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Molecular characterization of tick salivary gland glutaminyl cyclase.

ABSTRACT: Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control.

SUBMITTER: Adamson SW

PROVIDER: S-EPMC3740044 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Molecular characterization of tick salivary gland glutaminyl cyclase.

Adamson Steven W SW Browning Rebecca E RE Chao Chien-Chung CC Bateman Robert C RC Ching Wei-Mei WM Karim Shahid S

Insect biochemistry and molecular biology 20130613 9

Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapul ...[more]

PMID: 23770496

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Project description:IntroductionAlpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS.MethodsEight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs.ResultsCompared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature.ConclusionTSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.

Project description:The success of cattle tick fixation largely depends on the secretion of substances that alter the immune response of the host. The majority of these substances are expressed by the parasite salivary gland and secreted in tick saliva. It is known that hosts can mount immune responses against ticks and bovine European breeds, and bovine industrial crossbreeds are more susceptible to infestations than are Bos indicus cattle. To identify candidates for the development of novel control strategies for the cattle tick Rhipicephalus (Boophilus) microplus, a salivary gland transcriptome analysis of engorged females fed on susceptible or resistant hosts was performed. Using RNA-Seq, transcriptomes were de novo assembled and produced a total of 235,451 contigs with 93.3% transcriptome completeness. Differential expression analysis identified 137 sequences as differentially expressed genes (DEGs) between ticks raised on tick-susceptible or tick-resistant cattle. DEGs predicted to be secreted proteins include innexins, which are transmembrane proteins that form gap junction channels; the transporters Na+/dicarboxylate, Na+/tricarboxylate, and phosphate transporter and a putative monocarboxylate transporter; a phosphoinositol 4-phosphate adaptor protein; a cysteine-rich protein containing a trypsin inhibitor-like (TIL) domain; a putative defense protein 3 containing a reeler domain; and an F-actin-uncapping protein LRRC16A with a CARMIL_C domain; these genes were upregulated in ticks fed on tick-susceptible cattle. DEGs predicted to be non-secreted proteins included a small heat shock protein and the negative elongation factor B-like, both acting in a coordinated manner to increase HSP transcript levels in the salivary glands of the ticks fed on tick-susceptible cattle; the 26S protease regulatory subunit 6B and another chaperone with similarity to calnexin, also upregulated in ticks fed on tick-susceptible cattle; an EF-hand calcium binding protein and a serine carboxypeptidase (SCP), both involved in the blood coagulation cascade and upregulated in ticks fed on tick-susceptible cattle; and two ribosomal proteins, the 60S acidic ribosomal protein P2 and the 60S ribosomal protein L19. These results help to characterize cattle tick salivary gland gene expression in tick-susceptible and tick-resistant hosts and suggest new putative targets for the control of tick infestations, as those genes involved in the mechanism of stress response during blood feeding.

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Molecular characterization of tick salivary gland glutaminyl cyclase.

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Molecular characterization of tick salivary gland glutaminyl cyclase.

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