title: Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.
abstract: BACKGROUND
  Ticks and tick-borne diseases affect animal and human health worldwide and cause significant economic losses in the animal industry. Functional molecular research is important to understand the biological characteristics of ticks at the molecular level. Enzymes and enzyme inhibitory molecules play very important roles in tick physiology, and the cystatins are tight-binding inhibitors of papain-like cysteine proteases. To this end, a novel cystatin, designated RHcyst-1, was isolated from the tick Rhipicephalus haemaphysaloides.
  
  
  METHODS
  The full-length gene of RHcyst-1 was cloning by RACE. The recombinant protein of RHcyst-1 was expressed in a glutathione S-transferase (GST)-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. Expression analysis of RHcyst-1 at different tick stages was performed by quantitative reverse transcription - PCR (qRT-PCR). An RNAi experiment for RHcyst-1 was performed to determine its function for tick physiology.
  
  
  RESULTS
  The full-length cDNA of RHcyst-1 is 471 bp, including an intact open reading frame encoding an expected protein of 98 amino acids, without a signal peptide, having a predicted molecular weight of ~11 kDa and an isoelectric point of 5.66. A sequence analysis showed that it has significant homology with the known type 1 cystatins. The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-1 genes' expression profile showed that it was more richly transcribed in the embryo (egg) stage. A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching.
  
  
  CONCLUSIONS
  Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. 453425665@qq.com.|College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. 453425665@qq.com.
authors.forename: Yujian
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. zhyzhi@shvri.ac.cn.
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. gonghaiyan76@msn.com.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Gong
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. caojie@shvri.ac.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Cao
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. zhanghoushuang@msn.com.
authors.forename: Houshuang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. lixiangrui@njau.edu.cn.
authors.forename: Xiangrui
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China. jinlinzhou@shvri.ac.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China. jinlinzhou@shvri.ac.cn.
authors.forename: Jinlin
authors.initials: J
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001096:Arachnid Vectors / Q000235:genetics / Q000378:metabolism*; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D011817:Rabbits; D048494:Rhipicephalus / Q000235:genetics / Q000378:metabolism*
chemical_list: D060829:Arthropod Proteins; D015891:Cystatins; D018076:DNA, Complementary