A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

doi:10.1016/j.ibmb.2006.03.003

Insect Biochemistry and Molecular Biology

Volume 36, Issue 7, July 2006, Pages 527-535

https://doi.org/10.1016/j.ibmb.2006.03.003 Get rights and content

Abstract

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host–parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.

Introduction

Cystatins are tight-binding inhibitors of papain-like cysteine proteases and are widespread in plants and animals. On the basis of amino acid sequencing, this superfamily can be subdivided into three closely related families (Rawlings and Barrett, 1990; Turk and Bode, 1991). Family 1 cystatins are small polypeptides of about 100 amino acid residues and lack carbohydrate side chains and disulphide bridges. The members of family 2 are secretion-type proteins consisting of about 120 amino acid residues and having two disulphide bridges. Family 3 cystatins are kininogens, which are large multifunctional glycoproteins of blood plasma and synovial fluid. They contain three domains that are homologous with those of family 2 cystatins; two of them inhibit proteinases. The physiological function of cystatins is not well understood. However, the regulation of protein turnover and protection of plants against insects and pathogens have been proposed in plants (Turk and Bode, 1991). Mammalian cystatin C had also been suggested to participate in the defense against the invasion of pathogens (Olsson et al., 1999). Filarial cystatins participate in pathogenicity and are thought to play a key role in the balance of the host–parasite immune relationship (Schierack et al., 2003). In the tick, genes encoding cystatins have also been found in several ixodid ticks (Valenzuela et al., 2002; Karim et al., 2005). However, the function of tick cystatins remains unknown.

Ticks are important vectors of a wide variety of disease-causing bacteria, viruses, protozoa, and other pathogenic organisms. Despite the importance of ticks as vectors of disease, very little is known of their basic biology, particularly, immune system. Understanding vector immunity is important in determining the host–pathogen interactions that facilitate or limit disease transmission. The hard tick, Haemaphysalis longicornis, is distributed mainly in East Asia and Australia, where it transmits a wide range of pathogens, including bovine theileriosis (Theileria spp), bovine babesiosis (Babesia ovata), canine babesiosis (Babesia gibsoni) and human rickettsiosis (Rickettsia japonica) (Fujisaki et al., 1994; Jongejan and Uilenberg, 2004). In this study, we report the characterization of a secreted cystatin from tick H. longicornis and provide evidence that cystatin is involved in tick innate immunity.

Section snippets

Ticks and tissue collection

The parthenogenetic Okayama strain of the tick H. longicornis has been maintained by feeding on rabbits and mice for several generations in our laboratory (Fujisaki, 1978). For tissue collection, adult females of H. longicornis were infested on the ears of rabbit, ticks were recovered from the rabbit ears after 4 days, and the tissues were immediately dissected under the microscope (You et al., 2001). The sample materials were stored at −80 °C until used.

Construction of the tick midgut full-length cDNA library by vector capping and cDNA sequencing

A full-length cDNA library was made using

Construction of a full-length cDNA library using total RNA

The vector-capping method was applied to construct cDNA libraries from the total RNA of the midgut of the H. longicornis tick. After the transformation of E. coli cells, transformants were grown on agar plates without amplification in a liquid medium. The libraries were composed of approximately 10⁵ independent colonies. From the library, 10,000 colonies were randomly picked, and the 5′ ends of the cDNA of these clones were sequenced. Readable sequences of 8304 cDNA inserts were obtained, and

Discussion

The present study describes the sequence of a novel cystatin Hlcyst-2 from the tick H. longicornis. The characteristics of the putative amino acid sequence of Hlcyst-2 indicate that it is a member of family 2 cystatins. The family 2 cystatins represented by human cystatin C and chicken egg cystatin are secretory proteins with one cystatin domain and two characteristic disulphide bridges (Rawlings and Barrett, 1990). Various cystatins have been characterized regarding their capacity to inhibit

Acknowledgements

This work was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN), Grants-in-Aid for Scientific Research (A) from the Japan Society for the Promotion of Science, and a grant from the 21st Century COE program (A-1), Ministry of Education, Sports, Science, and Technology of Japan.

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A Coxiella-like endosymbiont (Coxiella-LE hereinafter) stably infects and influences Haemaphysalis longicornis development, indicating a mutualistic relationship of Coxiella-LE and ticks. To further elucidate the patterns of growth dynamics and tissue localization of Coxiella-LE in H. longicornis, 16S rRNA high-throughput sequencing, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) were used in this study. The density of Coxiella-LE varied among different tick life stages, and fed female ticks had the highest density, followed by unfed female and unfed larval ticks. In the four organs that were dissected from fed female ticks, the ovary carried the highest density of Coxiella-LE, which was significantly different from salivary glands, midgut and Malpighian tubules. The high abundance of Coxiella-LE in fed female ticks and in the ovaries of fed female ticks in the bacterial microbiota analyses further confirmed that Coxiella-LE rapidly proliferates in the ovary after blood feeding. The ovaries continued to develop after engorgement and oviposition began on day 5, with a significant decrease in the density of Coxiella-LE in the ovaries occurring on day 7. FISH results indicated that Coxiella-LE is mainly colonized in the cytoplasm of the oocyte and proliferates with oogenesis. Coxiella-LE was expelled from the body with the mature oocyte, ensuring its vertical transmission. In the Malpighian tubules at different days after engorgement, the white flocculent materials were increasing, and the density of Coxiella-LE raised significantly on day 7. Unlike the localization pattern in the ovary, Coxiella-LE was initially distributed in a mass and continually increased during the development of Malpighian tubules until it filled the Malpighian tubules. These findings provide new insights on the growth dynamics and tissue localization of Coxiella-LE in ticks and are useful for further investigation on the interactions of symbiont and ticks .
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A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases
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Cysteine peptidase inhibitors are classified into the cystatin superfamily based on their primary features (Barrett, 1985; Rawlings et al., 2018). In the past years, several type 2 cystatins from ticks were identified and associated with different physiological processes, such as blood feeding (Karim et al., 2005; Yamaji et al., 2009), parasite-host relationship (Kotsyfakis et al., 2006; Salat et al., 2010), blood digestion (Cardoso et al., 2017; Yamaji et al., 2010) and immune response (Lu et al., 2014; Zhou et al., 2006). On the other hand, only a handful of type 1 cystatins have been characterized.
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A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

Abstract

Introduction

Section snippets

Ticks and tissue collection

Construction of the tick midgut full-length cDNA library by vector capping and cDNA sequencing

Construction of a full-length cDNA library using total RNA

Discussion

Acknowledgements

A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning, and tissue localization

J. Biochem.

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site

J. Biol. Chem.

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

Growth inhibitory effect of triclosan on equine and bovine Babesia parasites

Am. J. Trop. Med. Hyg.

Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom

Biochem. J.

Friends and relations of the cystatin superfamily—new members and their evolution

Protein Sci

Nippocystatin, a cysteine protease inhibitor from Nippostrongylus brasiliensis, inhibits antigen processing and modulates antigen-specific immune response

Infect. Immun.

Structure and expression of the gene encoding cystatin D, a novel human cysteine inhibitor interaction

EMBO J

Development of acquired resistance and precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodoidea: Ixodidae)

Natl. Inst. Anim. Health Quart. (Tokyo)

The taxonomy of the bovine Theileria spp

Parasitol. Today

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

J. Vet. Med. Sci.

Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause

Gene

Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly 11) for cysteine endopeptidase inhibition

Biochem. J.

Modulation of host immune responses by nematode cystatins

Int. J. Parasitol.

The global importance of ticks

Parasitology