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Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host

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Abstract

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

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Data availability

The atomic coordinates and structure factors of the Ricistatin–cathepsin V complex has been deposited in the Protein Data Bank with accession code 7PK4, and the raw X-ray diffraction images has been deposited in the SBGrid Data Bank with accession code 1045.

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Funding

MK received funding received from the Grant Agency of the Czech Republic (Grant 19-38207247S) and ERD Funds, project CePaVip OPVVV (No. 384 CZ.02.1.01/0.0/0.0/16_019/0000759). The project was further supported by the Grant Agency of the Czech Republic (Grant 19-14704Y to JC). MM and MB were supported by ERD Fund project ChemBioDrug (CZ.02.1.01/0.0/0.0/16_019/0000729), by Grant LTAUSA19109 from the Ministry of Education of the Czech Republic, and by institutional project RVO 61388963.

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  1. Larissa A. Martins and Michal Buša have contributed equally to this work.

Authors and Affiliations

  1. Institute of Parasitology, Branišovská 1160/31, 37005, Ceske Budejovice, Czech Republic

    Larissa A. Martins, Jan Kotál, Mohamed Amine Jmel & Michail Kotsyfakis

  2. Laboratory of Neurological Infections and Immunity, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, 59840, USA

    Larissa A. Martins

  3. Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo N. 2, 16610, Prague, Czech Republic

    Michal Buša & Michael Mareš

  4. Department of Medical Biology, Faculty of Science, the University of South Bohemia in České Budějovice, Branišovská 1760C, 37005, Ceske Budejovice, Czech Republic

    Adéla Chlastáková, Jan Kotál, Zuzana Beránková & Jindřich Chmelař

  5. Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany

    Natascha Stergiou & Edgar Schmitt

  6. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, N. Plastira 100, 70013, Heraklion, Crete, Greece

    Michail Kotsyfakis

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Contributions

LAM, MB, AC, JK, ZB, MAJ, and NS performed experiments and analyzed data; LAM, MB, AC, and MK designed experiments; MM, ES, JC, and MK supervised the study; LAM, MB, MM, JC, and MK evaluated the data and revised the manuscript for publication. All authors contributed to the manuscript and approved the final version of the manuscript.

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Correspondence to Michael Mareš or Michail Kotsyfakis.

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All animal experiments were carried out in accordance with the Animal Protection Law of the Czech Republic No. 246/1992 Sb., ethics approval No. 34/2018, and the responsible committee of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and the Ministry of Education, Youth and Sports of the Czech Republic approved the protocol (No. 19085/2015-3).

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Martins, L.A., Buša, M., Chlastáková, A. et al. Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host. Cell. Mol. Life Sci. 80, 339 (2023). https://doi.org/10.1007/s00018-023-04993-4

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