Recognition of microbial glycans by human intelectin-1



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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Stowell, S.R. et al. Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16, 295–301 (2010).

    CAS  Article  Google Scholar 

  2. 2

    Vaishnava, S. et al. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science 334, 255–258 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Gallo, R.L. & Hooper, L.V. Epithelial antimicrobial defence of the skin and intestine. Nat. Rev. Immunol. 12, 503–516 (2012).

    CAS  Article  Google Scholar 

  4. 4

    Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A. & Gordon, J.I. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005).

    Article  Google Scholar 

  5. 5

    Varki, A. Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2009).

  6. 6

    Lis, H. & Sharon, N. Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem. Rev. 98, 637–674 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Weis, W.I. & Drickamer, K. Structural basis of lectin-carbohydrate recognition. Annu. Rev. Biochem. 65, 441–473 (1996).

    CAS  Article  Google Scholar 

  8. 8

    Turner, M.W. The role of mannose-binding lectin in health and disease. Mol. Immunol. 40, 423–429 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Fujita, T. Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2, 346–353 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Jack, D.L., Klein, N.J. & Turner, M.W. Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol. Rev. 180, 86–99 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Thomsen, T., Schlosser, A., Holmskov, U. & Sorensen, G.L. Ficolins and FIBCD1: soluble and membrane bound pattern recognition molecules with acetyl group selectivity. Mol. Immunol. 48, 369–381 (2011).

    CAS  Article  Google Scholar 

  12. 12

    Holmskov, U., Thiel, S. & Jensenius, J.C. Collectins and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21, 547–578 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Lehotzky, R.E. et al. Molecular basis for peptidoglycan recognition by a bactericidal lectin. Proc. Natl. Acad. Sci. USA 107, 7722–7727 (2010).

    CAS  Article  Google Scholar 

  14. 14

    Lee, J.K. et al. Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development. Glycobiology 7, 367–372 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Lee, J.K., Baum, L.G., Moremen, K. & Pierce, M. The X-lectins: a new family with homology to the Xenopus laevis oocyte lectin XL-35. Glycoconj. J. 21, 443–450 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Weis, W.I., Taylor, M.E. & Drickamer, K. The C-type lectin superfamily in the immune system. Immunol. Rev. 163, 19–34 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Tsuji, S. et al. Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J. Biol. Chem. 276, 23456–23463 (2001).

    CAS  Article  Google Scholar 

  18. 18

    French, A.T. et al. The expression of intelectin in sheep goblet cells and upregulation by interleukin-4. Vet. Immunol. Immunopathol. 120, 41–46 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Voehringer, D. et al. Nippostrongylus brasiliensis: identification of intelectin-1 and -2 as Stat6-dependent genes expressed in lung and intestine during infection. Exp. Parasitol. 116, 458–466 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Pemberton, A.D., Knight, P.A., Wright, S.H. & Miller, H.R. Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis. Proteomics 4, 1101–1108 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Datta, R. et al. Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite. Infect. Immun. 73, 4025–4033 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Kerr, S.C. et al. Intelectin-1 is a prominent protein constituent of pathologic mucus associated with eosinophilic airway inflammation in asthma. Am. J. Respir. Crit. Care Med. 189, 1005–1007 (2014).

    Article  Google Scholar 

  23. 23

    Kuperman, D.A. et al. Dissecting asthma using focused transgenic modeling and functional genomics. J. Allergy Clin. Immunol. 116, 305–311 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Suzuki, Y.A., Shin, K. & Lonnerdal, B. Molecular cloning and functional expression of a human intestinal lactoferrin receptor. Biochemistry 40, 15771–15779 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Tsuji, S. et al. Secretion of intelectin-1 from malignant pleural mesothelioma into pleural effusion. Br. J. Cancer 103, 517–523 (2010).

    CAS  Article  Google Scholar 

  26. 26

    de Souza Batista, C.M. et al. Omentin plasma levels and gene expression are decreased in obesity. Diabetes 56, 1655–1661 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Tsuji, S. et al. Differential structure and activity between human and mouse intelectin-1: human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer. Glycobiology 17, 1045–1051 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Pedersen, L.L. & Turco, S.J. Galactofuranose metabolism: a potential target for antimicrobial chemotherapy. Cell. Mol. Life Sci. 60, 259–266 (2003).

    CAS  Article  Google Scholar 

  29. 29

    Nassau, P.M. et al. Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase. J. Bacteriol. 178, 1047–1052 (1996).

    CAS  Article  Google Scholar 

  30. 30

    Wesener, D.A., May, J.F., Huffman, E.M. & Kiessling, L.L. UDP-galactopyranose mutase in nematodes. Biochemistry 52, 4391–4398 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Bishop, J.R. & Gagneux, P. Evolution of carbohydrate antigens: microbial forces shaping host glycomes? Glycobiology 17, 23R–34R (2007).

    CAS  Article  Google Scholar 

  32. 32

    Herget, S. et al. Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans. BMC Struct. Biol. 8, 35 (2008).

    Article  Google Scholar 

  33. 33

    Adibekian, A. et al. Comparative bioinformatics analysis of the mammalian and bacterial glycomes. Chem. Sci. 2, 337–344 (2011).

    CAS  Article  Google Scholar 

  34. 34

    Mann, D.A., Kanai, M., Maly, D.J. & Kiessling, L.L. Probing low affinity and multivalent interactions with surface plasmon resonance: ligands for concanavalin A. J. Am. Chem. Soc. 120, 10575–10582 (1998).

    CAS  Article  Google Scholar 

  35. 35

    Kiessling, L.L. & Grim, J.C. Glycopolymer probes of signal transduction. Chem. Soc. Rev. 42, 4476–4491 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Blixt, O. et al. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038 (2004).

    CAS  Article  Google Scholar 

  37. 37

    Tefsen, B., Ram, A.F., van Die, I. & Routier, F.H. Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact. Glycobiology 22, 456–469 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Stowell, S.R. et al. Microbial glycan microarrays define key features of host-microbial interactions. Nat. Chem. Biol. 10, 470–476 (2014).

    CAS  Article  Google Scholar 

  39. 39

    Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Garlatti, V. et al. Structural insights into the innate immune recognition specificities of L- and H-ficolins. EMBO J. 26, 623–633 (2007).

    CAS  Article  Google Scholar 

  41. 41

    Taha, H.A., Richards, M.R. & Lowary, T.L. Conformational analysis of furanoside-containing mono- and oligosaccharides. Chem. Rev. 113, 1851–1876 (2013).

    CAS  Article  Google Scholar 

  42. 42

    Richards, M.R., Bai, Y. & Lowary, T.L. Comparison between DFT- and NMR-based conformational analysis of methyl galactofuranosides. Carbohydr. Res. 374, 103–114 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Altona, C. & Sundaralingam, M. Conformational analysis of sugar ring in nucleosides and nucleotides: a new description using the concept of pseudorotation. J. Am. Chem. Soc. 94, 8205–8212 (1972).

    CAS  Article  Google Scholar 

  44. 44

    Angata, T. & Varki, A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem. Rev. 102, 439–469 (2002).

    CAS  Article  Google Scholar 

  45. 45

    Cartwright, K. Pneumococcal disease in western Europe: burden of disease, antibiotic resistance and management. Eur. J. Pediatr. 161, 188–195 (2002).

    CAS  Article  Google Scholar 

  46. 46

    Bentley, S.D. et al. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet. 2, e31 (2006).

    Article  Google Scholar 

  47. 47

    Black, S. et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr. Infect. Dis. J. 19, 187–195 (2000).

    CAS  Article  Google Scholar 

  48. 48

    Arnold, R.R., Cole, M.F. & McGhee, J.R. A bactericidal effect for human lactoferrin. Science 197, 263–265 (1977).

    CAS  Article  Google Scholar 

  49. 49

    Alexander, D.B., Iigo, M., Yamauchi, K., Suzui, M. & Tsuda, H. Lactoferrin: an alternative view of its role in human biological fluids. Biochem. Cell Biol. 90, 279–306 (2012).

    CAS  Article  Google Scholar 

  50. 50

    Arnold, R.R., Russell, J.E., Champion, W.J. & Gauthier, J.J. Bactericidal activity of human lactoferrin: influence of physical conditions and metabolic state of the target microorganism. Infect. Immun. 32, 655–660 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Asensio, J.L., Arda, A., Canada, F.J. & Jimenez-Barbero, J. Carbohydrate-aromatic interactions. Acc. Chem. Res. 46, 946–954 (2013).

    CAS  Article  Google Scholar 

  52. 52

    Pemberton, A.D., Rose-Zerilli, M.J., Holloway, J.W., Gray, R.D. & Holgate, S.T. A single-nucleotide polymorphism in intelectin 1 is associated with increased asthma risk. J. Allergy Clin. Immunol. 122, 1033–1034 (2008).

    CAS  Article  Google Scholar 

  53. 53

    Barrett, J.C. et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn′s disease. Nat. Genet. 40, 955–962 (2008).

    CAS  Article  Google Scholar 

  54. 54

    Schnaitman, C.A. & Klena, J.D. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol. Rev. 57, 655–682 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Willis, L.M. et al. Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens. Proc. Natl. Acad. Sci. USA 110, 7868–7873 (2013).

    CAS  Article  Google Scholar 

  56. 56

    Varghese, J.N., Mckimmbreschkin, J.L., Caldwell, J.B., Kortt, A.A. & Colman, P.M. The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins 14, 327–332 (1992).

    CAS  Article  Google Scholar 

  57. 57

    Kraschnefski, M.J. et al. Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein. Glycobiology 19, 194–200 (2009).

    CAS  Article  Google Scholar 

  58. 58

    Blanchard, H., Yu, X., Coulson, B.S. & von Itzstein, M. Insight into host cell carbohydrate-recognition by human and porcine rotavirus from crystal structures of the virion spike associated carbohydrate-binding domain (VP8*). J. Mol. Biol. 367, 1215–1226 (2007).

    CAS  Article  Google Scholar 

  59. 59

    Dormitzer, P.R., Sun, Z.Y.J., Wagner, G. & Harrison, S.C. The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J. 21, 885–897 (2002).

    CAS  Article  Google Scholar 

  60. 60

    Sauter, N.K. et al. Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography. Biochemistry 31, 9609–9621 (1992).

    CAS  Article  Google Scholar 

  61. 61

    Song, X., Lasanajak, Y., Xia, B., Smith, D.F. & Cummings, R.D. Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem. Biol. 4, 741–750 (2009).

    CAS  Article  Google Scholar 

  62. 62

    Heimburg-Molinaro, J., Song, X., Smith, D.F. & Cummings, R.D. Preparation and analysis of glycan microarrays. Curr. Protoc. Protein Sci. 64, 12.10 (2011).

    Google Scholar 

  63. 63

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  64. 64

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  65. 65

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  66. 66

    Afonine, P.V., Grosse-Kunstleve, R.W. & Adams, P.D. The Phenix refinement framework. CCP4 Newsl. 42, 8 (2005).

    Google Scholar 

  67. 67

    Schüttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

  68. 68

    Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  69. 69

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Laura L Kiessling.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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) Carbohydratebinding 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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wesener, D., Wangkanont, K., McBride, R. et al. Recognition of microbial glycans by human intelectin-1. Nat Struct Mol Biol 22, 603–610 (2015).

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing