a Glu (E)–to–Lys (K) mutation at position 627 (E627K) that is important in other viruses for respiratory droplet transmission among humans (10). Furthermore, the hemagglutinins (HAs) from most H7N9 human isolates have Leu at position 226 (H3 numbering) instead of Gln, which is conserved in avian H7 HAs (7, 8), as well as in other avian subtypes (11, 12). The Gln-to-Leu mutation is one of the hallmarks of the switch to human receptor binding specificity that occurred in the 1957 H2N2 and 1968 H3N2 human influenza virus pandemics, representing an adaptation believed to be required for efficient human-to-human transmission (11–13). Recent studies suggested that the H7N9 virus could efficiently transmit among experimental ferrets via direct contact (14–16), but respiratory droplet transmission, the mode of transmission relevant to human pandemics, is less efficient, as demonstrated by results from five independent studies (9, 14–18). Thus, it is of major interest for public health to understand the extent to which the current H7N9 viruses have evolved to acquire capabilities for human-to-human transmission. Most avian H7 viruses, including those associated with previous human outbreaks, contain highly conserved avian-specific residues, including Gln226, in their receptor binding sites that enable them to specifically recognize terminal sialic acids in an a2-3 linkage to galactose (19, 20). In contrast, whereas the first human H7N9 virus isolated contained Gln226 (A/Shanghai/1/2013, abbreviated as Sh1), most other human H7N9 viruses analyzed so far carry Leu226 (e.g., A/Shanghai/2/ 2013, Sh2), with a few isolates containing Ile226 (e.g., A/Hangzhou/1/2013, Hz1) (7). The Glnto-Leu mutation is associated with improved affinity for human receptors where sialic acid is a2-6–linked to galactose (7, 8). In avian H2 and 1 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 2Department of Cell and Molecular Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 3Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

*Corresponding author. E-mail: [email protected] (I.A.W.); [email protected] (J.C.P.)

H3 HA, the Q226L (Q, Gln; L, Leu) mutation by itself greatly decreases HA affinity for a2-3–linked glycans while substantially improving binding to a2-6–linked glycans (21, 22). Recent studies have showed that H7N9 viruses with Leu226 can bind to receptors on the human tracheal epithelium (23) and are able to replicate in the upper respiratory tract of ferrets (14, 15). Unlike previous H7 viruses isolated in humans (such as H7N7), which exhibit contact transmission between ferrets but no respiratory droplet transmission (24), the human H7N9 viruses exhibit limited transmission by respiratory droplets (9, 14–16, 18), heightening concern that receptor binding changes might support more efficient transmission in humans (24). We determined the crystal structure of Sh2 H7 HA at 2.7 Å resolution (Fig. 1A and table S1) and found that it is structurally similar to the HA from a highly pathogenic avian H7N7 virus that infected humans (A/Netherlands/219/2003, Neth219) [Protein Data Bank (PDB) entry 4DJ6] (20) [Ca root mean square deviation (RMSD) of 1.2 Å and only 0.4 Å for the receptor binding domain]. The main differences around the receptor binding site arise from the Q226L mutation and the absence of an N-linked glycosylation site at Asn133 in Sh2 because of a T135A (T, Thr; A, Ala) substitution (Fig. 1B).

A

Although studies evaluating receptor binding by using whole viruses (14–16, 25, 26) have reported that the Leu226 mutation increases binding of H7N9 viruses to human receptors, binding to avian- and human-type receptors is influenced by the neuraminidase, which preferentially cleaves avian-type receptors (15, 25). To directly examine the intrinsic receptor specificity of the H7N9 HA, we analyzed HA binding to sialosides in platebased assays and glycan arrays by using soluble recombinant trimeric HAs expressed in mammalian cells (Fig. 2). In the plate assay, Sh2 HA strongly prefers a2-3–sialylated di-N-acetyllactosamine (SLNLN), although weak binding to a2-6 SLNLN at high protein concentrations is also observed (Fig. 2A) (27). Substitution of Leu226 to Gln, as in Sh1, or Ile, as in Hz1 strains, slightly increases avidity for a2-3 SLNLN but completely abrogates binding to a2-6 SLNLN in this assay (Fig. 2A). The preferential binding of recombinant Sh2 HA to a2-3 sialosides in analogous plate assays or in biosensor-based assays has also been recently reported by other groups (28–30). The H7N9 HA receptor binding properties were further investigated by using a custom influenza receptor glycan microarray comprised of diverse a2-3 sialosides [numbers (nos.) 3 to 35] and a2-6 sialosides (nos. 36 to 56) that correspond to N- and O-linked glycans and linear

B Lys193

Asp158A

Glu190 Leu194

Asn240

His183

Tyr195 Leu155

Gln222

Trp153

Leu226

Asn133 Tyr98

HA2: Asn82

Arg131

Thr136

C

Asn38 Asn22

HA2: Asn154

Fig. 1. Crystal structure of the HA from a human H7N9 virus (A/Shanghai/2/2013, Sh2). (A) HA trimer is shown in cartoon representation. For one of the protomers, HA1 is colored in green and HA2 in cyan. The other two protomers (B and C) of the trimer are in yellow and magenta, respectively. N-glycosylation sites and N-linked glycans are highlighted in sticks and numbered at the Asn attachment site. (B) Human H7 HA has Leu226 in its receptor binding site. (C) Variation at the four HA positions that are known to mediate the switch in receptor binding specificity for human H1, H2, and H3 pandemic viruses and the corresponding sequences in H1, H2, H3, H5, and H7 subtypes in avian viruses in comparison with human H7N9. G, Gly; S, Ser.

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Similarly, in the crystal structure of Sh2 with human receptor analog LSTc at 2.9 Å resolution, only Sia-1 and Gal-2 could be modeled (Fig. 3C). Leu226 mediates hydrophobic interactions with the Gal-2 hydrophobic face through its C6 and C4 atoms. Although the specific interactions involving Gal-2 are similar in the human H2 HA complex with LSTc (PDB entry 2WR7) (Fig. 3E) (36), other structural features in H7N9 HA may prevent further stabilization of the glycan receptor analog beyond GlcNAc-3. Similar to avian H7 HA (20), the 150 loop of Sh2 H7 HA is closer to the receptor binding site than it is in H2 HA (Fig. 3, C and E). These Sh2 HA structures with receptor analogs (LSTa and LSTc) then suggest that Leu226

A

modifies HA recognition of glycans with an a2-3 linkage but does not adapt the HA receptor binding site for binding of a2-6–linked glycans (Fig. 3, B and C). These structural findings are generally consistent with other recent structural studies on human H7 (25, 29). The most notable difference is the cis configuration of a2-3 linkage in these structures, which may result from use of different receptor analogs (37). To further explore the structural basis for retention of avian-type receptor specificity, we determined crystal structures of Sh2 H7 HA at 2.5 to 2.85 Å resolution with glycans 3, 21, and 23, which were identified as specific binders on the microarray (Fig. 4 and table S2). In each case, additional HA-glycan contacts are created distal

B KY/07

KY/07

2-3 Linked 2-6 Linked 2-3/2-6 Linked

2-3 Linked 2-6 Linked

Sh2 L226

Sh2 L226I

Sh2 L226Q

Sh2 L226

Fluorescence (RFU x 103)

Absorbance (450 nm)

fragments of glycans on mammalian glycoproteins and glycolipids (see list in table S3). Sh2 with Leu226 shows highly restricted binding to a small number of a2-3–sialylated glycans, and no detectable binding to a2-6–sialylated glycans, consistent with the more stringent requirements for binding in this assay (31). This subset of a2-3– sialosides includes sulfated linear glycans (nos. 3, 4, and 7), linear and branched O-linked glycans (nos. 20, 21, 23, and 24), and biantennary N-linked glycans (nos. 25 to 27). Specificity for sulfated glycans nos. 3 {NeuAca2-3Galb1-4[6S]GlcNAc, where NeuAc is N-acetylneuraminic acid, Gal is galactose, and GlcNAc is N-acetylglucosamine} and 4 {NeuAca2-3Galb1-4(Fuca1-3) [6S]GlcNAc, where Fuc is fucose} are characteristic of H7 avian viruses, including those from poultry and aquatic birds (19, 24, 32). The L226I and L226Q (I, Ile) substitutions change the distribution of glycan receptors that Sh2 HA binds, but specificity for a2-3–sialylated glycans is maintained (Fig. 2B) (33). A similar strict specificity for a2-3–sialylated glycans was observed for recombinant H7 HAs expressed in a baculovirus expression system (figs. S1 and S2) (34). In summary, Sh2 HA with Leu226 still maintains strong avian-type receptor specificity, with only very weak binding to human-type a2-6 receptors. Previous structural analyses of HA-receptor recognition and specificity have been limited to a small group of linear glycan receptor analogs, including short synthetic fragments of glycans on glycoproteins and glycolipids (3′-SLN, NeuAca2-3Galb14GlcNAc, and 6′-SLN, NeuAca2-6Galb1-4GlcNAc) and related oligosaccharides from human milk [LSTa, NeuAca2-3Galb1-3GlcNAcb1-3Galb1-4Glc; LSTc, NeuAca2-6Galb1-4GlcNAcb1-3Galb1-4Glc; and LSTb, Galb1-4(NeuAca2-6)GlcNAcb1-3Galb14Glc] (Fig. 3A) (30, 35). Notwithstanding, these ligands have provided valuable information on how avian and human HAs differentially recognize a2-3– and a2-6–linked sialosides. We determined crystal structures of Sh2 H7 HA with avian receptor analog LSTa at 2.6 to 2.7 Å resolution and found that only sialic acid 1 (Sia-1) and Gal-2 of LSTa were ordered (Fig. 3B and fig. S3). A comparison of human and avian H7 HA structures reveals several differences in receptor recognition. In avian H7 from Neth219 virus (PDB entry 4DJ7) (20), preferential binding to avian receptors is mediated by Gln226, which hydrogen bonds with the Gal-2 O3 atom at the Sia-Gal linkage and the 4-hydroxyl group of Gal-2 (Fig. 3D). In addition, the Sia-1 carboxyl group is located within 3.5 Å of Gln226. This mode of recognition is highly conserved in other avian HA subtypes (35). In Sh2, the hydrophobic Leu226 does not support such a hydrogen-bonding network, and Gal-2 moves away from Leu226, which results in LSTa taking a slightly different trajectory exiting the binding pocket. No significant interactions were visualized with the glycan ligand beyond Sia-1, suggesting a weak interaction that was not improved by increased soaking time and concentration of LSTa (fig. S3 and table S1).

Sh2 L226I

Sh2 L226Q

HA (µg/ml)

Glycan

Fig. 2. Receptor binding specificity of H7N9 HAs determined by plate-based glycan binding and glycan microarray analyses. (A) Enzyme-linked immunosorbant assay (ELISA) plates coated with either a2-3– or a2-6–linked SLNLN-PAA were probed with recombinant HAs produced in human embryonic kidney (HEK293S GnTI–) cells. The HAs were human H1N1 seasonal strain KY/07 or H7N9 Sh2 wild type containing Leu (L) at position 226 and mutants at this position harboring Ile (I) or Gln (Q). Shown are data representative of three independent experiments, each done in triplicate, with binding to a2-6–glycans in solid circles and a2-3–glycans in open squares. Error bars indicate standard errors. (B) The same recombinant HA proteins were used for assessment of receptor binding specificity on a glycan array. The mean signal and standard error were calculated from six independent replicates on the array. a2-3 sialosides are shown in white bars (glycans 3 to 35), a2-6 sialosides in black (glycans 36 to 56), and striped bars denoted mixed biantennary glycans containing both a2-3– and a2-6– linked sialylated glycans (glycans 57 and 58). Glycans imprinted on the array are listed in table S3.

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A α3

β3

LSTa α3

β4

3’-SLN

β3

β4

α6

β4

β3

B

C

Gln222

Gln222

β4

Lys193

LSTc α6

by avian H7 viruses (19, 32) and that H7N9 HA is not optimally evolved for recognition of humanlike receptors. Because acquisition of humantype receptor specificity of HA is considered a risk factor for human-to-human transmission, reports of binding to human-type (a2-6) receptors (14–16, 23, 25, 26) have raised concern for the potential for H7N9 to become a pandemic virus. However, we and others have found that the human H7 HA is predominantly specific for avian-type (a2-3) receptors with only weak binding to human receptors (28–30). Thus, additional mutations will be needed for the HA to achieve specificity for human-type receptors as observed in previous human pandemic viruses. We suggest that the intrinsic weak avidity to human receptors in receptor assays is exaggerated in studies with whole virus by preferential cleavage of the avian-type receptors by the neuraminidase (15, 25) and the high valency of HA on the virus that can amplify binding to receptors of weaker affinity. This explanation is consistent with previous observations from analysis of a human H7N7 virus (A/NY/107/03); this recombinant H7 HA exhibited strong preference for avian-type receptors (39), but whole virus showed significant binding to human-type receptors and manifests contact transmission, but not respiratory droplet transmission, in ferrets (24, 39). The situation for human H7N9 viruses is also analogous to H5N1 viruses, where other mutations, in addition to Q226L, are required to achieve humantype receptor specificity that enables respiratory droplet transmission in ferrets (31). Thus, the weak avidity for human-type receptors may contribute

Siaa2-3Gal linkage adopts a cis configuration (Fig. 4E). Gal-2 rotates about 180° (Fig. 5B), bringing its 6-hydroxyl group into hydrogenbonding distance with the Gly225 carbonyl oxygen. The receptor analog exits over the 220 loop and turns away from the receptor binding site between GlcNAc-3 and GalNAc-4, where Gln222 mediates hydrogen bonds with O3 in the linkage as well as the acetamido carbonyls from GlcNAc-3 and GalNAc-4 (Figs. 4E and 5D). Glycan no. 23 is a biantennary structure with one arm identical to glycan no. 21 (Fig. 4, B and C). Only this arm is visualized in the electron density map and binds HA similarly to glycan no. 21 (Fig. 4, F and G). Sh2 HA also recognizes other a2-3–linked biantennary glycans (N-linked glycans 24 to 27), and their longer arms may allow the glycan to span HA trimers to improve avidity. The HA complexes with these three aviantype receptor glycans reveal different binding modes that highlight the enormous diversity in HA-glycan recognition. Preferential binding for these glycans is mediated by HA-receptor interactions that were not predictable without these crystal structures. These interactions can compensate for unfavorable contacts between Leu226 and the a2-3 linkage, allowing human H7N9 HA to effectively maintain avian-type receptor specificity. The combination of glycan microarrays and x-ray structural determination has enabled us to demonstrate that the HA from current human H7N9 viruses retains specificity for sulfated a2-3–linked sialylated glycans that are recognized

to the Sia-Gal linkage, so that efficient binding to a2-3–linked glycans is achieved despite Leu226 (Fig. 5). Most relevant is glycan no. 3 (NeuAca23Galb1-4[6S]GlcNAc), which has been demonstrated to be a receptor analog recognized by all avian H7 viruses (19, 32). Previous efforts to model sulfated glycans in the avian H7 structure positioned the sulfo group in the vicinity of Lys193 for favorable charge interactions (19, 32) and were supported by the crystal structure of H5 HA with a sulfated sialoside (38). Perhaps unexpectedly, we found that the sulfo group is located near the 220 loop between Leu226 and Val186 (Figs. 4D and 5C) and hydrogen bonds to the Ser 227 mainchain amide and Glu190. The Sia-Gal linkage adopts a trans configuration, similar to linear avian receptor analogs in avian H7 and Sh2 H7 structures (Fig. 5A). The glycan exits the receptor binding site above the 220 loop and follows a trajectory similar to those of avian receptors in H7 and other avian HAs. The sulfated GlcNAc-3, however, rotates its linkage with Gal-2 by about 180° to bring the sulfated 6hydroxyl group over to the side of the 190 helix rather than proximal to the 130 loop. Electron density for the sulfo group is substantially better defined than GlcNAc-3, suggesting that glycan no. 3 binding to HA is mainly anchored by Sia-1 and the sulfo group, with flexibility in the rest of the receptor. The O-linked glycans (nos. 21 and 23) share a common terminal sequence analogous to 3′-SLN. For glycan no. 21 (NeuAca2-3Galb1-4GlcNAcb13GalNAca−Thr, where GalNAc indicates N-acetyl galactosamine) in complex with Sh2 HA, the

β4

Glu190

Gal-2

Sia-1

Gly228

6’-SLN

Gal-2

Leu155

Leu226

Leu226

Thr136

Ala135

Ala135 Thr136

Sh2 H7 HA / LSTa

Sh2 H7 HA / LSTc

D

E

Gal-4 GlcNAc-3 Thr193

GlcNAc-3

Gal-2

Glu190 Gln222

Sia-1

Gly228

Leu155

Fig. 3. Crystal structures of human Sh2 H7N9 HA in complex with receptor analogs. (A) Glycan structures of four receptor analogs commonly used for structural study. Purple diamonds represent Sia, yellow circles represent Gal, blue squares represent GlcNAc, and blue circles represent glucose (Glc). (B) Avian receptor analog LSTa bound to human H7 HA. (C) Human receptor analog LSTc bound to human H7 HA. (D) Avian receptor analog 3′-SLN bound to avian H7 HA (PDB entry 4DJ7). (E) Human receptor analog LSTc bound to human H2 HA (PDB entry 2WR7).

Glu190

Gal-2

Glu190

Lys222

Sia-1

Gly228

Leu155

Ser228

Thr155

Sia-1 Leu226

Gln226

Thr136

Gly135 Ser136

Thr135

avian H7 HA / 3’-SLN

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A

F

glycan #3

GalNAc-4

6S

α3

β4

ethyl-N3

β

6-O-sulfo-GlcNAc-3 Val186

GlcNAc-3

Glu190

Glu190 Gln222

B

Sia-1

Gal-2

Ser227

Gln222

glycan #21

α3

β4

β3

α

Sia-1

Gal-2

Leu155

Leu155

Gly225

Leu226

Thr-NH2

Leu226 Ala135

Thr136

Ala135

Thr136

C α3

β4

α3

β4

G

E

glycan #23

β6

β3

α

GalNAc-4

Thr-NH2

GlcNAc-3

Glu190 190

Gln222

222

Sia-1

Gal-2

155

Leu155

226

Leu226 136 Thr136

Ala135

Fig. 4. Crystal structures of human Sh2 H7N9 HA in complex with three avian-type receptor glycans recognized in the glycan array. (A to C) Glycan compounds used for structural determination. The glycan portion that could be built in the final model is highlighted by a red dashed circle. Purple diamonds represent Sia, yellow circles represent Gal, and blue and yellow squares represent GlcNAc and

A

B

GalNAc, respectively. (D) Sulfated glycan no. 3 bound to human H7N9 HA. (E) Glycan no. 21 bound to human H7N9 HA. (F) Biantennary glycan no. 23 bound to H7N9 HA using one of its two arms. (G) Superposition of glycan no. 23 (colored in purple), glycan no. 21 (in blue), and glycan no. 3 (in green) in the HA receptor binding site. Glycans 21 and 23 share a similar mode of ligand recognition.

C

GalNAc-4

D GalNAc-4

GlcNAc-3

GlcNAc-3

GlcNAc-3: C6

GlcNAc-3

6-O-sulfo-GlcNAc-3

Gln222

Gal-2

Gal-2 Gal-2

GlcNAc-3

Gal-2: C6

Ser227

Val186 Leu226

Gal-2

6-O-sulfo-GlcNAc-3

Gly225 Leu226

Fig. 5. Preferential binding of human Sh2 H7N9 HA to avian-type a2-3–linked glycans is achieved by flexible twisting of the glycan rings that create additional HA-glycan contacts away from Leu226. (A) Superposition of glycan no. 3 (cyan) and 3′-SLN (gray, from PDB entry 4DJ7) in the HA receptor binding site. (B) Superposition of glycan no. 21 (cyan) and to poor transmission by respiratory droplet in the ferret model (14–16, 18) and the lack of sustained transmission in humans (7, 8). References and Notes 1. Centers for Disease Control and Prevention (CDC), MMWR Morb. Mortal. Wkly. Rep. 62, 366–371 (2013). 2. World Health Organization, “Overview of the emergence and characteristics of the avian influenza A (H7N9) virus” (2013), www.who.int/influenza/human_animal_interface/ influenza_h7n9/WHO_H7N9_review_31May13.pdf. 3. R. Gao et al., N. Engl. J. Med. 368, 1888–1897 (2013).

1234

Glu190

Sia-1

Sia-1

3′-SLN (gray) in the HA receptor binding site. (C) The sulfo group of glycan 3 is located near the amide nitrogen of Ser227 and within hydrogen-bonding distance of Glu190. (D) Preferential binding to glycan 21 is mediated by hydrophilic interactions between Gln222 and the glycosidic linkage between GlcNAc-3 and GalNAc-4.

4. Q. Li et al., N. Engl. J. Med., published online 24 April 2013 (10.1056/NEJMoa1304617). 5. D. M. Morens, J. K. Taubenberger, A. S. Fauci, N. Engl. J. Med. 368, 2345–2348 (2013). 6. R. E. Kahn, J. A. Richt, Vector Borne Zoonotic Dis. 13, 347–348 (2013). 7. D. Liu et al., Lancet 381, 1926–1932 (2013). 8. T. Kageyama et al., Euro Surveill. 18, 20453 (2013). 9. D. A. Steinhauer, Nature 499, 412–413 (2013). 10. E. K. Subbarao, W. London, B. R. Murphy, J. Virol. 67, 1761–1764 (1993). 11. M. Imai, Y. Kawaoka, Curr. Opin. Virol. 2, 160–167 (2012).

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12. M. Matrosovich, J. Stech, H. D. Klenk, Rev. Sci. Tech. 28, 203–217 (2009). 13. R. J. Connor, Y. Kawaoka, R. G. Webster, J. C. Paulson, Virology 205, 17–23 (1994). 14. H. Zhu et al., Science 341, 183–186 (2013); 10.1126/science.1239844. 15. T. Watanabe et al., Nature 501, 551–555 (2013). 16. J. A. Belser et al., Nature 501, 556–559 (2013). 17. Q. Zhang et al., Science 341, 410–414 (2013); 10.1126/science.1240532. 18. M. Richard et al., Nature 501, 560–563 (2013). 19. A. S. Gambaryan et al., J. Virol. 86, 4370–4379 (2012).

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REPORTS 20. H. Yang, P. J. Carney, R. O. Donis, J. Stevens, J. Virol. 86, 8645–8652 (2012). 21. R. Xu, R. McBride, J. C. Paulson, C. F. Basler, I. A. Wilson, J. Virol. 84, 1715–1721 (2010). 22. G. N. Rogers et al., J. Biol. Chem. 260, 7362–7367 (1985). 23. K. Tharakaraman et al., Cell 153, 1486–1493 (2013). 24. J. A. Belser et al., Proc. Natl. Acad. Sci. U.S.A. 105, 7558–7563 (2008). 25. X. Xiong et al., Nature 499, 496–499 (2013). 26. J. Zhou et al., Nature 499, 500–503 (2013). 27. As a control, the HA from a representative human seasonal H1N1 strain (A/Kentucky/UR06-0258/2007, KY/07) displays characteristic strong binding to a2-6 SLNLN and negligible binding to a2-3 SLNLN (Fig. 2A). 28. I. Ramos et al., J. Gen. Virol. 94, 2417–2423 (2013). 29. Y. Shi et al., Science 342, 243–247 (2013); 10.1126/ science.1242917. 30. H. Yang, P. J. Carney, J. C. Chang, J. M. Villanueva, J. Stevens, J. Virol. 87, 12433–12446 (2013). 31. J. C. Paulson, R. P. de Vries, Virus Res. 178, 99–113 (2013). 32. A. S. Gambaryan et al., Virol. J. 5, 85 (2008). 33. As a control, human HA KY/07 binds broadly to a2-6-linked glycans on the array (Fig. 2B), and avian H7 HA recognizes a large number and variety of a2-3-sialylated glycans (fig. S2).

34. The L226Q mutant produced in insect cells showed robust binding to the array, revealing stronger avidity to most a2-3-glycans but still no binding to a2-6-sialosides. 35. R. Xu, I. A. Wilson, in Structural Glycomics, E. Yuriev, P. A. Ramsland, Eds. (CRC Press, Boca Raton, FL, 2012), pp. 235–257. 36. J. Liu et al., Proc. Natl. Acad. Sci. U.S.A. 106, 17175–17180 (2009). 37. Xiong et al. used 3′-SLN as an avian-like receptor analog. Shi et al. used a2-3-SLNLN. In our study, we used LSTa. 38. X. Xiong et al., Virus Res. 178, 12–14 (2013). 39. K. Srinivasan, R. Raman, A. Jayaraman, K. Viswanathan, R. Sasisekharan, PLOS ONE 8, e49597 (2013). Acknowledgments: This work was supported in part by NIH R56 AI099275 (I.A.W.) and the Skaggs Institute for Chemical Biology, the Scripps Microarray Core Facility, and a contract from the Centers for Disease Control ( J.C.P.). R.P.d.V. is a recipient of a Rubicon grant from the Netherlands Organization for Scientific Research (NWO). Several glycans used for HA binding assays were provided by the Consortium for Functional Glycomics (www.functionalglycomics.org/) funded by National Institute of General Medical Sciences (NIGMS) grant GM62116 (J.C.P.). X-ray diffraction data were collected at the Advanced Photon Source beamline 23ID-B (GM/CA CAT) and the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2. GM/CA CAT is funded in whole or in part with federal funds

HCF-1 Is Cleaved in the Active Site of O-GlcNAc Transferase Michael B. Lazarus,1,4*† Jiaoyang Jiang,1*‡ Vaibhav Kapuria,2 Tanja Bhuiyan,2 John Janetzko,4 Wesley F. Zandberg,3 David J. Vocadlo,3,5 Winship Herr,2§ Suzanne Walker1§ Host cell factor–1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate–GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site. -linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a Ser/Thr (S/T) glycosyltransferase that O-GlcNAcylates nuclear and cytoplasmic proteins, thus influencing their activity, localization, and overall function (1–3). Because OGT activity is sensitive to uridine

O

1 Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. 2Center for Integrative Genomics, University of Lausanne, Génopode, 1015 Lausanne, Switzerland. 3Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. 4Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. 5Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

*These authors contributed equally to this work. †Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA. ‡Present address: School of Pharmacy, University of Wisconsin– Madison, Madison, WI 53705, USA. §Corresponding author. E-mail: [email protected]. edu (S.W.); [email protected] (W.H.)

diphosphate (UDP)–GlcNAc concentrations, OGT is proposed to regulate cellular responses to nutrient status (4–6). Human HCF-1 is a transcriptional coregulator involved in regulating cellcycle progression (7, 8). In an unusual proteolytic maturation process (9–11), any of six centrally located 20 to 26 amino acid sequence repeats called HCF-1PRO repeats (Fig. 1A) are cleaved by OGT in the presence of UDP-GlcNAc (12), providing a link between cell-cycle progression and nutrient levels. The HCF-1PRO repeats contain two essential regions for proteolysis: a threonine-rich region proposed to be an OGT-binding site and the cleavage site, which contains a conserved Cys-Glu-Thr (CET) sequence (10, 11). OGT can cleave a fragment of HCF-1, called HCF-1rep1, which contains the first HCF-1PRO repeat plus N-terminal HCF-1 sequences containing several O-GlcNAc sites (12) (Fig. 1A). To elucidate the cleavage process, we first analyzed the effect of amino acid substitutions in OGT (Fig. 1B and fig. S1) and the HCF-1PRO repeat

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from the National Cancer Institute (Y1-CO-1020) and the NIGMS (Y1-GM-1104). Use of the Advanced Photon Source was supported by the U.S. Department of Energy (DOE), Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. The SSRL is a Directorate of Stanford Linear Accelerator Center National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. DOE Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH, NIGMS (including P41GM103393) and the National Center for Research Resources (NCRR, P41RR001209). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS, NCRR, or NIH. This is publication 24079 from the Scripps Research Institute. Coordinates and structure factors are deposited in the PDB (4N5J, 4N5K, 4N60, 4N61, 4N62, 4N63, and 4N64).

Supplementary Materials www.sciencemag.org/content/342/6163/1230/suppl/DC1 Materials and Methods Figs. S1 to S5 Tables S1 to S3 References (40–47) 25 July 2013; accepted 28 October 2013 10.1126/science.1243761

(Fig. 1C) on cleavage and glycosylation. Three OGT active site residues implicated in S/T glycosylation were evaluated: K842, which is involved in binding and activation of UDP-GlcNAc for glycosyl-transfer; H498, which contacts the C2-N-acetyl group of UDP-GlcNAc; and H558, which contacts a backbone carbonyl of glycosylation substrates (13–17). Substitution of K842 with methionine prevented S/T glycosylation upstream of the proteolytic repeat as well as cleavage within the repeat region. Substitution of H498 or H558 with alanine decreased S/T glycosylation but had a negligible effect on the extent of cleavage after 16 hours. K842 is an essential residue for glycosylation (14, 15), and its importance in cleavage suggests that UDP-GlcNAc is involved in the cleavage mechanism. Next, we tested substitutions in the proteolytic repeat of HCF-1rep1. We previously showed that alanine substitution of glutamate E10 leads to loss of cleavage (11, 12). To probe the role of E10 in more detail, we substituted it with glutamine (E10Q), aspartate (E10D), and serine (E10S). All three substitutions blocked cleavage (Fig. 1C), indicating that the chemical nature of the glutamate residue is critical for OGT-mediated HCF-1PROrepeat cleavage. In contrast, the C9 position can tolerate alanine and serine substitution (12) (fig. S2). Because S/T glycosylation upstream of the cleavage site in HCF-1rep1 complicates study of the cleavage requirements, we identified a cleavage substrate consisting of the first three proteolytic repeats (HCF3R, Fig. 1A), which did not undergo substantial glycosylation. No cleavage products were observed when HCF3R was incubated with OGT alone or in the presence of UDP, but several products were observed in reactions containing both OGTand UDP-GlcNAc (Fig. 2A). These products did not form if HCF3R was incubated with a K842A OGT mutant incapable of

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