Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major.
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.
Hamasaki
Ryoichi
R
Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi, Japan.
Kato
Hirotomo
H
Terayama
Yoshimi
Y
Iwata
Hiroyuki
H
Valenzuela
Jesus G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001081:Apyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007303:Insect Vectors / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D018320:Leishmania major / Q000502:physiology*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D008969:Molecular Sequence Data; D010691:Phlebotomus / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012469:Salivary Glands / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D019476:Insect Proteins; D011994:Recombinant Proteins; D001081:Apyrase