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


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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Available from: Michael Galperin, Aug 10, 2015
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    • "Apyrases are enzymes that hydrolyze ADP, a key agonist for platelet aggregation. Sand-fly apyrases belong to the Cimex apyrase family of proteins and are distinct from the apyrases in mosquitoes, which belong to the 5′ nucleotidase family of proteins [27]. The predicted mw of P. papatasi apyrase (PPTSP36) is 36 kDa and the predicted pI is 9.03. "
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    • "CD39, also known as ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), is an integral plasma membrane protein with two transmembrane domains and a large heavily glycosylated extracellular region with nucleoside triphosphate diphosphohydrolase activity [18], [29], [30]. However, a novel and evolutionarily distinct apyrase, that differs from the CD39 family in amino acid sequence as well as its exclusive calcium-dependent functionality, has been identified in the salivary glands of blood-sucking bed bug Cimex lectularius [31]. A series of homologs to the Cimex gene were recently found in other blood-sucking insects, as well as in vertebrates, including humans, indicating that these enzymes represent an evolutionarily widespread family of proteins [31], [32], [33], [34], [35], [36], [37], [38]. "
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    ABSTRACT: Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.
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    • "Because ADP and ATP are important activators of platelet and neutrophils, apyrase activity removes these agonists of hemostasis and inflammation [78]. Different genes have been described for this activity such as members of the 5'-nucleotidase family in mosquitoes and triatomines [79-82], the Cimex-type apyrase family in bed bugs and sand flies [83,84] and the type CD-39 protein family in fleas [52]. Expression of this enzyme in mosquitoes has helped to understand the feeding preference in Anopheles, Aedes, and Culex genus [85]. "
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    ABSTRACT: Little is known about the composition and function of the saliva in black flies such as Simulium guianense, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity. Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the S. guianense sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in Simulium. Additionally, we found a novel peptide similar to kunitoxin with a structure distantly related to serine protease inhibitors. This study revealed a relative increase of transcripts of the SVEP protein family when compared with Simulium vittatum and S. nigrimanum sialotranscriptomes. We were able to extract coding sequences from 164 proteins associated with blood and sugar feeding, the majority of which were confirmed by proteome analysis. Our results contribute to understanding the role of Simulium saliva in transmission of Onchocerca volvulus and evolution of salivary proteins in black flies. It also consists of a platform for mining novel anti-hemostatic compounds, vaccine candidates against filariasis, and immuno-epidemiologic markers of vector exposure.
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