Mini reviewThe role of cystatins in tick physiology and blood feeding☆
Introduction
Cystatins comprise a large superfamily of reversible and tight-binding inhibitors that interact with papain-like cysteine proteases and legumains (Abrahamson et al., 2003). Cystatins are present in a wide range of organisms such as vertebrates, invertebrates, and plants as well as protozoa (Vray et al., 2002, Turk et al., 2008). They are involved in various vertebrate biological processes, e.g. in antigen presentation, immune system development, epidermal homeostasis, neutrophil chemotaxis during inflammation, or apoptosis (Reddy et al., 1995, Honey and Rudensky, 2003, Wille et al., 2004, Lombardi et al., 2005). As regulators of proteolysis, they are associated with cell/tissue homeostasis as well as proteolysis-related pathological conditions (Zavasnik-Bergant, 2008).
Cystatins are classified, according to certain sequence motifs and the number of conserved cystatin domains, into four subfamilies: the type 1 cystatins (also known as stefins), the type 2 cystatins, the type 3 cystatins (kininogens), and the type 4 cystatin-like proteins (fetuins, histidine-rich proteins) (Rawlings and Barrett, 1990). A recent classification was proposed that assigns each peptidase, or peptidase inhibitor family, with a unique identification number in the MEROPS classification system that is widely accepted in the proteolytic society (Rawlings et al., 2010). According to the MEROPS database, cystatins belong to the I25 family, and this family is further divided into the subfamilies I25A (stefins), I25B (type 2 cystatins and kinogens), and I25C (fetuins, histidine-rich proteins). In this review, we will refer to the different cystatins using the classical categorization and not the MEROPS code.
The first type 1 cystatin (stefin) was discovered in the cytosol of human polymorphonuclear granulocytes (Brzin et al., 1983) and named cystatin A (NCBI Accession No. P01040, Fig. 1). Shortly afterwards, the full protein sequence of cystatin B (NCBI: P04080) was determined, and this stefin was detected in the human liver (Ritonja et al., 1985) (Fig. 1). Both cystatins are potent and tight-binding inhibitors of papain, cathepsins L, S, and H, and they also inhibit cathepsin B, but with lower affinity (Turk et al., 2008). Stefins are cystatins of low molecular weight (about 11 kDa), and they possess a single cystatin domain. This domain consists of the conserved N-terminal glycine and two β-hairpin loops 1 and 2, with loop 1 possessing the conserved QXVXG motif (Fig. 1). These three cystatin regions form a wedge-like structure that interacts with the catalytic cleft of cysteine proteases (Grzonka et al., 2001). Stefins are mainly intracellular proteins without a signal peptide, lacking any carbohydrate side chains as well as disulfide bridges found in other cystatins (Abrahamson et al., 2003). Homologs of human cystatin A and cystatin B were discovered in different vertebrates such as mouse, rat, cat, pig, and cow and in invertebrates such as leeches where they regulate proteolysis (Lefebvre et al., 2004, Rawlings et al., 2006). Cystatins originating from parasites such as nematodes received immediate attention as candidate determinants of host–parasite interactions because of their potential interaction with cysteine proteases that play a role in immunity-mediated homeostatic mechanisms. Fg-stefin 1 from Fasciola gigantica (NCBI: ACS35603) inhibited cathepsin B, L, and S activities and was suggested to protect the intestine and tegumental surface of the parasite against extracellular proteolysis (Tarasuk et al., 2009). Sm-cystatin from Schistosoma mansoni (NCBI: AAQ16180) successfully inhibited papain (Morales et al., 2004) and might play a role in the digestion of hemoglobin since it is localized in the parasite's gut (Wasilewski et al., 1996).
The prototypical type 2 cystatin was isolated in 1968 from chicken egg white (Fossum and Whitaker, 1968). This cysteine protease inhibitor interacted with papain, ficin, and cathepsins B and C (Sen and Whitaker, 1973, Keilová and Tomásek, 1975). The name ‘cystatin’ was given to this protein to indicate its function as a cysteine protease inhibitor (Barrett, 1981). The differences in the protein sequence of chicken cystatin and human stefin A accounted for significant differences in their resolved structures. Like stefins, type 2 cystatins possess a single cystatin domain that bears the conserved N-terminal glycine and the QXVXG segment of loop 1 (both sequence motifs are shown in the alignment of Fig. 1, and the cystatin domain is seen in Fig. 2A). Unlike stefins, type 2 cystatins also possess a conserved PW dipeptide in the β-hairpin loop 2 as shown in the case of human cystatin C (NCBI: P01034). They are secreted proteins of 13–15 kDa molecular weight with a signal peptide and two intracellular disulfide bonds (Turk et al., 2008, Zavasnik-Bergant, 2008) (Fig. 1). Type 2 cystatins are generally non-glycosylated proteins (Dickinson, 2002); however, there are exceptions, for example, human cystatins F and E/M are glycosylated (Sotiropoulou et al., 1997, Halfon et al., 1998). Besides inhibiting C1 proteases, human cystatin C is also able to inhibit legumain/asparaginyl endopeptidases with a second conserved SND inhibitory domain (Fig. 1) located between the conserved N-terminal glycine and loop 1 (Zavasnik-Bergant, 2008).
In parasites, type 2 cystatins are widely studied rather than stefins. For example, in nematodes, onchocystatin of Onchocerca volvulus was discovered in the cuticle of some larval developmental stages, the adult stages and the eggshell of microfilariae (Lustigman et al., 1991, Lustigman et al., 1992). Onchocystatin inhibited human cathepsins L and S, suggesting that this cystatin modulates the moulting of the larval stages while it is also involved in developing microfilariae in the uterus. Furthermore, this cystatin also showed strong immunomodulatory properties; it affected the human peripheral blood mononuclear T cell proliferation, the induction of TNF-α, nitric oxide, and Il-10, and it was responsible for the downregulating activities of the MHC II complex and CD86 molecules (Schönemeyer et al., 2001, Hartmann and Lucius, 2003, Schierack et al., 2003).
Type 3 cystatins are also known as kininogens; they are large (60–120 kDa), secreted multidomain proteins of three repeated type 2-like cystatin domains, where only the last two domains account for the inhibitory activity of the protein (Ohkubo et al., 1984, Müller-Esterl et al., 1985). These cystatins are found in extracellular fluids, they are usually glycosylated, and they possess eight disulfide bridges. Kinogens are involved, for example, in the protection against leaking lysosomal cysteine proteases or proteases derived from invading microorganisms; they also coordinate adaptive immunity (Scharfstein et al., 2007). Fetuins and histidine-rich glycoproteins from the type 4 cystatin subfamily are also secreted proteins, but they lack the cystatin-inhibitory properties; thus they are only cystatin-like proteins possessing two tandem cystatin domains (Brown and Dziegielewska, 1997).
Ticks, as obligate hematophagous ectoparasites, greatly impact animal and human health because they transmit various pathogens worldwide. Family 1 and family 2 cystatins have been reported in various species of soft and hard ticks. Tick control measures are difficult (e.g. acaricide applications), but new strategies such as developing anti-tick vaccines are being implemented (de la Fuente et al., 2007). These vaccines are based on tick antigens that have immunomodulatory properties and interact with the vertebrate immune system. In this review, we present all currently described cystatins from hard and soft ticks, and we review their role in tick physiology. We describe their inhibitory activities on various cysteine proteases and their host immunomodulatory function during tick feeding. Particular emphasis is given to any demonstrated potential of tick cystatins as candidate tick antigens towards anti-tick vaccine development, and to their potential for developing novel pharmacological applications.
Section snippets
Type 1 cystatins in ticks
Tick cystatins are divergent from all the other described vertebrate, invertebrate, and plant cystatins. For example, in comparison to the prototypical human cystatins A, B and C, tick cystatins of type 1 and 2 only show up to 37% and 25% amino acid identity, respectively (Fig. 1). Currently, all known type 1 tick cystatins were discovered from five different hard tick species from four genera. All these stefins were described from transcriptome sequencing projects, and they are mostly found in
Type 2 tick cystatins
Most likely due to their secretory nature, type 2 cystatins have been intensively studied in ticks compared to stefins. In recent years, several studies in vector control research have focused on the pharmacological and immunomodulatory properties of saliva in order to understand arthropod blood feeding as well as pathogen transmission. Within this context, it is not surprising that most of the cystatins studied in ticks are secreted type 2 cystatins. A total of 14 type 2 cystatins and two
Conclusions
Worldwide, ticks are important vectors of infectious diseases impacting on human and animal health. Ticks are not only vectors of parasites of livestock that account for diseases such as babesiosis, theileriosis, or anaplasmosis, but they also mediate the transmission of human tick-borne encephalitis and Lyme disease (de Castro, 1997, Alciati et al., 2001). Over the last decades, increases in the tick abundance, such as of I. ricinus, the castor bean tick in central Europe, and its infection
Acknowledgements
The authors would like to acknowledge the financial support of the Academy of Sciences of the Czech Republic (Grant No. Z60220518). A.S. was funded by the Alexander von Humboldt Foundation (Feodor Lynen Research Fellowship). M.K. received support from the Academy of Sciences of the Czech Republic (Jan Evangelista Purkyne Fellowship), from the Grant Agency of the Czech Republic (Grant No. P502/12/2409), from the 7th Framework Programme of the European Union (Marie Curie Reintegration grant,
References (86)
- et al.
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) - et al.
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) Cystatin, the egg white inhibitor of cysteine proteinases
Methods Enzymol.
(1981)- et al.
Protein oxidation in aging, disease, and oxidative stress
J. Biol. Chem.
(1997) Endosomal proteases in antigen presentation
Curr. Opin. Immunol.
(2006)- et al.
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) Sustainable tick and tickborne disease control in livestock improvement in developing countries
Vet. Parasitol.
(1997)- et al.
Ficin and papain inhibitor from chicken egg white
Arch. Biochem. Biophys.
(1968) - et al.
An insight into the salivary transcriptome and proteome of the soft tick and vector of epizootic bovine abortion, Ornithodoros coriaceus
J. Proteomics
(2008) - et al.
An insight into the sialome of the soft tick, Ornithodoros parkeri
Insect Biochem. Mol. Biol.
(2008)
Modelling protein docking using shape complementarity, electrostatics and biochemical information
J. Mol. Biol.
Leukocystatin, a new class II cystatin expressed selectively by hematopoietic cells
J. Biol. Chem.
Modulation of host immune responses by nematode cystatins
Int. J. Parasitol.
RNAi-mediated gene silencing to assess the role of synaptobrevin and cystatin in tick blood feeding
Biochem. Biophys. Res. Commun.
Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis
J. Biol. Chem.
Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis
J. Biol. Chem.
Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus
Biochem. Biophys. Res. Commun.
Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms
Mol. Biochem. Parasitol.
Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus
J. Biol. Chem.
The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo
Mol. Biochem. Parasitol.
Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data
FEBS Lett.
Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells
Cell
An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks
Insect Biochem. Mol. Biol.
Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver
Biochem. Biophys. Res. Commun.
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
Some properties of a ficin-papain inhibitor from avian egg white
Arch. Biochem. Biophys.
IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus
Int. J. Parasitol.
Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer
J. Biol. Chem.
Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product
Mol. Biochem. Parasitol.
Ectoparasites: future challenges in a changing world
Vet. Parasitol.
Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo
Mol. Biochem. Parasitol.
Tick control: thoughts on a research agenda
Vet. Parasitol.
Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis
Parasitol. Int.
A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity
Insect Biochem. Mol. Biol.
Characterization of an intracellular cystatin homolog from the tick Haemaphysalis longicornis
Vet. Parasitol.
Cystatins Biochem. Soc. Symp.
Dipeptidyl peptidase activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis
J. Clin. Invest.
Human infections tick-transmitted
Panminerva Med.
Characterization of asparaginyl endopeptidase, legumain, induced by blood feeding in the ixodid tick Haemaphysalis longicornis
Insect Biochem. Mol. Biol.
An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus
BMC Genomics
Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae)
BMC Genomics
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
Friends and relations of the cystatin superfamily – new members and their evolution
Protein Sci.
Cited by (66)
Tick salivary protein Cystatin: structure, anti-inflammation and molecular mechanism
2024, Ticks and Tick-borne DiseasesSymbiont-regulated serotonin biosynthesis modulates tick feeding activity
2021, Cell Host and MicrobeCitation Excerpt :Saliva contains hundreds of bioactive molecules with vasodilator, anticoagulant, and immunosuppressive activity that ensure their unnoticed and long-term attachment to the host skin and facilitate blood flow (Šimo et al., 2017). Midgut, the primary tissue for blood storage and digestion, contains various molecules that are involved in antihemostatics, proteolysis, detoxification, and nutrient assimilation (Alim et al., 2009; Feng and Cheng, 2020; Galay et al., 2015; Schwarz et al., 2012). In addition, temperature, host species, tick density, and age also affect their feeding activity (Jones et al., 2015; Pollock et al., 2015).
A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases
2020, Ticks and Tick-borne DiseasesRhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus
2020, Ticks and Tick-borne DiseasesCitation Excerpt :Cystatins are among the most well studied protein family of peptidase inhibitors in ticks (Parizi et al., 2018). These proteins are involved in tick endogenous processes, parasite-host interaction, and pathogen survival/transmission (Schwarz et al., 2012). The broad physiologic roles of cystatins make these inhibitors potential antigens for inclusion in new generation anti-tick vaccines.
Immunosuppressive effects of sialostatin L1 and L2 isolated from the taiga tick Ixodes persulcatus Schulze
2020, Ticks and Tick-borne Diseases
- ☆
This paper was presented at the Ticks and Tick-Borne Pathogens Conference 7 (TTP7), held in Zaragoza (Spain), August 28th to September 2nd, 2011, and selected for submission to TTBD by the Scientific and Organizing Committees.