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Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota

Nature Chemical Biology volume 18, pages 841–849 (2022)Cite this article

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An Author Correction to this article was published on 05 August 2022

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Abstract

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

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Fig. 1: Schematic representation of sulfated host carbohydrates found in the colon.
Fig. 2: S1 carbohydrate sulfatases share a conserved α/β/α-fold, S site and catalytic apparatus.
Fig. 3: S1_46 members from the HGM require recognition of the N-acetyl group for activity.
Fig. 4: Aromatic stacking analysis at the 0 subsite in S1_16 members from the HGM.
Fig. 5: Specificity of S1_15 subfamily members for 6S-Gal and 6S-GalNAc is determined by the openness of the S and 0 subsites.
Fig. 6: Variations in the non-conserved region of S1_11 members, dictated by PUL context, drive recognition of N-sulfate or N-acetyl.

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Data availability

The crystal structure datasets generated have been deposited in the Protein Data Bank (PDB) under the following accession numbers: 7OZ8, 7OZ9, 7OZA, 7OZE, 7OZC, 7P26 and 7P24. Source data are provided with this paper.

Code availability

No new code was developed or compiled in this study.

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Acknowledgements

This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement number 748336 and Wilhelm och Martina Lundgrens Vetenskapsfond (2020.3597) awarded to A.S.L. Additional funding sources include The European Research Council ERC (694181), the Knut and Alice Wallenberg Foundation (2017.0028), Swedish Research Council (2017-00958) awarded to G.C.H., the National Institute of Health (R01 DK118024 and DK125445 and U01AI095473) award to E.C.M. and G.C.H. and the Academy of Medical Sciences/Wellcome Trust through the Springboard Grant (SBF005\1065 163470) awarded to A.C. We acknowledge access to the SOLEIL and Diamond Light sources via both the University of Liverpool and Newcastle University BAGs (proposals mx21970 and mx18598, respectively). We thank the staff of DIAMOND and SOLEIL and members of the Liverpool’s Molecular biophysics group for assistance with data collection. We are also grateful for E. Corre’s help regarding bioinformatics analyses (ABIMS platform, Station Biologique de Roscoff, France).

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA

    Ana S Luis & Eric C. Martens

  2. Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden

    Ana S Luis, Chunsheng Jin, Niclas G. Karlsson & Gunnar C. Hansson

  3. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

    Arnaud Baslé

  4. Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK

    Dominic P. Byrne, Gareth S. A. Wright, James A. London, Patrick A. Eyers, Edwin A. Yates & Alan Cartmell

  5. Faculty of Health Sciences, Department of Life Sciences and Health, Pharmacy, Oslo Metropolitan University, Oslo, Norway

    Niclas G. Karlsson

  6. Sorbonne Université, Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS, Roscoff, France

    Mirjam Czjzek & Tristan Barbeyron

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  1. Ana S Luis
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Contributions

A.S.L. and A.C. conceived and designed experiments. A.C., A.S.L. and E.C.M. wrote the draft manuscript. A.S.L. and A.C. cloned, expressed and purified sulfatases and performed the enzymatic assays. A.C., D.P.B. and J.A.L. performed and analyzed kinetic and binding experiments. E.A.Y. and J.A.L. performed labeling and NMR experiments. A.C. and A.B. performed structural biology experiments. M.C. and T.B. performed sulfatase phylogenetic analyses. J.C. and N.G.K. performed glycan analyses. G.S.A.W. performed light scattering experiments and size determination. A.C., A.S.L., G.C.H. and E.C.M. supervised and provided funding for the project. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Ana S Luis or Alan Cartmell.

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Nature Chemical Biology thanks Jan-Hendrick Hehemann, Nicolas Terrapon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Electron density maps of extracted ligands.

The 2mFobs-Fc maps are shown contoured at 1σ for all substrates and products co-crystallised with their respective sulfatase.

Extended Data Fig. 2 Biophysical, Specificity and phylogenetic analysis of BT19183S-GlcNAc and S1_46.

a, Radial version of the phylogenetic tree of representative sulfatases from subfamily S1_46. The tree comprises a total of 564 sequences with 250 being Firmicutes; 156 are Bacteroidetes; 54 are Actinobacteria; 25 are Proteobacteria; 20 are Lentisphaerae. For clarity all labels and sequence accession codes have been omitted. The annotations next to the colour code reveal the presence or absence of conserved residues crucial for substrate recognition by BT19183S-GlcNAc (acc-code Q8A6G6) in the following order: Y94, N174, R327 and Y408. These residues are invariant in HGM Bacteroidetes, whilst in Firmicutes from the HGM, the Y408 equivalent is not conserved. The residues are coloured as following: black means an equivalent residue is present; a grey and bold letter at any position means that the corresponding residue is replaced by that amino acid; a grey, bold and italic letter at any position means that the equivalent position is replaced by any type of amino acid; a bold grey letter followed by one-letter codes in parentheses indicates that the equivalent position can be substituted by any of those amino acids; the dash at the Y408-equivalent position indicates that no equivalent amino acid can be deduced from the multiple alignment. Branches of the same colour have the corresponding pattern in common. The red filled circle designates the sequence of the S1_46 sulfatase from B. thetaiotaomicron (See Supplementary Fig. 2 for full tree). b, Thin layer chromatography analysis of BT19183S,6S-GlcNAc versus 3S-glucosamine, 3 S,6S-glucosamine, and 3S,6S-N-acetylglucosamine, and QSI_25163S,6S-GlcNAc versus 3S-glucosamine. 3 S,6S-glucosamine, and 3S,6S-N-acetylglucosamie. All assays described were performed for 48 h at 37 °C, containing 6 mM substrate and 5 μM (BT19183S-GlcNAc) or 100 μM enzyme (QSI_25163S,6S-GlcNAc) and 3 mM HEPES pH 7.0, 45 mM NaCl and 5 mM CaCl2.

Source data

Extended Data Fig. 3 Activity and stability analysis of S1_16 sulfatases and their mutant variants.

a, DSF analysis of the effects of galactose and N-acetylgalactosamine on thermostability, with a positive-shift indicative of substrate binding. b, Normalised DSF melt curves of BT30574S-Gal/GalNAc and BT37964S-Gal/GalNAc. c, DSF melt curves of purified monomer and dimer species (left), monomer species in the presence of galactose and N-acetylgalactosamine (middle), or dimer species in the presence of galactose and N-acetylgalactosamine (right). d, Thin layer chromatography (TLC) analysis of wild-type (WT) and mutant S1_16 sulfatases. Asterisks are placed above lanes where sulfatase activity is observed. e, High pressure anion exchange chromatography (HPAEC) of WT and mutants. A grey block highlights the desulfated product. Both TLC and HPAEC reactions utilised 6 mM substrate and 1 μM enzyme, except for W109A variants where 10 μM was used, with 3 mM HEPES, 45 mM NaCl, and 5 mM CaCl2. Reactions incubated at 37 °C for 48 h. Control represents the substrate incubated in same conditions without adding enzyme. Experiments are technical triplicates and error bars represent SEM.

Source data

Extended Data Fig. 4 Radial phylogenetic tree of representative sulfatases from subfamily S1_16.

The tree comprises a total of 1368 sequences of which 854 are Bacteroidetes; 211 are Planctomycetes; 107 are Kiritimatiellaeota; 64 are Verrucomicrobia; 53 are Lentisphaerae. For clarity all labels and sequence accession codes have been omitted. The annotations next to the colour code reveal the presence or absence of conservation of the critical residues in substrate recognition by BT30574S-Gal/GalNAc (acc-code Q8A397) in the following order: W109, H182, and H423. Sequences coded by orange branches contain an additional W332 present in BT37964S-Gal/GalNAc (acc-code Q8A171) but absent in other sequences. For simplification the residue numbers have been omitted, except for W332. The residues are coloured as following: black means an equivalent amino acid is present; a grey and bold letter at any position means that the corresponding residue is replaced by that amino acid; a grey and italic letter at any position means that the equivalent position is replaced by any type of amino acid; a bold grey letter followed by one-letter codes in parentheses indicates that the equivalent position can be substituted by any of those amino acids; the dash at the H-equivalent position indicates that no equivalent amino acid can be deduced from the multiple alignment. When two patterns are indicated separated by a comma (that is W - H, W H -) both have been given the same colour code. Branches having the same colour have the corresponding pattern in common. Red filled circles designate sequences of S1_16 sulfatases from B. thetaiotaomicron (See Supplementary Fig. 4 for full tree).

Extended Data Fig. 5 Analysis of the activity and stability of BT16246S-Gal/GalNAc and its mutant variants.

a, Thin layer chromatography (TLC) analysis of wild-type (WT) BT16246S-Gal/GalNAc and its mutants. Asterisks are placed above lanes where activity is observed. b, High pressure anion exchange chromatography (HPAEC) of wild-type BT16246S-Gal/GalNAc WT and its mutants. The desulfated product is highlighted by a grey box. All TLC (a) HPAEC (b) reactions utilised 6 mM substrate and 5 μM enzyme, with 3 mM HEPES, 45 mM NaCl, and 5 mM CaCl2. Reactions incubated for 48 h at 37 °C. Control represents the substrate incubated in same conditions without adding enzyme. c, DSF analysis showing relative thermostability of BT16246S-Gal/GalNAc mutant proteins with respect to the WT enzyme. d, DSF analysis of the effects of alanine scanning on the ability of BT16246S-Gal/GalNAc to bind galactose, with the Tm of the protein shown above the bar. The experiments were performed using 5 μM of protein and 324 mM of galactose in 100 mM BTP pH 7.0 and 150 mM NaCl. Experiments are technical triplicates and error bars represent SEM.

Source data

Extended Data Fig. 6 Radial phylogenetic tree of S1_15 showing the conservation of the galacto- recognition triad and N-acetyl-D-galactosamine specificity features.

The tree comprises a total of 1906 sequences of which 1424 are Bacteroidetes; 172 are Planctomycetes; 119 are Kiritimatiellaeota; 57 are Proteobacteria; 53 are Verrucomicrobia. The annotations next to the colour code concern the presence or absence of conservation of the BT16246S-Gal/GalNAc (acc-code Q8A7A1) indicated residues and in this order: I100, D170, R171, H220, K461 and A462. These residues are crucial in substrate recognition and D170, R171, and H220 represent the galacto-recognition triad within S1_15 subfamily. For simplification the residue numbers have been omitted. For example, an I in black means an equivalent isoleucine is present; a grey and bold letter at any position means that the corresponding residue is replaced by that amino acid; a grey and italic letter at any position means that the equivalent position can be replaced by any type of amino acid; a bold grey letter followed by one-letter codes in parentheses indicates that the equivalent position is substituted by any of those amino acids. Branches having the same colour have the corresponding pattern in common. For clarity all labels and sequence accession codes have been omitted. Red filled circles designate sequences of S1_15 sulfatases from B. thetaiotaomicron (See Supplementary Fig. 6 for full tree).

Extended Data Fig. 7 Conservation of the N-acetyl-D-galactosamine specificity features (Y463/W464) in S1_15 enzymes within PULs targeting chondroitin sulfate.

Schematic representation of PULs targeting chondroitin sulfate aligned by orthologues of BT33336S-GalNAc. Light green background shows orthologues with Y463/W464, a dark green background highlights orthologues with F463/W464, a light blue background highlights orthologues with H463/W464, and a purple background highlights orthologues with Q463/W464. The numbering used corresponds to the sequence of BT33336S-GalNAc. A red background highlights the presence of GH88 and S1_27 (an endo 4S-chrondroitin sulfatase), which is encoded by a discrete genetic region not always physically localised next to the core PUL. A black background highlights a core block observed in CS PULs containing BT33336S-GalNAc orthologues. HP (protein of unknown function), S1 (sulfatase S1 with the respective subfamily number superscript), GHXX (glycoside hydrolase with X representing the family number), PL (polysaccharide lyase), DUF (domain of unknown function), HTCS (hybrid two-component system), SusC (starch utilization system C-like), SusD (starch utilization system D-like), SGBP (surface glycan binding protein).

Extended Data Fig. 8 Analysis of the activity and stability of BT31776S-GlcNAc and mutant variants.

a, High pressure anion exchange chromatography (HPAEC) of wild-type BT31776S-GlcNAc wild-type (WT) and substituted variants. The produced product is highlighted by a grey box. b, Thin layer chromatography (TLC) analysis of WT BT31776S-GlcNAc and its mutants. Asterisks are placed above lanes where activity is observed. Both HPAEC (a) and TLC (b) reactions utilised 6 mM substrate and 5 μM enzyme, with 3 mM HEPES, 45 mM NaCl, and 5 mM CaCl2 over a 48 h period at 37 °C. Control represents the substrate incubated in same conditions without enzyme. c, DSF analysis showing relative thermostability of mutant proteins of BT31776S-GlcNAc in comparison to the WT enzyme. Experiments are technical triplicates and error bars represent SEM.

Source data

Extended Data Fig. 9 Radial phylogenetic tree of S1_11 showing the conservation of the substrate recognition triad and N-sulfate specificity features.

The tree comprises a total of 2178 sequences of which 1190 are Bacteroidetes; 233 are Verrucomicrobia;184 are Planctomycetes;143 are Ascomycota (fungi); 100 are Actinobacteria.The annotations next to the colour code concern the presence or absence of conservation of the indicated residues and in this order: R290, W273, D385, R387 and H471. These residues are required for substrate recognition by BT46566S-GlcNAc/GlcNS (acc-code Q89YS5). D385, R387, and H471 represent the recognition triad, whilst the presence of W or R at positions 273 and 290, respectively, represent N-sulfate specificity features. Residue numbers have been omitted for simplicity. For example, an R in black means an equivalent arginine is present; a grey and bold letter at this position means that the corresponding residue is replaced by that amino acid; the grey and italic R at this position means that the R-equivalent position is replaced by any type of amino acid; a bold grey R followed by one-letter codes in parentheses indicates that the R-equivalent position can be substituted by any of those amino acids; the dash at the R-equivalent position indicates that no equivalent amino acid can be deduced from the multiple alignment. Branches having the same colour have the corresponding pattern in common. Red filled diamonds designate sequences of S1_11 sulfatases from B. thetaiotaomicron. All sequences in the specific branch that contains BT46566S-GlcNAc/GlcNS are found within a conserved heparan sulfate PUL. For clarity, all labels and sequence accession codes have been omitted (See Supplementary Fig 7 for full tree).

Extended Data Fig. 10 Conservation of the N-sulfate targeting features, W273/R290, in S1_11 enzymes within PULs targeting heparan sulfate.

PULs targeting heparan sulfate (HS) aligned by orthologues of BT46566S-GlcNAc/GlcNS. Orthologues of BT46566S-GlcNAc/GlcNS with W273/R290 and W273/Q290 are highlighted with a green and blue background, respectively. A black background highlights a core block observed in HS PULs containing BT46566S-GlcNAc/GlcNS orthologues. HP (protein of unknown function), S1 (sulfatase S1 with the respective subfamily number superscript), GHXX (glycoside hydrolase with X representing the family number), PL (polysaccharide lyase), DUF (domain of unknown function), HTCS (hybrid two-component system), SusC (starch utilization system C-like), SusD (starch utilization system D-like), SGBP (surface glycan binding protein), MFS (major facilitator superfamily), ROK (repressor, ORF, kinase superfamily).

Supplementary information

Source data

Source Data Fig. 3

Raw and processed kinetic data.

Source Data Fig. 4

Raw and processed kinetic data.

Source Data Fig. 5

Raw and processed DSF data.

Source Data Fig. 6

Raw and processed DSF data.

Source Data Extended Data Fig. 2

Unmodified TLC gels.

Source Data Extended Data Fig. 3

Unmodified TLC gels and DSF data.

Source Data Extended Data Fig. 5

Unmodified TLC gels and DSF data.

Source Data Extended Data Fig. 8

Unmodified TLC gels and DSF data.

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Luis, A.S., Baslé, A., Byrne, D.P. et al. Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota. Nat Chem Biol 18, 841–849 (2022). https://doi.org/10.1038/s41589-022-01039-x

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