Characterization of phosphodiesterase-5 as a surface protein in the tegument of Schistosoma mansoni

Abstract

Schistosoma mansoni is a major causative agent of schistosomiasis, an important parasitic disease that constitutes a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection and the development of an effective vaccine still remains the most desirable means of control for this disease. In this work we describe the cloning and characterization of a S. mansoni nucleotide pyrophosphatase/phosphosdiesterase type 5 (SmNPP-5), previously identified in the tegument by proteomic studies. In silico analysis predicts an N-terminal signal peptide, three N-glycosylation sites and a C-terminal transmembrane domain similar to that described for mammalian isoforms. Real-time quantitative RT-PCR and Western blot analyses determined that SmNPP-5 is significantly upregulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages; additionally, the native protein was demonstrated to be N-glycosylated. Immunolocalization experiments and tegument surface membrane preparations confirm the protein as a tegument surface protein. Furthermore, the ectolocalization of this enzyme was corroborated through the hydrolysis of the phosphodiesterase specific substrate (rho-Nph-5'-TMP) by living adult and 21-day-old worms. Interestingly, pre-incubation of adult and 21-day-old worms with anti-rSmNPP-5 antibody was able to reduce by 50-60% the enzyme activity. These results suggest that SmNPP-5 is closely associated with the new tegument surface generation after cercarial penetration, and being located at the host-parasite interface, is a potential target for immune intervention.

Tegumental Phosphodiesterase SmNPP-5 Is a Virulence Factor for Schistosomes

“…This tegumental localization of SmNPP-5 has been reported by other research groups using proteomic approaches (7,33) and immunofluorescence localization (26); also, as noted earlier, phosphodiesterese activity in tegument-enriched fractions of on May 7, 2018 by guest http://iai.asm.org/ adult worms was previously reported (10,11,23,25). The localization of SmNPP-5 as determined using immunogold electron microscopy and reported here confirms and extends those data.…”

“…We proposed earlier that tegumental phosphodiesterase may participate with other schistosome tegumental phosphatase homologs in the catabolism of extracellular nucleotides (5). However, sequence analysis of this tegumental enzyme shows that it clearly belongs to the pyrophosphatase/phosphosdiesterase-5 (NPP-5) family (26), and members of this family have not been reported to metabolize nucleotides (30,31). In agreement with this sequence analysis, in our unpublished work, we found that SmNPP-5-suppressed parasites, though impaired in their ability to cleave the synthetic substrate p-nitrophenyl 5Ј-dTMP, are not impaired in their ability to degrade exogenous ATP, ADP, or AMP (data not shown).…”

“…Its presence had been earlier implied by the detection of phosphodiesterase activity in schistosome tegumental extracts (11). Recently, the cDNA encoding this enzyme was cloned and characterized and its sequence was shown to exhibit greatest homology with sequences of members of the nucleotide pyrophosphatase-phosphosdiesterase 5 (NPP-5) family (26).…”

“…To measure phosphodiesterase activity in live parasites, ϳ500 SmNPP-5-suppressed and control schistosomula (processed in triplicate) were incubated in assay buffer (50 mM Tris-HCl buffer [pH 8.9], 120 mM NaCl, 5.0 mM KCl, 60 mM glucose) containing 0.5 mM phosphodiesterase chromogenic substrate p-nitrophenyl 5Ј-dTMP (p-Nph-5Ј-TMP) (Sigma-Aldrich, MO), as described previously (26). Changes in optical density at 405 nm were monitored continuously for ϳ3 h.…”

Cloning, expression, and characterization of Schistosoma japonicum tegument protein phosphodiesterase-5

“…It is suggested that the combined use of chemotherapy and vaccination is the basis for a novel, more versatile method to control schistosomiasis (Bergquist et al 2005). The identification of appropriate schistosome antigens which could reduce pathology and limit parasite transmission is particularly important in the case of schistosomiasis (Rofatto et al 2009). …”