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A family of di-glutamate mucin-degrading enzymes that bridges glycan hydrolases and peptidases

Nature Catalysis volume 7, pages 386–400 (2024)Cite this article

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Abstract

Microbes utilize polysaccharides to protect their surfaces and build biofilms, whereas metazoans employ large mucins densely decorated with O-glycans to protect surfaces and keep microbes at a distance. However, gut microbes in mucus also feed on host mucins, thus imposing a need for continuous renewal to maintain protection, clearance and mucus homeostasis. Glycopeptidases that can cleave mucins are known, but mucinases that specifically cleave mucins are not. Here we report the discovery of such microbial mucinases that cleave mucins with trimmed glycans, recognize dense clusters of O-glycans, and employ a structural fold and catalytic machinery reminiscent of glycan hydrolases and peptidases. These di-glutamate mucinases are also found in eukaryotes, and we propose that they are designed to clear mucins following scavenging of O-glycans to promote healthy gut–microbiome homeostasis.

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Fig. 1: Discovery of a mucinase family sharing a β-sandwich catalytic domain (X414/DUF3472).
Fig. 2: The HC7 mucinase cleaves mucins with dense clusters of truncated O-glycans.
Fig. 3: Crystal structure of the HC7ΔCBM mucinase complexed to GalNAc glycopeptides.
Fig. 4: Structural features of the active site and catalytic mechanism of HC7.
Fig. 5: Topology analysis of similar catalytic domains and eukaryotic mucinases discovery.

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Data availability

The crystal structure of HC7ΔCBM-apo, as well as those of the HC7ΔCBM-E200A–triglycopeptide and HC7ΔCBM-E200A–tetraglycopeptide complexes, have been deposited at the RCSB PDB with accession codes 8PMU, 8PN5 and 8PN3, respectively. Previously published PDB structures used in this study are available under accession codes 4QHX, 1ZM1, 7ZVB, 2IFR, 1U0A, 1Y43 and 3ZSJ. Data files of the classical MD simulation and QM/MM metadynamics simulations have been deposited in Zenodo (https://doi.org/10.5281/zenodo.10103923). The full uncollapsed tree is available as Supplementary Data on Zenodo (https://doi.org/10.5281/zenodo.10103923) as well as the raw treefile used to generate this figure. Other data are available from the corresponding author upon request. The kinetics and computational data generated in this study are provided as source data. Any other data may be provided by the corresponding authors on request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Novo Nordisk Foundation (NNF22OC0076899 to H.J.J., NNF20SA0067193 to B.H. and NNF210071658 to H.C.), the Danish National Research Foundation (DNRF107), the Lundbeck Foundation, the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 787684 (to C.B.), the Spanish Ministry of Science, Innovation and Universities (BFU2016-75633-P, PID2019-105451GB-100, PID2022-136362NB-100, PID2021-127622OB-100, PID2020-118893GB-100 and CEX2021-001202-M), Gobierno de Aragón (E34_R17 and LMP58_18) with FEDER (2014–2020) funds for ‘Building Europe from Aragón’ for financial support (to R.H.-G.), and the Agency for Management of University and Research Grants of Catalonia (AGAUR, 2021-SGR-00680 to C.R.). We thank ALBA (Barcelona, Spain) synchrotron beamline XALOC, and Red Española de Supercomputación (RES-BSC) for computer time in CTE-Power and MareNostrum IV. Q.L. thanks the EU for a Marie-Skłodowska Curie fellowship (MCSA-IF-2020, agreement no. 101025071). R.H.-G. thanks M. del Carmen Hurtado-Lago for help with the design of Fig. 5c.

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Author notes

  1. These authors contributed equally: Yoshiki Narimatsu, Christian Büll, Víctor Taleb, Qinghua Liao.

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark

    Yoshiki Narimatsu, Christian Büll, Lars Hansen, Henrik Clausen, Hiren J. Joshi & Ramon Hurtado-Guerrero

  2. GlycoDisplay ApS, Copenhagen, Denmark

    Yoshiki Narimatsu

  3. Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands

    Christian Büll

  4. The Institute for Biocomputation and Physics of Complex Systems (BIFI), Zaragoza, Spain

    Víctor Taleb, David Sánchez-Navarro & Ramon Hurtado-Guerrero

  5. Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica) and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Barcelona, Spain

    Qinghua Liao & Carme Rovira

  6. Departamento de Química, Universidad de La Rioja, Instituto de Investigación en Química de la Universidad de La Rioja (IQUR), Logroño, Spain

    Ismael Compañón & Francisco Corzana

  7. Architecture et Fonction des Macromolecules Biologiques, Centre National de la Recherche Scientifique and Aix-Marseille University, Marseille, France

    Fabien Durbesson, Renaud Vincentelli & Bernard Henrissat

  8. Institució Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Spain

    Carme Rovira

  9. DTU Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark

    Bernard Henrissat

  10. Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

    Bernard Henrissat

  11. Fundación ARAID, Zaragoza, Spain

    Ramon Hurtado-Guerrero

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  1. Yoshiki Narimatsu
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  2. Christian Büll
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  3. Víctor Taleb
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  4. Qinghua Liao
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Y.N., C.B., B.H., H.C., H.J.J. and R.H.-G. conceived and directed the study. Y.N. and C.B. performed most of the experimental analysis. B.H., H.J.J. and L.H. performed the bioinformatics. F.D. and R.V. contributed to recombinant expression and purification. V.T. and D.S.-N. expressed and purified the HC7ΔCBM and Mucor mucinases. V.T. crystallized the HC7ΔCBM and HC7ΔCBM-E200A, and R.H.-G. solved and built the crystal structures. F.C. performed the MD simulations. Q.L. and C.R. performed MD and QM/MM enhanced sampling simulations. I.C. synthetized the glycopeptides. Y.N., H.C. and R.H.-G. wrote the paper, and all authors edited and approved the final version.

Corresponding authors

Correspondence to Yoshiki Narimatsu or Ramon Hurtado-Guerrero.

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Competing interests

The University of Copenhagen has filed a patent application on the cell-based display platform (US20190330601A1). GlycoDisplay Aps, Copenhagen, Denmark, has obtained a licence to the field of the patent application. Y.N. and H.C. are co-founders of GlycoDisplay Aps and hold ownership in the company. The other authors declare no competing interests.

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Nature Catalysis thanks Elisa Fadda and Shinya Fushinobu for their contribution to the peer review of this work.

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Supplementary Figs. 1–28, Tables 1 and 2 and Methods.

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Source Data Figs. 1c, 2b,c,e,f, 3a and 4e

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Narimatsu, Y., Büll, C., Taleb, V. et al. A family of di-glutamate mucin-degrading enzymes that bridges glycan hydrolases and peptidases. Nat Catal 7, 386–400 (2024). https://doi.org/10.1038/s41929-024-01116-5

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