Triatoma infestans Apyrases Belong to the 5′-Nucleotidase Family

Abstract

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADPinduced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.Hematophagy in triatomines (Hemiptera: Reduviidae) is associated with the presence of biochemical compounds in the salivary glands that are essential for obtaining blood meals. Indeed, most blood-feeding arthropods have salivary components with vasodilatory, anti-clotting, and anti-platelet aggregation activities that are capable of inhibiting hemostatic reactions of the host (1-4).Host platelet aggregation is considered to be an important hemostatic barrier against insect feeding, because it can stop the bleeding of small blood vessels regardless of other clotting factors (5, 6). Rhodnius prolixus, a triatomine, neutralizes and overrides platelet aggregation induced by collagen, thrombin, thromboxane A 2 , and ADP (7-10). Similarly, collagen-and thrombin-induced platelet aggregation is inhibited by, respectively, pallidipin and triabin, both of which are present in the saliva of Triatoma pallidipennis (11,12). However, the importance of ADP as a common mediator of platelet aggregation pathways is evidenced by the presence on the vascular endothelium surface of the CD39 apyrase, which limits platelet aggregation by hydrolyzing ADP, thus preventing thrombus formation (13). Thus, studying insect apyrases may lead to alternative strategies against the diseases they transmit, as well as new pharmaceutical tools for platelet aggregation-associated disorders. Apyrase removes inorganic phosphate from ATP and ADP, and thus prevents platelet aggregation (6,8,14). Apyrase activity has been characterized in the saliva of Anopheles st...

Cellular function and molecular structure of ecto-nucleotidases

“…Hydrolysis of ATP and ADP prevents platelet aggregation and thus facilitates feeding. Examples of insect salivary apyrases include the mosquito Aedes aegypti [293], Anopheles gambiae [294], the triatomine bug Triatoma infestans [295], the sand fly Lutzomyia longipalpis [296], or a variety of ticks [297]. Figure 7 depicts the phylogenetic relationship of select members of the 5′-nucleotidase family and their substrate specificity.…”

Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands

“…Apyrase removes inorganic phosphate from ATP and ADP and thus prevents platelet aggregation (6,8,16). Apyrase activity has been found in the saliva of many arthropods including Anopheles stephensi, Aedes albopictus, Aedes aegypti, Cimex lectularius, and Triatoma infestans, respectively (4,24,26,48). These apyrases limit platelet aggregation by hydrolyzing ADP, thus preventing thrombus formation.…”

“…Effects on the contraction of isolated rat femoral artery were followed the method reported by Taká c et al (3). Apyrase activities were measured according to previous methods (24,25). Peroxidase activities were according to the methods described (26).…”

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas’ disease

“…This enzyme was described before in T. infestans (SG)s (Faudry et al, 2004), where it helps the acquisition of blood meals by the degradation of adenosine diphosphate (ADP), a mediator of platelet aggregation and inflammation .…”

“…2D-Gel electrophoresis/Mass spectrometry proteomic investigation-The (SG)s of T. infestans were shown to contain proteins such as apyrase and the pore-forming protein trialysin Faudry et al, 2004). To obtain further information on the salivary proteins of T. infestans, electrophoresis of saliva was performed by two-dimensional (2D) SDS-PAGE followed by mass spectrometry.…”