Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis

Abstract

Background: Ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine.

Results: The two sulfotransferase genes were coded as Ixosc SULT 1 & 2 and corresponding proteins were referred as Ixosc Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that Ixosc SULT1 was statistically increased upon blood feeding while Ixosc SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins Ixosc Sult 1(R) and 2(R) showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in I. scapularis.

Conclusions: Collectively, these results suggest that a function of Ixosc Sult 1 and Sult 2 in Ixodid tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine.

Figure 1

Sequence Alignment of Ixosc Sult 1 and 2 with selected known cytosolic sulfotransferases. Ixosc Sult 1 and 2 are designated as q4pma2_Ixosc_Sult1_307aa and q4pma1_Ixosc_Sult2_320aa, respectively. Human sulfotransferases are listed with a protein data bank (PDB) code for a matching x-ray crystal structure. C. elegans and B. mori sulfotransferases are listed with their UniProt code. SULT_MOTIF indicates the residues which make up the cytosolic sulfotransferase amino acid sequence motif regions.

Figure 2

Quantitative analysis of Ixosc SULT 1 and SULT 2 mRNA Expression. Total RNA was extracted from different developmental stages and PCR amplified with gene specific primers. Quantitative transcriptional expression was determined as described in Materials and Methods. The PCR rates of SULT 1 (A) and SULT 2 (B), in various stages of the tick life cycle were normalized to the rate of synthesis of β-actin, included as the endogenous control. Data were plotted as copy numbers per reaction to samples at various level of feeding. Bars indicate PCR values as the Mean ± SD of three replicated experiments. Abbreviations: UL - Unfed Larvae, FL - Fed Larvae, UN - Unfed Nymph, FN - Fed Nymph.

Figure 3

Analysis of Ixosc Sult 1 protein expression during feeding. (A) Ixosc Sult 1 detected by Western blot, (B) Quantitation of band intensity. Tissue homogenates from different developmental stages were analyzed by Western blot using anti-Sult 1 (R) raised antibody. Lanes: 1. Magic Marker, 2. Unfed larvae, 3. Fed larvae, 4. Unfed nymph, 5. Fed nymph. SULT 1 gene products were quantified using Kodak Digital Science 1D image analysis software and the net intensity/band intensity was plotted against the different tick stages.

Figure 4

Analysis of Ixosc Sult 2 protein expression during feeding. (A) Ixosc Sult 2 detected by Western blot, (B) Quantitation of band intensity. Tissue homogenates from different developmental stages were analyzed by Western blot using anti-Sult 2 (R) raised antibody. Lanes: 1. Magic Marker, 2. Unfed larvae, 3. Fed larvae, 4. Unfed nymph, 5. Fed nymph. SULT 2 gene products were quantified using Kodak Digital Science 1D image analysis software and the net intensity/band intensity was plotted against the different tick stages.

Figure 5

SDS-PAGE of expressed Ixosc Sult 1 (R) (A), Sult 2 (R)(B). The expressed purified recombinant proteins were analyzed and stained with Coomassie brilliant blue. Lanes: 1. Prestained molecular weight marker, 2. Purified protein.

Figure 6

Dopamine, octopamine, and p-nitrophenol as substrates for Ixosc Sult 1 (R) and Sult 2 (R). TLC separation of ³⁵S-labeled product from PAP³⁵S (4 μM) after incubation of 10 μM of noted compounds with expressed purified Ixosc Sult 1 (R) or Sult 2 (R)as described in Experimental Procedures.

Figure 7

Mass Spectra of dopamine-sulfate and octopamine-sulfate formed by incubating Ixosc Sult 1(R) or Ixosc Sult 2 (R). A: Sult 1 (R) with PAP³⁵S (Control), B: Sult 1(R) + PAP³⁵S + Dopamine, C: Sult 1 (R) + PAP³⁵S + Octopamine, D: Sult 2 (R) + PAP³⁵S (Control), E: Sult 2 (R) + PAP³⁵S + Dopamine, F: Sult 2 (R) + PAP³⁵S + Octopamine.