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.

Laboratory of Genomics and Proteomics of Disease Vectors Institute of Parasitology Biology Centre AS CR v v i Ceske Budejovice Czech Republic

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Abrahamson M, Alvarez-Fernandez M, Nathanson CM. Cystatins Biochem. Soc Symp. 2003;70:179–199. PubMed

Adkison AM, Raptis SZ, Kelley DG, Pham CT. Dipeptidyl peptidase activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest. 2002;109:363–371. PubMed PMC

Alciati S, Belligni E, Del Colle S, Pugliese A. Human infections tick-transmitted. Panminerva Med. 2001;43:295–304. PubMed

Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Motobu M, Fujisaki K. Characterization of asparaginyl endopeptidase, legumain, induced by blood feeding in the ixodid tick Haemaphysalis longicornis. Insect Biochem Mol Biol. 2007;37:911–922. PubMed

Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Hatta T, Fujisaki K. HlLgm2, a member of asparaginyl endopeptidases/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion. J Insect Physiol. 2008;54:573–585. PubMed

Alim MA, Tsuji N, Miyoshi T, Islam MK, Hatta T, Fujisaki K. Legumains from the hard tick Haemaphysalis longicornis play modulatory roles in blood feeding and gut cellular remodelling and impact on embryogenesis. Int J Parasitol. 2009;39:97–107. PubMed

Anatriello E, Ribeiro JMC, de Miranda-Santos IKF, Brandão LG, Anderson JM, Valenzuela JG, Maruyama SR, Silva JS, Ferreira BR. An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus. BMC Genomics. 2010;11:450. PubMed PMC

Anderson JM, Sonenshine DE, Valenzuela JG. Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae) BMC Genomics. 2008;9:552. PubMed PMC

Barrett AJ. Cystatin, the egg white inhibitor of cysteine proteinases. Methods Enzymol. 1981;80:771–778.

Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem. 1997;272:20313–20316. PubMed

Björk I, Brieditis I, Raub-Segall E, Pol E, Håkansson K, Abrahamson M. The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases. Biochemistry. 1996;35:10720–10726. PubMed

Brown WM, Dziegielewska KM. Friends and relations of the cystatin superfamily –new members and their evolution. Protein Sci. 1997;6:5–12. PubMed PMC

Brzin J, Kopitar M, Turk V, Machleidt W. Protein inhibitors of cysteine proteinases. I Isolation and characterization of stefin, a cytosolic protein inhibitor of cysteine proteinases from human polymorphonuclear granulocytes. Hoppe Seylers Z Physiol Chem. 1983;364:1475–1480. PubMed

Chapman HA. Endosomal proteases in antigen presentation. Curr Opin Immunol. 2006;18:78–84. PubMed

Daniel M, Danielová V, Kříž B, Kott I. An attempt to elucidate the increased incidence of tick-borne encephalitis and its spread to higher altitudes in the Czech Republic. Int J Med Microbiol. 2004;293(Suppl 37):55–62. PubMed

de Castro JJ. Sustainable tick and tickborne disease control in livestock improvement in developing countries. Vet Parasitol. 1997;71:77–97. PubMed

de la Fuente J, Kocan KM. Strategies for development of vaccines for control of ixodid tick species. Parasite Immunol. 2006;28:275–283. PubMed

de la Fuente J, Kocan KM, Blouin EF. Tick vaccines and the transmission of tick-borne pathogens. Vet Res Commun. 2007;31(Suppl 1):85–90. PubMed

Dickinson DP. Salivary (SD-type) cystatins: over one billion years in the making – but to what purpose? Crit Rev Oral Biol Med. 2002;13:485–508. PubMed

Fossum K, Whitaker JR. Ficin and papain inhibitor from chicken egg white. Arch Biochem Biophys. 1968;125:367–375. PubMed

Francischetti IM, Meng Z, Mans BJ, Gudderra N, Hall M, Veenstra TD, Pham VM, Kotsyfakis M, Ribeiro JM. An insight into the salivary transcriptome and proteome of the soft tick and vector of epizootic bovine abortion, Ornithodoros coriaceus. J Proteomics. 2008a;71:493–512. PubMed PMC

Francischetti IM, Mans BJ, Meng Z, Gudderra N, Veenstra TD, Pham VM, Ribeiro JM. An insight into the sialome of the soft tick, Ornithodoros parkeri. Insect Biochem Mol Biol. 2008b;38:1–21. PubMed PMC

Gabb HA, Jackson RM, Sternberg MJE. Modelling protein docking using shape complementarity, electrostatics and biochemical information. J Mol Biol. 1997;272:106–120. PubMed

Grunclová L, Horn M, Vancová M, Sojka D, Franta Z, Mares M, Kopácek P. Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity. Biol Chem. 2006;387:1635–1644. PubMed

Grzonka Z, Jankowska E, Kasprzykowski F, Kasprzykowska R, Lankiewicz L, Wiczk W, Wieczerzak E, Ciarkowski J, Drabik P, Janowski R, Kozak M, Jaskólski M, Grubb A. Structural studies of cysteine proteases and their inhibitors. Acta Biochim Pol. 2001;48:1–20. PubMed

Halfon S, Ford J, Foster J, Dowling L, Lucian L, Sterling M, Xu Y, Weiss M, Ikeda M, Liggett D, Helm A, Caux C, Lebecques S, Hannum C, Menon S, Mc Clanahan T, Gorman D, Zurawski G. Leukocystatin, a new class II cystatin expressed selectively by hematopoietic cells. J Biol Chem. 1998;273:16400–16408. PubMed

Hartmann S, Lucius R. Modulation of host immune responses by nematode cystatins. Int J Parasitol. 2003;33:1291–1302. PubMed

Honey K, Rudensky AY. Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol. 2003;3:472–482. PubMed

Jacot MS. PhD thesis. Institute of Zoology, Faculty of Sciences, University of Neuchâtel; Neuchâtel, Switzerland: 2003. Etude des gènes exprimés au cours du repas sanguin dans l’intestin de la tique Ixodes ricinus; p. 104.

Kadokura H, Katzen F, Beckwith J. Protein disulfide bond formation in prokaryotes. Annu Rev Biochem. 2003;72:111–135. PubMed

Karim S, Miller NJ, Valenzuela JG, Sauer JR, Mather TN. RNAi-mediated gene silencing to assess the role of synaptobrevin and cystatin in tick blood feeding. Biochem Biophys Res Commun. 2005;334:1336–1342. PubMed

Keilová H, Tomásek V. Inhibition of cathepsin C by papain inhibitor from chicken egg white and by complex with cathepsin B1. Coll Czech Chem Commun. 1975;40:218–224.

Kotsyfakis M, Sá-Nunes A, Francischetti IM, Mather TN, Andersen JF, Ribeiro JM. Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis. J Biol Chem. 2006;281:26298–26307. PubMed

Kotsyfakis M, Karim S, Andersen JF, Mather TN, Ribeiro JM. Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis. J Biol Chem. 2007;282:29256–29263. PubMed

Kotsyfakis M, Anderson JM, Andersen JF, Calvo E, Francischetti IM, Mather TN, Valenzuela JG, Ribeiro JM. Cutting edge: Immunity against a “silent” salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed. J Immunol. 2008;181:5209–5212. PubMed PMC

Kotsyfakis M, Horka H, Salat J, Andersen JF. The crystal structures of two salivary cystatins from the tick Ixodes scapularis and the effect of these inhibitors on the establishment of Borrelia burgdorferi infection in a murine model. Mol Microbiol. 2010;77:456–470. PubMed PMC

Kozakov D, Hall DR, Beglov D, Brenke R, Comeau SR, Shen Y, Li K, Zheng J, Vakili P, Paschalidis IC, Vajda S. Achieving reliability and high accuracy in automated protein docking: Cluspro, PIPER, SDU, and stability analysis in CAPRI rounds 13–19. Proteins: Struct Funct Bioinf. 2010;78:3124–3130. PubMed PMC

Lefebvre C, Cocquerelle C, Vandenbulcke F, Hot D, Huot L, Lemoine Y, Salzet M. Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity. Biochem J. 2004;380:617–625. PubMed PMC

Lenarčič B, Ritonja A, Sali A, Kotnik M, Turk V, Machleidt W. In: Cysteine Proteinases and their Inhibitors. Turk V, editor. Walter de Gruyter; Berlin: 1986. pp. 473–487.

Lima CA, Sasaki SD, Tanaka AS. Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus. Biochem Biophys Res Commun. 2006;347:44–50. PubMed

Lombardi G, Burzyn D, Mundiñano J, Berguer P, Bekinschtein P, Costa H, Castillo LF, Goldman A, Meiss R, Piazzon I, Nepomnaschy I. Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number. J Immunol. 2005;174:7022–7032. PubMed

Lustigman S, Brotman B, Huima T, Prince AM. Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms. Mol Biochem Parasitol. 1991;45:65–75. PubMed

Lustigman S, Brotman B, Huima T, Prince AM, McKerrow JH. Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus. J Biol Chem. 1992;267:17339–17346. PubMed

Machleidt W, Borchart U, Fritz H, Brzin J, Ritonja A, Turk V. Protein inhibitors of cysteine proteinases. II Primary structure of stefin, a cytosolic protein inhibitor of cysteine proteinases from human polymorphonuclear granulocytes. Hoppe Seylers Z Physiol Chem. 1983;364:1481–1486. PubMed

Morales FC, Furtado DR, Rumjanek FD. The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo. Mol Biochem Parasitol. 2004;134:65–73. PubMed

Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, Popovic T, Turk V, Towatari T, Katunuma N, Bode W. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991;10:2321–2330. PubMed PMC

Müller-Esterl W, Fritz H, Machleidt W, Ritonja A, Brzin J, Kotnik M, Turk V, Kellermann J, Lottspeich F. Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data. FEBS Lett. 1985;182:310–314. PubMed

Ohkubo I, Kurachi K, Takasawa T, Shiokawa H, Sasaki M. Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry. 1984;23:5691–5697. PubMed

Pham CT, Ley TJ. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc Natl Acad Sci USA. 1999;96:8627–8632. PubMed PMC

Pierre P, Mellman I. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell. 1998;93:1135–1145. PubMed

Rawlings ND, Barrett AJ. Evolution of proteins of the cystatin superfamily. J Mol Evol. 1990;30:60–71. PubMed

Rawlings ND, Morton FR, Barrett AJ. MEROPS: the peptidase database. Nucleic Acids Res. 2006;34:D270–272. PubMed PMC

Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database. Nucleic Acids Res. 2010;38:D227–233. PubMed PMC

Reddy VY, Zhang QY, Weiss SJ. Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages. Proc Natl Acad Sci USA. 1995;92:3849–3853. PubMed PMC

Ribeiro JM, Alarcon-Chaidez F, Francischetti IM, Mans BJ, Mather TN, Valenzuela JG, Wikel SK. An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks. Insect Biochem Mol Biol. 2006;36:111–129. PubMed

Ribeiro JM, Anderson JM, Manoukis NC, Meng Z, Francischetti IM. A further insight into the sialome of the tropical bont tick, Amblyomma variegatum. BMC Genomics. 2011;12:136. PubMed PMC

Ritonja A, Machleidt W, Barrett AJ. Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver. Biochem Biophys Res Commun. 1985;131:1187–1192. PubMed

Sá-Nunes A, Bafica A, Antonelli LR, Choi EY, Francischetti IM, Andersen JF, Shi GP, Chavakis T, Ribeiro JM, Kotsyfakis M. The immunomodulatory action of sialostatin L on dendritic cells reveals its potential to interfere with autoimmunity. J Immunol. 2009;182:7422–7429. PubMed PMC

Salát J, Paesen GC, Rezácová P, Kotsyfakis M, Kovárová Z, Sanda M, Majtán J, Grunclová L, Horká H, Andersen JF, Brynda J, Horn M, Nunn MA, Kopácek P, Kopecký J, Mares M. Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata. Biochem J. 2010;429:103–112. PubMed PMC

Scharfstein J, Schmitz V, Svensjö E, Granato A, Monteiro AC. Kininogens coordinate adaptive immunity through the proteolytic release of bradykinin, an endogenous danger signal driving dendritic cell maturation. Scand J Immunol. 2007;66:128–136. PubMed

Schönemeyer A, Lucius R, Sonnenburg B, Brattig N, Sabat R, Schilling K, Bradley J, Hartmann S. Modulation of human T cell responses and macrophage functions by onchocystatin, a secreted protein of the filarial nematode Onchocerca volvulus. J Immunol. 2001;167:3207–3215. PubMed

Schierack P, Lucius R, Sonnenburg B, Schilling K, Hartmann S. Parasite-specific immunomodulatory functions of filarial cystatin. Infect Immun. 2003;71:2422–2429. PubMed PMC

Schwarz A, Maier WA, Kistemann T, Kampen H. Analysis of the distribution of the tick Ixodes ricinus L. (Acari: Ixodidae) in a nature reserve of western Germany using Geographic Information Systems. Int J Hyg Environ Health. 2009;212:87–96. PubMed

Sen LC, Whitaker JR. Some properties of a ficin-papain inhibitor from avian egg white. Arch Biochem Biophys. 1973;158:623–632. PubMed

Sojka D, Hajdusek O, Dvorák J, Sajid M, Franta Z, Schneider EL, Craik CS, Vancová M, Buresová V, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopácek P. IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol. 2007;37:713–724. PubMed PMC

Sotiropoulou G, Anisonowicz A, Sager R. Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem. 1997;272:903–910. PubMed

Stubbs MT, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 1990;9:1939–1947. PubMed PMC

Tarasuk M, Vichasri Grams S, Viyanant V, Grams R. Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product. Mol Biochem Parasitol. 2009;167:60–71. PubMed

Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. PubMed PMC

Tsuji N, Miyoshi T, Battsetseg B, Matsuo T, Xuan X, Fujisaki K. A cysteine protease is critical for Babesia spp. transmission in Haemaphysalis ticks. PLoS Pathog. 2008;4:e1000062. PubMed PMC

Turk V, Stoka V, Turk D. Cystatins: Biochemical and structural properties, and medical relevance. Front Biosci. 2008;13:5406–5420. PubMed

Valenzuela JG, Francischetti IM, Pham VM, Garfield MK, Mather TN, Ribeiro JMC. Exploring the sialome of the tick Ixodes scapularis. J Exp Biol. 2002;205:2843–2864. PubMed

Vray B, Hartmann S, Hoebeke J. Immunomodulatory properties of cystatins. Cell Mol Life Sci. 2002;59:1503–1512. PubMed PMC

Wall R. Ectoparasites: future challenges in a changing world. Vet Parasitol. 2007;148:62–74. PubMed

Wasilewski MM, Lim KC, Phillips J, McKerrow JH. Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo. Mol Biochem Parasitol. 1996;81:179–189. PubMed

Willadsen P, Riding GA, McKenna RV, Kemp DH, Tellam RL, Nielsen JN, Lahnstein J, Cobon GS, Gough JM. Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus. J Immunol. 1989;143:1346–1351. PubMed

Willadsen P. Tick control: thoughts on a research agenda. Vet Parasitol. 2006;138:161–168. PubMed

Wille A, Gerber A, Heimburg A, Reisenauer A, Peters C, Saftig P, Reinheckel T, Welte T, Bühling F. Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells. Biol Chem. 2004;385:665–670. PubMed

Yamaji K, Tsuji N, Miyoshi T, Islam MK, Hatta T, Alim MA, Anisuzzaman M, Takenaka A, Fujisaki K. Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis. Parasitol Int. 2009a;58:232–237. PubMed

Yamaji K, Tsuji N, Miyoshi T, Islam MK, Hatta T, Alim MA, Anisuzzaman M, Kushibiki S, Fujisaki K. A salivary cystatin, HlSC-1, from the ixodid tick Haemaphysalis longicornis play roles in the blood-feeding processes. Parasitol Res. 2009b;106:61–68. PubMed

Yamaji K, Tsuji N, Miyoshi T, Hatta T, Alim MA, Anisuzzaman M, Kushibiki S, Fujisaki K. Hlcyst-1 and Hlcyst-2 are potential inhibitors of HlCPL-A in the midgut of the ixodid tick Haemaphysalis longicornis. J Vet Med Sci. 2010;72:599–604. PubMed

Zavasnik-Bergant T, Turk B. Cysteine cathepsins in the immune response. Tissue Antigens. 2006;67:349–355. PubMed

Zavasnik-Bergant T. Cystatin protease inhibitors and immune functions. Front Biosci. 2008;13:4625–4637. PubMed

Zhou J, Ueda M, Umemiya R, Battsetseg B, Boldbaatar D, Xuan X, Fujisaki K. A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. Insect Biochem Mol Biol. 2006;36:527–535. PubMed

Zhou J, Liao M, Ueda M, Gong H, Xuan X, Fujisaki K. Characterization of an intracellular cystatin homolog from the tick Haemaphysalis longicornis. Vet Parasitol. 2009;160:180–183. PubMed

Zhou J, Liao M, Gong H, Xuan X, Fujisaki K. Characterization of Hlcyst-3 as a member of cystatins from the tick Haemaphysalis longicornis. Exp Appl Acarol. 2010;51:327–333. PubMed

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