title: Sequence characterization and immunogenicity of cystatins from the cattle tick Rhipicephalus (Boophilus) microplus.
abstract: Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil.
authors.forename: Luís F
authors.initials: LF
authors.lastname: Parizi
authors.forename: Naftaly W
authors.initials: NW
authors.lastname: Githaka
authors.forename: Carolina
authors.initials: C
authors.lastname: Acevedo
authors.forename: Uruguaysito
authors.initials: U
authors.lastname: Benavides
authors.forename: Adriana
authors.initials: A
authors.lastname: Seixas
authors.forename: Carlos
authors.initials: C
authors.lastname: Logullo
authors.forename: Satoru
authors.initials: S
authors.lastname: Konnai
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ohashi
authors.forename: Aoi
authors.initials: A
authors.lastname: Masuda
authors.forename: Itabajara
authors.initials: I
authors.lastname: da Silva Vaz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001938:Brazil; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology / Q000469:parasitology*; D019295:Computational Biology; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D048494:Rhipicephalus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013984:Tick Infestations / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*
chemical_list: D015891:Cystatins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins
keywords: Cystatin; Inhibitor; Rhipicephalus (Boophilus) microplus; Tick