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Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.

Wang Y, Zhou Y, Gong H, Cao J, Zhang H, Li X, Zhou J - Parasit Vectors (2015)

Bottom Line: The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases' enzyme activities.A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching.Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.

View Article: PubMed Central - PubMed

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.

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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Related in: MedlinePlus

Expression of a novel type 1 cystatin,RHcyst-1,isolated from the tickRhipicephalushaemaphysaloides,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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Fig5: Expression of a novel type 1 cystatin,RHcyst-1,isolated from the tickRhipicephalushaemaphysaloides,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.

Mentions: Unfed adult ticks were injected with RHcyst-1 or luciferase dsRNA and then allowed to feed on a rabbit. Gene-specific silencing was confirmed by real-time PCR. Within 5 days of dsRNA treatment, 95.6% of the RHcyst-1 transcripts in ticks were silenced as shown in Figure 5. The attachment rate 24 h after putting adult ticks onto the host ears, the engorgement rate, the death rate, and the larval hatching rate were measured to evaluate the RHcyst-1 function in tick physiology. Compared with luciferase-injected ticks, there was a significant effect on the attachment and hatching rates, but no significant effect on engorgement or death rates. Table 2 shows that the attachment and hatching rates in the RHcyst-1 dsRNA group were 48.6% and 69.1%, respectively, whereas those in the control group were 63.8% and 80.4%, respectively.Figure 5


Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.

Wang Y, Zhou Y, Gong H, Cao J, Zhang H, Li X, Zhou J - Parasit Vectors (2015)

Expression of a novel type 1 cystatin,RHcyst-1,isolated from the tickRhipicephalushaemaphysaloides,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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352250&req=5

Fig5: Expression of a novel type 1 cystatin,RHcyst-1,isolated from the tickRhipicephalushaemaphysaloides,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.
Mentions: Unfed adult ticks were injected with RHcyst-1 or luciferase dsRNA and then allowed to feed on a rabbit. Gene-specific silencing was confirmed by real-time PCR. Within 5 days of dsRNA treatment, 95.6% of the RHcyst-1 transcripts in ticks were silenced as shown in Figure 5. The attachment rate 24 h after putting adult ticks onto the host ears, the engorgement rate, the death rate, and the larval hatching rate were measured to evaluate the RHcyst-1 function in tick physiology. Compared with luciferase-injected ticks, there was a significant effect on the attachment and hatching rates, but no significant effect on engorgement or death rates. Table 2 shows that the attachment and hatching rates in the RHcyst-1 dsRNA group were 48.6% and 69.1%, respectively, whereas those in the control group were 63.8% and 80.4%, respectively.Figure 5

Bottom Line: The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases' enzyme activities.A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching.Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus