Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides

• Published in: Experimental & applied acarology Vol. 67; no. 2; pp. 289 - 298
• Main Authors: Wang, Yujian, Yu, Xinmao, Cao, Jie, Zhou, Yongzhi, Gong, Haiyan, Zhang, Houshuang, Li, Xiangrui, Zhou, Jinlin
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.10.2015; Springer Nature B.V
• Subjects: acarology; adults; Amino Acid Sequence; Amino acids; Animal Ecology; Animal Genetics and Genomics; Animal Systematics/Taxonomy/Biogeography; Animals; Arthropod Proteins - chemistry; Arthropod Proteins - genetics; Arthropod Proteins - metabolism; Base Sequence; Biomedical and Life Sciences; cathepsin L; complementary DNA; cystatins; Cystatins - chemistry; Cystatins - genetics; Cystatins - metabolism; DNA, Complementary - chemistry; DNA, Complementary - genetics; DNA, Complementary - metabolism; E coli; eggs; Entomology; Enzymatic activity; enzyme activity; Escherichia coli; genes; glutathione; isoelectric point; Ixodidae; Larva - genetics; Larva - metabolism; Life Sciences; midgut; Molecular Sequence Data; molecular weight; Nymph - genetics; Nymph - metabolism; open reading frames; Ovum - metabolism; papain; recombinant proteins; reverse transcriptase polymerase chain reaction; Rhipicephalus; Rhipicephalus - genetics; Rhipicephalus - growth & development; Rhipicephalus - metabolism; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; saliva; Sequence Alignment; sequence analysis; signal peptide; ticks; Tissue Distribution; Western blotting; Cystatin; Inhibitory activity
• ISSN: 0168-8162; 1572-9702
• DOI: 10.1007/s10493-015-9946-8

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773 bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13 kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-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. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases’ enzyme activities. An investigation of the RHcyst-2 genes’ expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.