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. 2015 Mar 3:8:140.
doi: 10.1186/s13071-015-0725-5.

Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides

Yujian Wang  1   2 , Yongzhi Zhou  3 , Haiyan Gong  4 , Jie Cao  5 , Houshuang Zhang  6 , Xiangrui Li  7 , Jinlin Zhou  8   9
Affiliations

Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides

Yujian Wang et al. Parasit Vectors. .

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.

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Figures

Figure 1
Figure 1
Analysis of the structure of a novel type 1 cystatin, RHcyst-1, isolated from the tick Rhipicephalus haemaphysaloides . (A) cDNA and putative amino acid sequences. (B) Putative amino acid alignment of RHcyst-1 with other tick type 1 cystatins: Bmcystatin of Boophilus microplus (GenBank accession number: ABG36931); DvM602 of Dermacentor variabilis (GenBank accession number: ACF35512); and Hlcyst-1 of Haemaphysalis longicornis (GenBank accession number: ABZ89553). The conserved cystatin active sites are boxed (1: N-terminal conserved glycine; 2: QXVXG conserved motif).
Figure 2
Figure 2
Expression and purification of the recombinant protein of RHcyst- 1, a novel type 1 cystatin isolated from the tick Rhipicephalus haemaphysaloides, in a GST- fused soluble form, rRHcyst- 1- GST, in Escherichia coli . A total of ~10 μg recombinant protein or bacterial lysate was electrophoresed in a 12% SDS-polyacrylamide gel, and then stained with Coomassie blue. Lane 1: The supernatant of uninduced cell lysate; lane 2: The supernatant of induced cell lysate; and lanes 3 and 4: purified RHcyst-1 recombinant protein.
Figure 3
Figure 3
Inhibition of protease activities by the recombinant protein of RHcyst- 1, a novel type 1 cystatin isolated from the tick Rhipicephalus haemaphysaloides . Cathepsin L, B, C, H, and S and papain were incubated with each of the substrates in the presence of different concentrations of rRHcyst-1. Incubation of cathepsins without rRHcyst-1 resulted in 100% enzyme activity.
Figure 4
Figure 4
Gene expression patterns of a novel type 1 cystatin, RHcyst- 1, isolated from the tick Rhipicephalus haemaphysaloides in different developmental stages. The relative expression (%) refers to adult stage RHcyst-1 expression (1 unit). Each column represents the mean and standard deviation (error bars) of three analyses.
Figure 5
Figure 5
Expression of a novel type 1 cystatin, RHcyst- 1, isolated from the tick Rhipicephalus haemaphysaloides, dsRNA or luciferase dsRNA treatments in female ticks. The relative expression (%) refers to the RHcyst-1 expression (1 unit) in RHcyst-1 dsRNA treated female ticks. Each column represents the mean and standard deviation (error bars) of three analyses.
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