Valenzuela JG, Charlab R, Galperin MY, Ribeiro JMPurification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme. J Biol Chem 273:30583-30590

Medical Entomology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/1998; 273(46):30583-30590. DOI: 10.1074/jbc.273.46.30583

ABSTRACT An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying
apyrase (EC activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purifiedC. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of ∼40 kDa that inhibited ADP-induced platelet aggregation
and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectulariuscDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase
pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The
processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product
of the cDNA clone with nativeC. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against
a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized bothC. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing
gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum.C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the
genome of the nematode C. elegansand in mouse and human expressed sequence tags from fetal and tumor EST libraries.

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    • "). Sandfly apyrases belong to the Cimex family, which includes human homologues (Valenzuela et al. 1998). Therefore, any vaccine faces the challenge of using apyrase to prevent Leishmania establishment without undermining host haemostasis. "
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    ABSTRACT: Vaccine development is informed by a knowledge of genetic variation among antigen alleles, especially the distribution of positive and balancing selection in populations and species. A combined approach using population genetic and phylogenetic methods to detect selective signatures can therefore be informative for identifying vaccine candidates. Parasitic Leishmania species cause the disease leishmaniasis in humans and mammalian reservoir hosts after inoculation by female phlebotomine sandflies. Like other arthropod vectors of disease agents, sandflies use salivary peptides to counteract host haemostatic and immunomodulatory responses during bloodfeeding, and these peptides are vaccine candidates because they can protect against Leishmania infection. We detected no contemporary adaptive selection on one salivary peptide, apyrase, in 20 populations of Phlebotomus ariasi, a European vector of Leishmania infantum. Maximum likelihood branch models on a gene phylogeny showed apyrase to be a single copy in P. ariasi but an ancient duplication event associated with temporary positive selection was observed in its sister group, which contains most Mediterranean vectors of L. infantum. The absence of contemporary adaptive selection on the apyrase of P. ariasi may result from this sandfly’s opportunistic feeding behaviour. Our study illustrates how the molecular population genetics of arthropods can help investigate the potential of salivary peptides for disease control and for understanding geographical variation in vector competence.
    Evolutionary Applications 04/2012; 5(3). DOI:10.1111/j.1752-4571.2011.00226.x · 4.57 Impact Factor
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    • "This difference may be due to the bird feeding preferences of Culex mosquitoes, which do not face the platelet barrier of mammal feeding mosquitoes (Ribeiro, 2000). In sand flies the apyrase activity results from a completely different protein family (Charlab et al., 1999; Valenzuela et al., 2001b), which was first discovered in bed bug salivary glands, constituting a new family of nucleotide binding enzymes (Valenzuela et al., 1998). In the sand fly Lutzomyia longipalpis there also exists a 5'-nucleotidase enzyme that was shown to hydrolyze not only AMP, but also UDP-Glucose, thus acting as a phosphodiesterase (Ribeiro et al., 2000b). "
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    ABSTRACT: Within the Diptera and outside the suborder Brachycera, the blood-feeding habit occurred at least twice, producing the present day sand flies, and the Culicomorpha, including the mosquitoes (Culicidae), black flies (Simulidae), biting midges (Ceratopogonidae) and frog feeding flies (Corethrellidae). Alternatives to this scenario are also discussed. Successful blood-feeding requires adaptations to antagonize the vertebrate's mechanisms of blood clotting, platelet aggregation, vasoconstriction, pain and itching, which are triggered by tissue destruction and immune reactions to insect products. Saliva of these insects provides a complex pharmacological armamentarium to block these vertebrate reactions. With the advent of transcriptomics, the sialomes (from the Greek word sialo = saliva) of at least two species of each of these families have been studied (except for the frog feeders), allowing an insight into the diverse pathways leading to today's salivary composition within the Culicomorpha, having the sand flies as an outgroup. This review catalogs 1288 salivary proteins in 10 generic classes comprising over 150 different protein families, most of which we have no functional knowledge. These proteins and many sequence comparisons are displayed in a hyperlinked spreadsheet that hopefully will stimulate and facilitate the task of functional characterization of these proteins, and their possible use as novel pharmacological agents and epidemiological markers of insect vector exposure.
    Insect biochemistry and molecular biology 11/2010; 40(11):767-84. DOI:10.1016/j.ibmb.2010.08.002 · 3.42 Impact Factor
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    • "these molecules include apyrase (Valenzuela et al., 1998), an inhibitor of Factor Xa of the coagulation cascade (Valenzuela et al., 1996), and a nitrophorin (NP) that transports NO (Valenzuela et al., 1995). Together these molecules are able to prevent vasoconstriction , coagulation and platelet aggregation at the feeding site. "
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    ABSTRACT: The bedbugs Cimex lectularius and Cimex hemipterus are obligate hematophages in all their nymphal instars as well as in the adult stage. The efficiency with which the insects obtain blood from their hosts is directly related to their population dynamics. In the present study we compared the feeding process and salivary content in individuals of these two species when fed on different blood sources or host sites, using a cibarial pump electromyogram. Females ingested more blood than males but needed longer contact time with the host to complete the meal. The bedbug C. lectularius was more efficient than C. hemipterus in obtaining blood from mice and pigeons. With regard to the feeding site on mice, it was easier for the insects to obtain blood from the skin of the belly than that of the back. Individuals of C. hemipterus were able to maintain the cibarial pump functioning at higher frequencies for longer periods when fed on pigeons treated with anticoagulant. Although saliva from C. lectularius contained more hemeproteins and showed more anti-clotting activity its total protein content was similar to that of C. hemipterus. Overall, C. lectularius obtains a bloodmeal more efficiently from its hosts, which may have enabled this species to reach higher levels of infestation than C. hemipterus.
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