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

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. The aim of this study was the detection of the cathepsins in Rhipicephalus [Boophilus] annulatus larvae extract. In the current research, cysteine proteases extracted from Rhipicephalus [Boophilus] annulatus larvae were studied by one- and two-dimensional zymography. 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 DTTwas 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. 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

Expressão e atividade enzimática de glutationa s-transferase em tecidos de fêmeas de Boophilus microplus / Expression and enzymatic activity of glutathione s-transferase in tissues of Boophilus microplus females

Cellular detoxification and excretion enzymes are important to the maintenance of cellular homeostasis. In this work mRNA transcription, protein expression and enzymatic activity of Glutathione S-transferases (GSTs), enzymes involved in the excretion of endo and xenobiotic compounds were analyzed. These parameters are elements believed to protect cells against chemical toxicity and oxidative stress in different tissues (salivary gland, ovary and synganglion) from partially engorged females and engorged females of Boophilus microplus. The results presented showed elevated GST activity in partially engorged females. The enzymatic activity decreased during the preoviposition period in engorged females. GST mRNA transcription was detected in salivary glands and synganglion from partially engorged and engorged females, but not in ovary. The results of this work help to elucidate the role of GST in tick development and assist in the understanding of the importance of GST in tick females during the preparation for oviposition.

Enzimas de detoxificação e excreção celular são importantes para a manutenção da homeostase celular. Neste trabalho foi caracterizada a transcrição de mRNA, a expressão da proteína e a atividade enzimática de glutationa S-transfersases (GSTs), enzimas que atuam em rotas de excreção de substâncias endo e xenobióticas, protegendo as células contra toxicidade química e estresse, em diferentes tecidos (glândula salivar, ovário e singânglio) de fêmeas adultas semi-ingurgitadas e ingurgitadas do carrapato do bovino Boophilus microplus. Os resultados mostraram que a atividade de GST é mais alta em fêmeas semi-ingurgitadas e diminui em fêmeas ingurgitadas de acordo com o final do período de pré-postura. A expressão de mRNA de GST foi detectada em glândulas salivares e singânglios de fêmeas adultas semi-ingurgitadas e ingurgitadas, mas não em ovários. Estes dados podem ajudar a compreender melhor o papel de enzimas antioxidantes durante a preparação das fêmeas do carrapato para a postura.