Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

Abstract

Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

“…Optimal activity was seen at alkaline pH and the K m of the recombinant SmATPDase1 for ATP was 0.4±0.02 mM and for ADP, 0.25±0.02 mM. The work confirmed the ability of tegumental SmATPDase1, expressed on the tegumental surface of live intravascular life stages of the parasite, to hydrolyze exogenous ATP and ADP [12].…”

“…The ability of SmATPDase1 to hydrolyze ATP and ADP was confirmed following the functional characterization of enzymatically active, full-length recombinant SmATPDase1 in CHO-S cells [12]. The enzyme-degraded ATP and ADP in a cation-dependent manner.…”

“…Medium containing ATP and lacking parasites was incubated as above and served as a control. To look for the release of ATPase activity from cultured parasites, groups of adults (12)(13)(14)(15) were first incubated in medium for 55 h at 5 % CO 2 and 37°C. Next, the parasites were removed and ATP (to 10 mM) was added to the medium.…”

“…These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP-diphosphohydrolase (SmATPDase1) [9]. By comparing the ATP-hydrolyzing abilities of worms that have had each of these genes suppressed, it has recently been shown that only the last enzyme in this list, i.e., SmATPDase1, is actually capable of cleaving exogenous ATP [12].…”

“…The three ectoenzymes at the host/parasite interface that were listed above have each been hypothesized to also catabolize ADP [9]. However, as for ATP cleavage, it has been shown that SmATPDase1 is the only one of the three that is actually capable of cleaving exogenous ADP [12].…”