title: Ornithodoros savignyi: soft tick apyrase belongs to the 5'-nucleotidase family.
abstract: Salivary apyrases are nucleotide-metabolising enzymes that blood-feeding parasites utilise for modulation of extracellular nucleotides to prevent platelet activation and aggregation. In this study a 5'-nucleotidase specific degenerate primer was used to identify homologous transcripts from Ornithodoros savignyi salivary gland cDNA. Two 5'-nucleotidase isoforms that share significant sequence identity to putative apyrases from Rhipicephalus appendiculatus and Ixodes scapularis were identified. Structure prediction showed a tertiary structure similar to periplasmic ecto-5'-nucleotidase from Escherichia coli, with high conservation of functional residues. The O. savignyi 5'-nucleotidase isoform I was recombinantly expressed in Pichia pastoris. Cross-reactivity was demonstrated with polyclonal anti-apyrase antisera produced against O. savignyi apyrase. Subsequent Edman sequencing and MS/MS analysis of purified O. savignyi apyrase identified peptide sequence fragments that shared sequence identity with both newly identified 5'-nucleotidase isoforms. It was concluded that wild-type apyrase is a mixture of the isoforms identified from the salivary glands of O. savignyi. These results represent the first confirmation of a soft (argasid) tick apyrase that belongs to the 5'-nucleotidase family of enzymes.
authors.affiliation: Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Christian
authors.initials: C
authors.lastname: Stutzer
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Mans
authors.forename: Anabella R M
authors.initials: AR
authors.lastname: Gaspar
authors.forename: Albert W H
authors.initials: AW
authors.lastname: Neitz
authors.forename: Christine
authors.initials: C
authors.lastname: Maritz-Olivier
mesh_terms: D015720:5'-Nucleotidase / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000829:Animals, Domestic / Q000469:parasitology; D001081:Apyrase / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D026861:Ornithodoros / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology; D012469:Salivary Glands / Q000201:enzymology; D017421:Sequence Analysis; D012822:Silicon Dioxide; D013019:South Africa; D053719:Tandem Mass Spectrometry; D013984:Tick Infestations / Q000469:parasitology / Q000662:veterinary
chemical_list: D007527:Isoenzymes; D012822:Silicon Dioxide; D015720:5'-Nucleotidase; D001081:Apyrase