Molecular cloning of Schistosoma mansoni calcineurin subunits and immunolocalization to the excretory system.
In order to explain the schistosomicidal effect of cyclosporin A, the hypothesis was advanced that the drug, complexed with cyclophilin, inhibits the phosphatase activity of parasite calcineurin (CN), with mechanisms similar to those operating in its immunosuppressive action. As a preparatory step to the testing of this hypothesis, we report the molecular cloning of both CN subunits in Schistosoma mansoni. The catalytic (A) subunit has a predicted sequence of 607 amino acids and shows substantial similarity to other cloned CNs, except for the carboxy-terminal end that is highly divergent. The regulatory (B) subunit consists of 169 amino acids that are 86% identical to those of the human counterpart and, from its anomalous electrophoretic mobility, it appears to be myristoylated. The results of Southern blotting experiments are compatible with the existence of multiple genes for CNA and a single gene for CNB. Western blots showed that both subunits are present at all stages of the parasite life cycle and can be detected both in the soluble and in the membrane fraction. Immunofluorescence confocal microscopy revealed a striking concentration of the anti-CNA reactivity in 6-8 discrete spots in the schistosomula and in distinct spots along the body of the adult parasite, corresponding to the expected localization of flame cells. Both patterns were confirmed by a perfect co-localization of the anti-CNA signal with that of a previously characterized anti-flame cell monoclonal antibody. The preferential confinement of schistosome CN to the protonephridial system suggests that the enzyme in the parasite may fulfil similar functions to those performed in mammalian kidneys.
Mecozzi
B
B
Institute of Cell Biology, Consiglio Nazionale delle Ricerche, Rome, Italy.
Rossi
A
A
Lazzaretti
P
P
Kady
M
M
Kaiser
S
S
Valle
C
C
Cioli
D
D
Klinkert
M Q
MQ
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015139:Blotting, Southern; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004064:Digestive System / Q000201:enzymology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment
D018076:DNA, Complementary; D019703:Calcineurin