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.

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

Institute for Immunology University Medical Center of the Johannes Gutenberg University Mainz Langenbeckstrasse 1 55131 Mainz Germany

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

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

Institute of Parasitology Branišovská 1160 31 37005 Ceske Budejovice Czech Republic

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

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