Characterization of phosphodiesterase-5 as a surface protein in the tegument of Schistosoma mansoni.
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.
Rofatto
Henrique K
HK
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
Tararam
Cibele A
CA
Borges
William C
WC
Wilson
R Alan
RA
Leite
Luciana C C
LC
Farias
Leonardo P
LP
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000378:metabolism; D003001:Cloning, Molecular; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D015801:Helminth Proteins / Q000737:chemistry / Q000378:metabolism*; D008018:Life Cycle Stages / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D000909:Antibodies, Helminth; D015801:Helminth Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5