Study of cathepsins involved in haemoglobin and vitellin digestion in Rhipicephalus (Boophilus) annulatus larvae by one- and two-dimensional zymography

Authors

1 Rastegar Reference Laboratory, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran

2 Department of Parasitology and Research Center of Ticks and Tick Borne Disease, Faculty of Veterinary Medicine, University of Tehran, Tehran- Iran

3 Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran

Abstract

BACKGROUND: Enzymatic digestion of proteins in ticks is a complex process and the study of functional proteomics of these enzymes can help to select them as possible vaccine candidates. Blood protein changes (e.g. haemoglobin to vitellin) occur in female mature ticks. Vitellin digestion, as an amino acid and energy source, is one of the vital and important processes in development and evolution of tick eggs and larval stage of unengorged ticks. Several studies reveal a network of proteolytic enzymes involved in haemoglobin and vitellin digestion. These enzymes are mostly cysteine and aspartic peptidases. OBJECTIVES: The aim of this study was the detection of the cathepsins in Rhipicephalus (Boophilus) annulatus larvae extract. METHODS: In the current research, cysteine proteases extracted from Rhipicephalus (Boophilus) annulatus larvae were studied by one- and two-dimensional zymography. RESULTS: Findings from one dimensional zymography showed a transparent band with 28 KDa. In two-dimensional zymography transparent area are seen in the dark gel background distributed in 21 to 65 KDa zones related to cathepsins. When DTT was added to incubation buffer (10 mM acetate buffer, pH= 4), the proteolytic activity of some enzymes was increased and appeared as more clear transparent bands in one-dimentional zymography compared with samples incubated in buffer without DTT. CONCLUSIONS: As the pH of incubation buffer was acidic and adding DTT resulted in increased activity of the enzymes, therefore, some of these proteolytic enzymes are assumed to be cysteine proteases.
 

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