Background: Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.
Methodology/principal findings: Here we report on the functional characterisation of a 5'nucleotidase-related (5'Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5'Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5'nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5'Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K(m) and V(max) values of 43+/-4 microM and 684+/-49 nmol Pi/min xmg for ATPase and 49+/-11 microM and 177+/-37 nmol Pi/min xmg for the ADPase activity. In addition, recombinant 5'Nuc was found to bind to GPIIb/IIIa with an apparent K(D) of 92+/-25 nM. Consistent with these features, 5'Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5'Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.
Conclusions/significance: These data show that this 5'nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.