Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides

Particle and Fibre Toxicology

Jinlin Zhou²  Xiangrui Li³  Houshuang Zhang¹  Jie Cao¹  Haiyan Gong¹  Yongzhi Zhou¹  Yujian Wang³ 

[1] Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

关键词: Embryonic development;    Inhibitory activity;    Cystatin;    Rhipicephalus haemaphysaloides;   

Others  :  1146799
DOI  :  10.1186/s13071-015-0725-5

 received in 2014-11-17, accepted in 2015-02-09,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Wang et al.; licensee BioMed Central.

【 图 表 】

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