Ornithodoros savignyi: Soft tick apyrase belongs to the 5′-nucleotidase family

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.