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.

• Keywords
• Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
• MeSH
• Cystatins * pharmacology   MeSH
• Cysteine metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Cathepsins metabolism   MeSH
• Ixodes * chemistry   MeSH
• Vertebrates MeSH
• Peptide Hydrolases metabolism   MeSH
• Salivary Cystatins chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins * MeSH
• Cysteine MeSH
• Endopeptidases MeSH
• Cathepsins MeSH
• Peptide Hydrolases MeSH
• Salivary Cystatins MeSH

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

• Keywords
• cystatin, cysteine cathepsin, helminth parasite, protease inhibitor, protein evolution, protein structure, stefin,
• MeSH
• Cystatins * genetics   chemistry   MeSH
• Disulfides MeSH
• Fasciola hepatica * genetics   MeSH
• Phylogeny MeSH
• Helminth Proteins chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cystatins * MeSH
• Disulfides MeSH
• Helminth Proteins MeSH

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

• Keywords
• Ixodes ricinus, cathepsin, crystal structure, cysteine protease, digestion, midgut, parasite,
• MeSH
• Cystatins metabolism   MeSH
• Phylogeny MeSH
• Cathepsin L metabolism   MeSH
• Ticks metabolism   MeSH
• Ixodes metabolism   MeSH
• Blood Proteins metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteolysis MeSH
• Amino Acid Sequence MeSH
• Digestive System metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins MeSH
• Cathepsin L MeSH
• Blood Proteins MeSH

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

• Keywords
• cysteine protease inhibitor, diversification, parasite, phylogenetic analysis, protein structure, signal peptide, stefin,
• Publication type
• Journal Article MeSH

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

• Keywords
• Cathepsin, Crystal structure, Immune responses, Ixodes ricinus, Saliva,
• MeSH
• Cystatins classification   genetics   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Epoxy Compounds metabolism   MeSH
• Phylogeny MeSH
• Immunosuppressive Agents chemistry   metabolism   pharmacology   MeSH
• Ixodes chemistry   genetics   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Macrophages drug effects   metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Arthropod Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Salivary Cystatins chemistry   genetics   pharmacology   MeSH
• T-Lymphocytes drug effects   metabolism   MeSH
• Tyrosine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• cathestatin C MeSH Browser
• Cystatins MeSH
• Cytokines MeSH
• Epoxy Compounds MeSH
• Immunosuppressive Agents MeSH
• Nitric Oxide MeSH
• Arthropod Proteins MeSH
• Salivary Cystatins MeSH
• Tyrosine MeSH

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

• Keywords
• DPP1, cathepsin C, cathepsin S, cystatin OmC2, dendritic cells, dipeptidyl peptidase 1, lysosomal proteases, tick saliva,
• MeSH
• B7-2 Antigen MeSH
• Antigens, Differentiation, B-Lymphocyte MeSH
• Cell Line MeSH
• Cystatins metabolism   MeSH
• Dendritic Cells immunology   metabolism   MeSH
• Epoxy Compounds immunology   metabolism   MeSH
• Genes, MHC Class II immunology   MeSH
• Cathepsin C metabolism   MeSH
• Cathepsins chemistry   immunology   metabolism   MeSH
• Ticks enzymology   MeSH
• Humans MeSH
• Lysosomes enzymology   MeSH
• Histocompatibility Antigens Class II MeSH
• Ornithodoros enzymology   MeSH
• Recombinant Proteins MeSH
• Saliva enzymology   MeSH
• Tyrosine analogs & derivatives   immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• B7-2 Antigen MeSH
• Antigens, Differentiation, B-Lymphocyte MeSH
• cathepsin S MeSH Browser
• cathestatin C MeSH Browser
• CTSC protein, human MeSH Browser
• Cystatins MeSH
• Epoxy Compounds MeSH
• invariant chain MeSH Browser
• Cathepsin C MeSH
• Cathepsins MeSH
• Histocompatibility Antigens Class II MeSH
• Recombinant Proteins MeSH
• Tyrosine MeSH

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

• Keywords
• cystatins, immunomodulation, protease inhibitors, serpins, tick-host interaction,
• MeSH
• Cystatins physiology   therapeutic use   MeSH
• Immunomodulation MeSH
• Protease Inhibitors classification   metabolism   therapeutic use   MeSH
• Serine Proteinase Inhibitors physiology   therapeutic use   MeSH
• Host-Parasite Interactions MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Serpins physiology   therapeutic use   MeSH
• Saliva enzymology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cystatins MeSH
• Protease Inhibitors MeSH
• Serine Proteinase Inhibitors MeSH
• Serpins MeSH

Th17 cells constitute a subset of CD4(+) T lymphocytes that play a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here, we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of the interleukin-6 (IL-6)/STAT-3 signaling pathway. Following activation, mature dendritic cells produce an array of cytokines, including the pleiotropic cytokine IL-6, which triggers the IL-6 signaling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. The results presented extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune responses.

• MeSH
• Borrelia immunology   MeSH
• Cell Differentiation drug effects   MeSH
• Th17 Cells drug effects   physiology   MeSH
• Dendritic Cells drug effects   physiology   MeSH
• Interleukin-6 antagonists & inhibitors   metabolism   MeSH
• Ixodes MeSH
• Mice, Inbred C57BL MeSH
• Serpins metabolism   MeSH
• Signal Transduction drug effects   MeSH
• STAT3 Transcription Factor antagonists & inhibitors   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• interleukin-6, mouse MeSH Browser
• Interleukin-6 MeSH
• Serpins MeSH
• Stat3 protein, mouse MeSH Browser
• STAT3 Transcription Factor MeSH

Ticks, as obligate hematophagous ectoparasites, impact greatly on animal and human health because they transmit various pathogens worldwide. Over the last decade, several cystatins from different hard and soft ticks were identified and biochemically analyzed for their role in the physiology and blood feeding lifestyle of ticks. All these cystatins are potent inhibitors of papain-like cysteine proteases, but not of legumain. Tick cystatins were either detected in the salivary glands and/or the midgut, key tick organs responsible for blood digestion and the expression of pharmacologically potent salivary proteins for blood feeding. For example, the transcription of two cystatins named HlSC-1 and Sialostatin L2 was highly upregulated in these tick tissues during feeding. Vaccinating hosts against Sialostatin L2 and Om-cystatin 2 as well as silencing of a cystatin gene from Amblyomma americanum significantly inhibited the feeding ability of ticks. Additionally, Om-cystatin 2 and Sialostatin L possessed strong host immunosuppressive properties by inhibiting dendritic cell maturation due to their interaction with cathepsin S. These two cystatins, together with Sialostatin L2 are the first tick cystatins with resolved three-dimensional structure. Sialostatin L, furthermore, showed preventive properties against autoimmune diseases. In the case of the cystatin Hlcyst-2, experimental evidence showed its role in tick innate immunity, since increased Hlcyst-2 transcript levels were detected in Babesia gibsoni-infected larval ticks and the protein inhibited Babesia growth. Other cystatins, such as Hlcyst-1 or Om-cystatin 2 are assumed to be involved in regulating blood digestion. Only for Bmcystatin was a role in tick embryogenesis suggested. Finally, all the biochemically analyzed tick cystatins are powerful protease inhibitors, and some may be novel antigens for developing anti-tick vaccines and drugs of medical importance due to their stringent target specificity.

• MeSH
• Cystatins pharmacology   MeSH
• Cysteine Proteinase Inhibitors pharmacology   MeSH
• Ticks drug effects   physiology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Feeding Behavior drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cystatins MeSH
• Cysteine Proteinase Inhibitors MeSH