Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni.
A Schistosoma mansoni G-protein coupled receptor (SmGPCR) was previously cloned and shown to be activated by the biogenic amine, histamine. Here we report a first investigation of the receptor's subunit organization, tissue distribution and expression levels in different stages of the parasite. A polyclonal antibody was produced in rabbits against the recombinant third intracellular loop (il3) of SmGPCR. Western blot studies of the native receptor and recombinant protein expressed in HEK293 cells showed that SmGPCR exists both as a monomer (65 kDa) and an apparent dimer of approximately 130 kDa These species were verified by immunoprecipitation of SmGPCR from S. mansoni extracts, using antibody that was covalently attached to agarose beads. Further investigation determined that the SmGPCR dimer was resistant to treatment with various detergents, 4 M urea and 0.1 M DTT but could be made to dissociate at acidic pH, suggesting the dimer is non-covalent in nature. Confocal immunofluorescence studies revealed significant SmGPCR immunoreactivity in sporocysts, schistosomula and adult worms but not miracidia. SmGPCR was found to be most widely expressed in the schistosomula, particularly the tegument, the subtegumental musculature and the acetabulum. In the adult stage we detected SmGPCR immunofluorescence mainly in the tubercles of male worms and, to a lesser extent, the body wall musculature. Localization in sporocysts was mainly confined to the tegument and cells within parenchymal matrices. A real-time quantitative reverse-transcription PCR analysis revealed that SmGPCR is upregulated at the mRNA level in the parasitic stages compared to the free-living miracidium and cercariae, and it is particularly elevated during early sporocyst and schistosomula development. The results identify SmGPCR as an important parasite receptor with potential functions in muscle and the tegument of S. mansoni.
El-Shehabi
Fouad
F
Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9.
Vermeire
Jon J
JJ
Yoshino
Timothy P
TP
Ribeiro
Paula
P
D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis / Q000276:immunology; D001700:Biomphalaria; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011817:Rabbits; D017982:Receptors, Biogenic Amine / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
D000909:Antibodies, Helminth; D012333:RNA, Messenger; D017982:Receptors, Biogenic Amine; D043562:Receptors, G-Protein-Coupled