The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

Abstract

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.The name apyrase (ATP diphosphohydrolase, EC 3.6.1.5) was first used by Meyerhof (1) to describe a yeast enzyme that hydrolyzed the pyrophosphate bonds of nucleoside di-and triphosphates. Apyrase activity was found also in vertebrate tissues, but the existence of apyrase was challenged when Kalckar (2) showed that the activity in muscle resulted from the concerted action of an ATPase and adenylate kinase. However, true apyrase activity was purified to apparent homogeneity from plants, especially potato tubers and pea cotyledons (3), and more recently, apyrase has been recognized in a variety of vertebrate tissues (4-6) and in the salivary secretions of many hematophagous insects (7,8). The functional significance of most of these enzymes remains unknown, but insect salivary apyrases inhibit platelet aggregation by destroying adenosine di-and triphosphates released from injured cells or by activated platelets.The secreted salivary apyrase of the mosquito Aedes aegypti shares a number of similarities with vertebrate pancreatic and endothelial apyrases, including molecular weight, pH sensitivity, divalent cation dependence, and immunological crossreactivity (9). However, the gene family to which this enzyme belongs was previously unknown. In this report, the salivary apyrase of A. aegypti is characterized as a member of the 5'-nucleotidase gene family.1 MATERIALS AND METHODS Mosquitoes. Adult mosquitoes (A. aegypti, Rockefeller strain) were reared in a 27°C controlled environment room at 80% relative humidity with a 16 hr light/8 hr dark cycle. All animals used in these experiments were 2-to 7-day-old adult females. Mosquitoes were fed a 10% sucrose solution.Purification, Activity, and Peptide Sequencing of Salivary Gland Apyrase. Salivary glands from adult female A. aegypti mosquitoes were dissected in phosphate-buffered saline (PBS), transferred to 10 mM Tris (pH 7.5), and stored frozen at -80°C. To purify active apyrase, 100 pairs of glands were homogenized with a Branson So...

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

“…In addition, endostatin could hydrolyze ADP, but not AMP ( Fig. 2A), showing that endostatin can cleave both b-g and a-b phosphodiester bonds, similar to apyrase and ecto-ATPase families (35,36). Concordant with these results, endostatin hydrolyzed ATP-g-S at a similar rate relative to ADP.…”

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.…”

“…GI accession numbers and references for functional studies-A. aegypti (mosquito): gi556272 [293] (Table 5). The sequence identity of human eN to 5NTs of known spatial structure is 39 % (Thermus thermophilus 5NT, Tt5NT), 30 % (H. influenzae NAD nucleotidase, HiNadN), 26 % (E. coli c4898), 25 % (E. coli 5NT, Ec5NT), 22 % (Staphylococcus aureus 5NT), and 20 % (C. albicans 5NT).…”

The enhancement of arbovirus transmission and disease by mosquito saliva is associated with modulation of the host immune response

“…Mosquitoes, like all haematophagous arthropods, have evolved mechanisms to effectively neutralize host haemostatic responses, based upon the release of saliva into the feeding site. Saliva is a pharmacologic cocktail of secreted molecules, principally proteins, that can affect vascular constriction, blood coagulation, platelet aggregation, inflammation, immunity, and angiogenesis (Arca et al 1999, Arca et al 2002, Arca et al 2005, Billingsley et al 2006, Calvo et al 2004, Calvo et al 2006a, Calvo et al 2006b, Calvo and Ribeiro 2006, Champagne 2004, Champagne et al 1995, Cross et al 1994, James et al 1991, Kerlin and Hughes 1992, Mellink and Vos 1977, Ribeiro 1987, 1989b, 1992, Ribeiro et al 2000, Ribeiro and Francischetti 2001, 2003, Ribeiro and Nussenzveig 1993, Ribeiro et al 1994, Stark and James 1998, Suwan et al 2002, Valenzuela et al 2002, Wanasen et al 2004, Wasserman et al 2004, Zeidner et al 1999. The saliva of all species of haematophagous arthropod analyzed to date contains at least one anticlotting, one anti-platelet, and one vasodilatory substance.…”