Monoamine Neurotransmitters as Substrates for Novel Tick Sulfotransferases, Homology Modeling, Molecular Docking, and Enzyme Kinetics

Abstract

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern and upper Midwestern US. Current research focuses on elucidating biochemical pathways which may be disrupted to prevent pathogen transmission, thereby preventing disease. A genome screening process reported transcripts coding for two putative sulfotransferases in salivary glands of adult Ixodes scapularis and 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 these two sulfotransferases (designated Ixosc Sult 1 and Ixosc Sult 2), and tested the predictions with biochemical assays. Amino acid sequence analysis showed that Ixosc Sult 1 and 2 are members of the cytosolic sulfotransferase superfamily. Homology models of 3D-protein structure were prepared for each tick sulfotransferase based on sulfotransferase X-ray crystal structures. 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 a catalytic binding mode. Other molecules including 17β-estradiol, pregnenolone and serotonin, did not dock in catalytically active position to either protein. The predictions were tested and confirmed using cytosolic fractions of extracts of whole ticks. Dopamine was found to be 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. Octopaminesulfonation was only detected after feeding when gene expression data suggests that only Ixosc Sult 1 is present. These results agree with the docking predictions that octopamine is sulfonated only by Ixosc Sult 1, whereas dopamine is sulfonated by both Ixosc Sult 1 and Sult 2. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function/s of Ixosc Sult 1 or Sult 2 in the Ixodid tick may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine. 15/2010). This tick species serves as the vector for the pathogen, Borrelia burgdorferi, which is a spirochete bacterium responsible for Lyme disease in humans. Small animals such as wild mice, chipmunks and other wild rodents provide the reservoir and seem not to be adversely affected by presence of the bacterium. The spirochete can be passed to humans after a tick, which has previously fed on an infected small animal, subsequently feeds on an individual. Blacklegged ticks (also known as deer ticks) have three life stages (larval, nymphal, adult) and feed once at each life stage. A single infect...

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