Similar mechanisms regulate protein exocytosis from the salivary glands of ixodid and argasid ticks.

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding in response to an external stimulus. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast-feeding ticks) and Ixodidae (slow-feeding ticks), but differ in their sensitivity to prostaglandin E(2). In both cases, dopamine is the external signal for inducing exocytosis. Dopamine-induced exocytosis was shown to be strongly calcium dependant. Firstly, it requires extracellular calcium via a L-type voltage-gated calcium channel located on the plasma membrane and, secondly, intracellular calcium which is released presumably in response to inositol 1,4,5-triphosphate (IP(3)). Pathways such as the activation of phospholipase C, inositol-phosphate kinases, G-proteins, GTPases and Na(+)-K(+)-ATPases have been shown to be essential.

Comparative properties of a three-dimensional model of Plasmodium falciparum ornithine decarboxylase.

The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal alpha/beta-barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon alpha-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structure-based approach for the design of novel malaria-specific inhibitors.

Amino acid sequence and structure modeling of savignin, a thrombin inhibitor from the tick, Ornithodoros savignyi.

The full-length gene of savignin, a potent thrombin (E.C. 3.4.21.5) inhibitor from the tick Ornithodoros savignyi has been cloned and sequenced. Both 5' and 3' UTR's, a signal peptide from the translated amino acid sequence and an unusual poly-adenylation signal (AATACA) has been identified. The translated protein sequence shows high identity (63%) with ornithodorin, the thrombin inhibitor from the tick, Ornithodoros moubata. Molecular modeling using the structure of ornithodorin as reference gave a structure with an RMSD of 0.25 A for the full-length protein, 0.11 A for the N-terminal BPTI-like domain and 0.11 A for the C-terminal BPTI-like domain, indicating that maximum deviation occurs in the mobile bridge (0.18 A) between the two domains. Docking of savignin to thrombin shows that the interaction is similar to the ornithodorin-thrombin complex. The N-terminal amino acid residues of savignin bind inside the active site cleft, while the C-terminal domain of savignin has a net negative electrostatic potential and interacts with the basic fibrinogen recognition exosite of thrombin through hydrogen bonds and hydrophobic interactions. These results correlate with kinetic data obtained, which showed that savignin is a competitive, slow, tight-binding inhibitor that requires thrombin's fibrinogen-binding exo-site for optimal inhibition.

Biochemical perspectives on paralysis and other forms of toxicoses caused by ticks.

Tick toxicoses, of which paralysis is the most widespread and dominant form, are important elements of pathogenesis induced by ticks. Tick paralysis is the most widespread and dominant form of tick toxicoses. Non-paralytic forms of tick toxicoses do occur and evidence suggests that these forms of toxicoses are not evolutionary related. While functional significance has been suggested for tick toxins, the advantages for tick survival in general are not clear. This review considers the molecular nature of tick toxins, the possibility that tick toxins have originated more than once independently and whether these toxins could have unrecognized benign functions.

Identification of extrinsic blood coagulation pathway inhibitors from the tick Ornithodoros savignyi (Acari: Argasidae).

The salt BaSO(4) selectively adsorbs two proteins from crude Ornithodoros savignyi salivary gland extract. They co-purify during reversed-phase HPLC, but can be separated by hydrophobic-interaction chromatography. Their molecular masses are 9333 and 9173Da. The 9.3kDa protein was designated BSAP1 and the 9.1kDa protein BSAP2. Their amino acid compositions show significant differences, in particular the presence of seven and eight cysteine residues in BSAP1 and BSAP2, respectively. The proteins do not contain gamma-carboxyglutamic acid, hydroxyproline, or hydroxylysine. The proteins do not inhibit the intrinsic coagulation cascade, but inhibit the extrinsic pathway. The observed inhibition is not due to inhibition of factor VII. Both proteins bind to membranes. BSAP1 binds neutral and negatively charged membranes more strongly than BSAP2. Its affinity for negative membranes is, however, much lower than for neutral membranes. In contrast, BSAP2 binds both membranes equally strongly. The binding of the proteins to the membranes was significantly lowered upon pre-incubation with Ca(2+).