Faudry Eric, Lozzi Silene P, Santana Jaime M, D'Souza-Ault Marian, Kieffer Sylvie, Felix Carlos R, Ricart Carlos A O, Sousa Marcelo V, Vernet Thierry, Teixeira Antonio R L
Chagas' Disease Multidisciplinary Research Laboratory, Department of Pathology, Faculty of Medicine, University of Brasília, Brazil 70.910-900.
J Biol Chem. 2004 May 7;279(19):19607-13. doi: 10.1074/jbc.M401681200. Epub 2004 Feb 25.
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 ADP-induced 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.
腺苷三磷酸双磷酸酶是存在于多种生物体中的核苷三磷酸二磷酸水解酶（EC 3.6.1.5）。在吸血昆虫唾液中发现的腺苷三磷酸双磷酸酶活性与吸血过程中防止宿主因二磷酸腺苷诱导的血小板聚集有关。通过寡聚（dT）-纤维素亲和层析和凝胶过滤层析从锥蝽（Triatoma infestans）唾液中纯化腺苷三磷酸双磷酸酶活性。分离得到的组分包括5种表观分子量分别为88、82、79、68和67 kDa的N-糖基化多肽。分离得到的腺苷三磷酸双磷酸酶混合物完全抑制人血小板聚集。用三磷酸腺苷底物类似物5'-对氟磺酰苯甲酰腺苷标记表明这5种物质具有功能性腺苷三磷酸双磷酸酶的三磷酸腺苷结合特性。此外，串联质谱肽段测序表明这5种物质与埃及伊蚊的腺苷三磷酸双磷酸酶以及其他物种的5'-核苷酸酶具有序列相似性。克隆了79-kDa酶的完整cDNA，其序列证实它编码一种属于5'-核苷酸酶家族的腺苷三磷酸双磷酸酶。导致锥蝽唾液中腺苷三磷酸双磷酸酶出现异常多样性的基因倍增，除了逃避宿主免疫反应外，可能是昆虫向吸血进化过程中放大该酶表达的重要机制，从而增强这种恰加斯病锥蝽传播媒介获取食物的能力。
