Characterization of Rhipicephalus microplus Glycogen Synthase Kinase 3β Protein by Inhibition Assays

Background: Rhipicephalus microplus is a monogenetic, hematophagous ectoparasite that has a large economic impact due to associated losses in the cattle industry. Glycogen synthase kinase 3 is a highly conserved and ubiquitously expressed protein in several species. It has been identified as GSK-3 beta isoform in the cattle tick, and is involved in the modulation of glycogen synthase activity, as a regulator of glycogen synthesis with a role in energy metabolism of R. microplus. It is a fundamental kinase for embryo development, and is directly linked with R. microplus reproductive process. Thus, the aim of this study was to investigate the role of GSK-3 beta in R. microplus physiology by inhibiting its activity. Materials, Methods & Results: In vitro and in vivo assays were carried out to test the effects of immunological and chemical GSK-3 beta inhibition. Synthetic peptides were designed by an in silico analysis of the protein antigenic index. Rabbit antibodies were raised against the designed synthetic peptides based on R. microplus GSK-3 beta sequence (anti-SRm0218 and anti-SRm86100). To access if the inhibition of GSK-3 beta would result in a decrease in fertility and hatching, the purified IgG from rabbit sera was used to feed partially engorged tick females. Results show that the antibodies were not capable of affect egg production. The same antibodies were used in an in vitro assay to test if they were able to inhibit GSK activity and to cause an effect on cell viability or morphology in an embryonic cell line (BME26) and again inhibition was not reached. GSK3 beta expression in BME26 cell line was confirmed by western blot. GSK-3 beta inhibition was reached when R. microplus embryo cells were cultivated with a specific inhibitor GSK3 beta XI. Three different inhibitor concentrations were tested for up to 8 days: 32 nM, 64 nM or 96 nM in BME26 and MDBK cultures (used as a host GSK-3 beta control) MDBK cells did not show any significant decrease in cellular viability (P < 0.05), while tick embryo cells showed up to 48% cellular viability, when incubated with 32 nM of GSK3 beta XI and a similar cellular viability decrease when incubated with 64 nM of inhibitor. This assay suggest that the specific GSK-3 beta inhibition by GSK3 beta XI might have more effect in the parasite than the host enzyme Discussion: GSK-3 beta was initially defined as a regulator of glycogen metabolism. It phosphorylates residues on glycogen synthase, inhibiting this enzyme and consequently modulating glycogen synthesis. However, GSK-3 beta is a multifunctional protein that affects cell cycle, gene transcription, apoptosis and embryo development, besides its role in carbohydrate metabolism. In this work, we demonstrated the ability of antibodies to interact with native and recombinant proteins although they were not able to inhibit GSK-3 beta activity. The chemical GSK-3 beta inhibition with alsterpaullone has been reported elsewhere resulting in egg viability alteration, reducing R. microplus oviposition and egg hatching, and demonstrating the important role played by GSK-3 beta in embryogenesis. In a similar manner chemical GSK3 beta XI inhibited GSK-3 beta activity. This results support the idea that this protein is a potential target to develop a new R. microplus cattle tick control method, especially since it was only observed an effect in the tick cells and not in the host cells. The different viability results between the cells lines are probably due to a specific inhibition of GSK-3 beta isoform without effect on the hosts other GSK-3 beta isoforms.