Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.

• Keywords
• Iripin-4, Ixodes ricinus, X-ray structure, cleaved conformation, native conformation, serpins,
• MeSH
• Arginine MeSH
• Ixodes * MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Serpins * chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Serpins * MeSH

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

• Keywords
• Hyalomma dromedarii, anticoagulants, salivary glands, serpin, thrombin inhibitor,
• MeSH
• Anticoagulants chemistry   metabolism   MeSH
• Phylogeny MeSH
• Blood Coagulation drug effects   MeSH
• Ixodidae metabolism   MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Computer Simulation MeSH
• Amino Acid Sequence MeSH
• Serpins chemistry   metabolism   pharmacology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anticoagulants MeSH
• Serpins MeSH

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

• Keywords
• Iripin-5, Ixodes ricinus, X-ray structure, serine protease inhibitors, serpins, tick saliva,
• MeSH
• Anti-Inflammatory Agents * chemistry   isolation & purification   MeSH
• Erythrocytes MeSH
• Enzyme Inhibitors * chemistry   isolation & purification   MeSH
• Ixodes metabolism   MeSH
• Rabbits MeSH
• Cells, Cultured MeSH
• Macrophages MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Neutrophils MeSH
• Serpins * chemistry   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Inflammatory Agents * MeSH
• Enzyme Inhibitors * MeSH
• Serpins * MeSH

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

• Keywords
• Ixodes ricinus, blood coagulation, crystal structure, parasite, saliva, serpin, tick,
• MeSH
• Complement Activation drug effects   immunology   physiology   MeSH
• Erythrocytes metabolism   MeSH
• Gene Expression genetics   MeSH
• Blood Coagulation drug effects   physiology   MeSH
• Ixodes enzymology   genetics   metabolism   MeSH
• Complement System Proteins metabolism   MeSH
• Lyme Disease MeSH
• Nymph MeSH
• Arthropod Proteins metabolism   MeSH
• Gene Expression Regulation genetics   MeSH
• Serpins metabolism   ultrastructure   MeSH
• Salivary Glands metabolism   MeSH
• Saliva chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Complement System Proteins MeSH
• Arthropod Proteins MeSH
• Serpins MeSH

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

• Keywords
• Ixodes ricinus, X-ray crystallography, adaptive immunity, blood coagulation, inflammation, saliva, serpin, tick,
• MeSH
• Adaptive Immunity drug effects   MeSH
• Lymphocyte Activation drug effects   MeSH
• Anticoagulants isolation & purification   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Blood Coagulation drug effects   MeSH
• Insect Proteins isolation & purification   pharmacology   MeSH
• Immunologic Factors isolation & purification   pharmacology   MeSH
• Protease Inhibitors isolation & purification   pharmacology   MeSH
• Ixodes metabolism   MeSH
• Rabbits MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Lymphocytes drug effects   immunology   metabolism   MeSH
• Guinea Pigs MeSH
• Mice, Inbred C3H MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Proliferation drug effects   MeSH
• Spleen drug effects   immunology   metabolism   MeSH
• Salivary Proteins and Peptides isolation & purification   pharmacology   MeSH
• Saliva metabolism   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Guinea Pigs MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anticoagulants MeSH
• Cytokines MeSH
• Insect Proteins MeSH
• Immunologic Factors MeSH
• Protease Inhibitors MeSH
• Salivary Proteins and Peptides 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

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

• MeSH
• Platelet Aggregation genetics   immunology   MeSH
• Chymases immunology   metabolism   MeSH
• Gene Expression MeSH
• Insect Proteins genetics   immunology   metabolism   MeSH
• Cathepsin G immunology   metabolism   MeSH
• Ixodes genetics   immunology   metabolism   MeSH
• Crystallization MeSH
• Protein Structure, Quaternary MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Molecular Sequence Data MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, Protein MeSH
• Serpins genetics   immunology   metabolism   MeSH
• Salivary Proteins and Peptides genetics   immunology   metabolism   MeSH
• Inflammation immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chymases MeSH
• Insect Proteins MeSH
• Cathepsin G MeSH
• serpin-2 MeSH Browser
• Serpins MeSH
• Salivary Proteins and Peptides MeSH