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 inhibit...
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| Format: | Article |
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BMC
2015-03-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-015-0725-5 |
| _version_ | 1827933804376883200 |
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| author | Yujian Wang Yongzhi Zhou Haiyan Gong Jie Cao Houshuang Zhang Xiangrui Li Jinlin Zhou |
| author_facet | Yujian Wang Yongzhi Zhou Haiyan Gong Jie Cao Houshuang Zhang Xiangrui Li Jinlin Zhou |
| author_sort | Yujian Wang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T07:29:56Z |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-13T07:29:56Z |
| publishDate | 2015-03-01 |
| publisher | BMC |
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| series | Parasites & Vectors |
| spelling | doaj.art-e08e459757004ceab2316b493889962c2023-06-04T11:12:22ZengBMCParasites & Vectors1756-33052015-03-01811710.1186/s13071-015-0725-5Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloidesYujian Wang0Yongzhi Zhou1Haiyan Gong2Jie Cao3Houshuang Zhang4Xiangrui Li5Jinlin Zhou6Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesKey Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesKey Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesKey Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesKey Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesCollege of Veterinary Medicine, Nanjing Agricultural UniversityKey Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural SciencesAbstract 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.https://doi.org/10.1186/s13071-015-0725-5Rhipicephalus haemaphysaloidesCystatinInhibitory activityEmbryonic development |
| spellingShingle | Yujian Wang Yongzhi Zhou Haiyan Gong Jie Cao Houshuang Zhang Xiangrui Li Jinlin Zhou Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides Parasites & Vectors Rhipicephalus haemaphysaloides Cystatin Inhibitory activity Embryonic development |
| title | Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides |
| title_full | Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides |
| title_fullStr | Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides |
| title_full_unstemmed | Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides |
| title_short | Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides |
| title_sort | functional characterization of a cystatin from the tick rhipicephalus haemaphysaloides |
| topic | Rhipicephalus haemaphysaloides Cystatin Inhibitory activity Embryonic development |
| url | https://doi.org/10.1186/s13071-015-0725-5 |
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