Abstract
Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17β-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (K(m) = 0.1-0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.
Highlights
Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the United States
Relative expression levels of the two sulfotransferase proteins in the tissue was found to be altered upon feeding of larval and nymphal stage ticks (Pichu et al, unpublished results), which suggests the function of the Ixosc Sult 1 and Sult 2 may be important within the process of tick feeding
Based on current knowledge of sulfotransferase function in other species [5,6,7], these enzymes could function to control (i) blood clotting in the host, (ii) protein secretion in the tick, or (iii) steroid, neurotransmitter, or thyroid activity in the host or tick
Summary
Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the United States. This paper describes comparative analyses of structure and sequence amongst diverse sulfotransferases with preparation of 3D-models of Ixosc Sult 1 and 2 in order to predict ligand binding and the interaction between the ligand and Ixosc Sult 1 and 2. Our approaches include global and local amino acid sequence similarity, 3D structure conservation among sulfotransferases, and protein structure modeling with ligand docking.
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