Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major

Abstract

Two transcripts coding for proteins homologous to apyrases were identified by massive sequencing of a Phlebotomus (P.) duboscqi salivary gland cDNA library. The sequence analysis revealed that the amino acids important for enzymatic activity including nucleotidase activity and the binding of calcium and nucleotides were well conserved in these molecules. A recombinant P. duboscqi salivary apyrase was expressed in Escherichia coli and purified. The resulting protein efficiently hydrolyzed ADP and ATP, but not AMP, GDP, CDP or UDP, in a calcium-dependent manner. Further, the recombinant protein inhibited ADP- and collagen-induced platelet aggregation. The results indicated that this salivary protein plays an important role in the blood-feeding process in P. duboscqi. Its unique enzymatic activity makes the salivary apyrase an attractive candidate as a therapeutic agent for the treatment of thrombotic pathologies as well as a reagent for a wide variety of research purposes.

Desbuquois dysplasia type I and fetal hydrops due to novel mutations in the CANT1 gene

“…The two frameshift mutations were found in compound heterozygous form in case 2, and the c.228_229insC mutation in homozygous form in the severely hydropic fetus of case 3. The third mutation, found homozygously in the case with parental consanguinity, involves Asp 112 , an amino acid that is highly conserved up to maize (Supplementary Figure s3c 19 show that the D112 is indeed a key residue for nucleotide binding by mutagenic experiments. This missense mutation was also predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) by an in silico analysis, using the PolyPhen-2 software (v2.0.23r349, http://genetics.bwh.harvard.edu/pph2/).…”

Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis

“…Although ayadualin also inhibited kallikrein, FIXa and FXa activities, the inhibition was lower than that of FXIIa. These and other insects [16,33,[45][46][47]. On the other hand, only a few structurally unique proteins identified from sand fly saliva have been functionally characterized; maxadillan as a strong vasodilator [48] and lufaxin as a factor Xa inhibitor [49], both from Lu.…”

“…Platelet aggregation was measured by using a microplate method as described previously [33]. Briefly, 100 μl of PRP was mixed with 30 After incubation for 1 hr at 37˚C, the wells were washed three times with PBS-T, and a mouse anti-human CD41 (GPIIb) monoclonal antibody (Exbio, Praha, Czech Republic) was added.…”

Cloning, expression, and characterization of salivary apyrase from Aedes albopictus

“…Apyrases are capable of metabolizing nucleoside tri-and diphosphates into organophosphate and mononucleotides; they have been reported in vertebrates (Leal et al 2005), arthropods (Hamasaki et al 2009), and plants (Handa and Guidotti 1996). Nucleotide-hydrolyzing enzymes were divided into three groups based on substrate specificities and amino acid sequences: (1) 5′ nucleotidases associated with the CD73 lymphocyte protein family, (2) ectonucleoside triphosphate diphosphohydrolyases (E-type NTPDases), and (3) enzymes belonging to hematophagous arthropods (Valenzuela et al 1998;Valenzuela et al 2001).…”

“…Blood-feeding insects are known to have the capability to secrete apyrases which deplete adenosine diphosphates (ADP) for the inhibition of platelet activation that facilitates feeding (Valenzuela et al 1998;Valenzuela et al 2001). Nucleotide-metabolizing enzymes may directly play a role in the regulation of extracellular adenosine triphosphate (ATP) levels which affect purinergic signalling and adjustment of host immune status (Bhardwaj and Skelly 2009;Hamasaki et al 2009). …”