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Recognition of microbial glycans by human intelectin-1

Nature Structural & Molecular Biology volume 22, pages 603–610 (2015)Cite this article

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Abstract

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. We used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol–modified glycans, heptoses, D-glycero- D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D- manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in human glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. This ligand selectivity suggests that hIntL-1 functions in microbial surveillance.

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Figure 1: hIntL-1 selectivity for monosaccharides.
Figure 2: Glycan selectivity of hIntL-1, assessed by glycan microarrays.
Figure 3: Structure of hIntL-1 bound to allyl-β-D-Galf.
Figure 4: Models for hIntL-1 interacting with relevant saccharide epitopes from humans (α-Neu5Ac) or microbes (α-KDO).
Figure 5: hIntL-1 binds to S. pneumoniae serotypes producing capsular polysaccharides with terminal vicinal diols.
Figure 6: Structures of the 20 most prevalent monosaccharides that are unique to bacterial glycans.

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Acknowledgements

This research was supported by the US National Institutes of Health (NIH) (R01GM55984 and R01AI063596 (L.L.K.)). D.A.W. thanks the US National Science Foundation (NSF) and the NIH Chemistry-Biology Interface Training Program (T32 GM008505) for fellowships. K.W. was supported by a fellowship from the Development and Promotion of Science and Technology Talents Project of Thailand. M.B.K. and H.L.H. were supported by the NIH (F32 GM100729 to M.B.K. and T32 GM008505 to H.L.H.). L.C.Z. was supported by a UW–Madison Hilldale Fellowship. R.A.S. thanks the American Chemical Society Division of Medicinal Chemistry for a fellowship. The glycan array experiments were made possible by the Consortium for Functional Glycomics (NIH NIGMS GM062116 and GM98791 (J.C.P.)), which supported the Glycan Array Synthesis Core at The Scripps Research Institute and the Protein-Glycan Interaction Resource (Emory University School of Medicine). These resources assisted with analysis of samples on the array. Printing and processing the furanoside array was supported through the US National Center for Functional Glycomics supported by NIH NIGMS (P41GM103694 (R.D.C.)). SPR experiments were performed at the University of Wisconsin (UW)−Madison Biophysics Instrumentation Facility, which is supported by UW–Madison, NSF grant BIR-9512577 and NIH grant S10 RR13790. Flow cytometry data were obtained at the UW–Madison Carbone Cancer Center (P30 CA014520), and microscopy images were acquired at the UW−Madison W.M. Keck Laboratory for Biological Imaging (1S10RR024715). The UW−Madison Chemistry NMR facility is supported by the NSF (CHE-9208463 and CHE-9629688) and NIH (1s10 RR08389). Use of the Advanced Photon Source at the Argonne National Laboratory was supported by the US Department of Energy (contract DE-AC02-06CH11357), and the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). We thank J.M. Fishman for assistance in preparing the synthetic methods and M.R. Levengood, A.H. Courtney and D.R. McCaslin for thoughtful discussions. We thank M.R. Richards (University of Alberta) for helpful discussions.

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  1. Matthew B Kraft & Rebecca A Splain

    Present address: Present Addresses: Gilead Sciences, Foster City, California, USA (M.B.K.) and Global API Chemistry, GlaxoSmithKline, King of Prussia, Pennsylvania, USA (R.A.S.).,

Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin−Madison, Madison, Wisconsin, USA

    Darryl A Wesener, Lucas C Zarling & Laura L Kiessling

  2. Department of Chemistry, University of Wisconsin−Madison, Madison, Wisconsin, USA

    Kittikhun Wangkanont, Matthew B Kraft, Heather L Hodges, Rebecca A Splain & Laura L Kiessling

  3. Department of Cell and Molecular Biology, Scripps Research Institute, La Jolla, California, USA

    Ryan McBride & James C Paulson

  4. Department of Chemical Physiology, Scripps Research Institute, La Jolla, California, USA

    Ryan McBride & James C Paulson

  5. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA

    Xuezheng Song, David F Smith & Richard D Cummings

  6. Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA

    Xuezheng Song, David F Smith & Richard D Cummings

  7. Department of Bacteriology, University of Wisconsin−Madison, Madison, Wisconsin, USA

    Katrina T Forest

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Contributions

D.A.W. and L.L.K. conceived the project. D.A.W., K.W. and L.L.K. planned the experiments, analyzed the data and wrote the paper, with input from all the other authors. Cloning, protein expression and biochemical experiments were performed by D.A.W. and L.C.Z. Microscopy was performed by H.L.H. Baculovirus was made by K.W. The carbohydrate ligands were synthesized and characterized by M.B.K. and R.A.S. The furanoside glycan microarray was constructed and analyzed with the mammalian glycan microarray by X.S., D.F.S. and R.D.C. The microbial glycan array was constructed and analyzed by R.M. and J.C.P. Protein crystallization and structure determination were performed by K.W. and K.T.F.

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Correspondence to Laura L Kiessling.

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Integrated supplementary information

Supplementary Figure 1 Expression, purification and carbohydrate binding activity of hIntL-1.

(a) Reducing SDS-PAGE analysis of HEK 293T culture medium from hIntL-1 transfected cells. Samples were analyzed by silver stain 48 hours post transfection. An arrow indicates the band corresponding to the molecular weight of a hIntL-1 reduced monomer.

(b) Coomassie stained gels of samples subjected to reducing and nonreducing SDS-PAGE analysis of hIntL-1 purified on an immobilized β-Galf column. The molecular weight of the sample analyzed under non-reducing conditions corresponds to that of a disulfide-linked hIntL-1 homotrimer.

(c) Schematic of streptavidin-based, ELISA-like carbohydrate binding assay developed for assessing hIntL-1 ligand specificity. Biotin-functionalized carbohydrate is immobilized. Bound hIntL-1 is detected the enzyme horseradish peroxidase (HRP) conjugated to an antibody (either a secondary or directly conjugated primary), and a chromogenic HRP substrate.

(d) Carbohydrate−binding activity of HEK 293T cell conditioned culture medium following transfection with hIntL-1 expression plasmid. The calcium ion dependence was tested by the addition of 25 mM EDTA. Data are presented as the mean (n=2 of a technical replicate and is representative of >3 independent experiments).

(e) Complete data set of hIntL-1 SPR analysis presented in Fig. 1c. β-Ribofuranose and β-arabinofuranose were included as they were reported to be ligands of hIntL-1 (Tsuji, S., et al. J. Biol. Chem., 276, 23456-63, 2001). α-Rhamnose was included as a non-human monosaccharide.

Supplementary Figure 2 Human IntL-1 binding specificity as determined from the microbial glycan microarray (MGMv2).

(a) Results of the Microbial Glycan Microarray organized by genus and species, alphabetically. The fluorescence values are identical to those presented in Fig. 2b. The chemical epitope that is proposed to be a hIntL-1 ligand is depicted. The chart identification number from this graph is provided in parenthesis below the graphically depicted ligand. Data are presented as the mean ± s.d. (n=4 of a technical replicate for each immobilized glycan). The complete data for this experiment are available in Supplementary Table 3.

(b) Chemical structures of terminal α-Galf containing glycans that failed to bind hIntL-1. The Galf residues in each glycan are depicted in red. The BPS number (BPS #) that references each glycan (Stowell, S.R., et al. Nat. Chem. Biol., 10, 470-6, 2014), and the hIntL-1 signal (from Fig. 2b) are shown.

Supplementary Figure 3 Structural alignment of hIntL-1 and human L-ficolin (PDB 2J3U).

(a) Primary protein sequence and secondary structure comparison of hIntL-1 and L-ficolin (PDB: 2J3U; Garlatti, V., et al. EMBO J., 26, 623-633, 2007.) generated using ESPript 3.0 (Robert, X. & Gouet, P. Nucleic Acids Res., 42, W320-W324, 2014.). The figure was produced from a Clustal W alignment of hIntL-1 (residues 29-313) and L-ficolin (Residues 96-313). The residues depicted correspond to those that were resolvable in each protein structure. This alignment omits the collagen-like domain of L-ficolin. The box denotes the proposed fibrinogen-like domain (FBD) of each molecule. A red box highlights identical residues. The cysteine residues from hIntL-1 that are involved in intermolecular trimerization are identified with an arrow.

(b) A hIntL-1 monomer (wheat) aligned to a L-ficolin monomer (PDB: 2J3U) (grey) using Gesamt v6.4 (Krissinel, E. J. Mol. Biochem., 1, 76-85, 2012.). Reported RMSD=3.6 Å for 165 superimposable Cα atoms between the two structures. After the first 165 Cα atoms, the structures are too divergent to assign Cα atoms as superimposable, and they are not included in this calculation. The co-crystallized carbohydrate ligands are depicted to highlight differences in ligand binding sites. The hIntL-1 ligand is shown in black and the L-ficolin ligand is shown in red. Calcium ions are shown in green. Human IntL-1 binds three calcium ions, while L-ficolin binds one. The N-termini are highlighted with an N.

(c) The alignment shown in panel b, except that L-ficolin is translated by 45 Å for clarity. The N-terminus of each monomer is denoted with an N.

Supplementary Figure 4 hIntL-1 bound to allyl-β-d-Galf.

(a) Structure of the ligand-binding site in Apo-hIntL-1 (4WMQ). Calcium ions are shown in green, and ordered water molecules in red. Dashed lines highlight functional groups important for the heptavalent coordination of the ligand binding site calcium ion.

(b) Close-up view of the ligand-binding site of the β-Galf−hIntL-1 protein structure (4WMY). This image is the same as depicted in Fig. 3b, although surface mesh is depicted around the β-Galf ligand to highlight the ligand electron density. Mesh represents an difference density map (mFo-DFc, 3σ). Calcium ions are depicted in green and ordered waters are shown in red. The ligand O(5) and O(6) hydroxyl groups coordinate to the calcium ion and displace two ordered water molecules.

(c) Structural comparison of the crystallized allyl-β-d-Galf ligands. The molecule from Chain A is shown in wheat, while the molecule shown in Chain B is shown in grey. The furanosides were overlaid using the C(2)-C(3) bond and translated apart by 8 Å.

(d) Table summarizing Chain A and Chain B in the β-Galf−hIntL-1 protein structure (4WMY).

Supplementary Figure 5 hIntL-1 exhibits specificity for microbial glycan epitopes bearing terminal 1,2-diols.

(a) hIntL-1 binding to immobilized α-Neu5Ac assayed by the ELISA-like carbohydrate-binding assay (Supplementary Fig. 1c). Data are fit to a one site binding equation (solid lines). Data are presented as the mean (n=2 of a technical replicate and is representative of three independent experiments).

(b) Inhibition of hIntL-1 binding to immobilized β-Galf. Four compounds (glycerol, 1-phosphoglycerol, the methyl-α-glycoside of Neu5Ac, and the methyl-α-D-mannopyranoside) were dissolved in binding buffer and included during the hIntL-1 incubation. Binding data shown are relative to a control where no competitor was added to the binding buffer. Data are presented as the mean (n=2 of a technical replicate and is representative of three independent experiments).

Supplementary Figure 6 hIntL-1 binding to S. pneumoniae.

(a) Strep-tagged hIntL-1 binding to different S. pneumoniae serotypes (8, 20, 70, 43) (Fig. 4b shows a subset of these data and the structures of the glycans on these serotypes are shown in Fig 4a). Human IntL-1 was visualized with the anti-Strep-tag antibody conjugate (red) and DNA visualized with Hoechst (blue). The addition of EDTA inhibits hIntL binding, supporting a calcium ion mediate mechanism of binding. The addition of a competitive ligand, glycerol, also inhibits hInL-1 binding. In the anti-Strep control, Strep-hIntL-1 was omitted. Images are representative of greater than five fields of view. Scale bar, 5 μm.

(b) Specificity of Strep-hIntL-1 for S. pneumoniae serotypes. The full data set from Fig. 4d is shown. The addition of EDTA and glycerol abrogate binding, supporting a role for calcium ions in 1,2 exocyclic diol recognition. In the anti-Strep control sample, recombinant Strep-hIntL-1 was omitted. All data were collected with identical instrument settings.

Supplementary Figure 7 Mouse intelectin-1 binding to immobilized carbohydrates.

Purified Strep-mIntL-1 binding to immobilized carbohydrates monitored using SPR. Addition of EDTA prevents carbohydrate binding, supporting a role for calcium ions in carbohydrate binding. Data are referenced to the biotin channel.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Note (PDF 2360 kb)

Supplementary Table 1

Human IntL-1 binding to furanoside glycan array (XLS 405 kb)

Supplementary Table 2

Human IntL-1 binding to mammalian glycan array (XLS 690 kb)

Supplementary Table 3

Human IntL-1 binding to microbial glycan array (XLS 2484 kb)

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Wesener, D., Wangkanont, K., McBride, R. et al. Recognition of microbial glycans by human intelectin-1. Nat Struct Mol Biol 22, 603–610 (2015). https://doi.org/10.1038/nsmb.3053

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