Brugia malayi: localisation and differential expression of extracellular and cytoplasmic CuZn superoxide dismutases in adults and microfilariae.
We have determined the levels of superoxide dismutase (SOD) in different stages of the lymphatic filarial nematode parasite of man, Brugia malayi. Adult male worm extracts showed the highest levels of enzyme activity at 34.5 U mg-1, and there was no significant difference in the overall levels of SOD in extracts of adult female worms and microfilariae (27.1 and 26.7 U mg-1, respectively). SOD activity was detected in the culture medium of parasites maintained in vitro, with particularly high levels of specific activity in media in which males and females were maintained (357 and 339 U mg-1, respectively), indicative of active secretion. In all cases, this was accounted for predominantly by CuZn SOD, assessed by potassium cyanide inhibition. Northern blots with cDNA probes specific for cytoplasmic and extracellular CuZn SODs indicated that levels of mRNA for the cytoplasmic form were similar between adults and microfilariae, whereas expression of the extracellular form was 10x higher in adult worms. Western blots with an antibody to recombinant CuZn SOD demonstrated that higher levels of the extracellular protein were present in adult male worms, whereas the cytoplasmic form was present in roughly equivalent amounts in males, females, and microfilaria. Iodination and immunoprecipitation experiments indicated that the extracellular enzyme was accessible to surface labeling of both male and female adult worms, but not microfilaria. Immuno-electron microscopy showed that CuZn SOD was localised predominantly in the hypodermis of adult parasites, with an asymmetric distribution in the intercordal regions suggestive of compartmentalisation into several distinct syncytia. No labeling was evident in the cuticle, and thus the accessibility of the extracellular enzyme to extrinsic iodination in adult worms remains unclear. No binding of antibody was demonstrable in the glandular region of the oesophagus or the uterus of females, presumed to be major sites of synthesis for secreted proteins. Dense labeling was observed in the seminal fluid surrounding spermatazoa in the vas deferens of male parasites. These data also suggest that, as observed in mammals, nematode spermatazoa are particularly susceptible to oxidative damage and are protected during storage by secreted anti-oxidant enzymes.
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Wellcome Research Centre for Parasitic Infections, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Tang
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McCrossan
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Henkle-Dührsen
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Selkirk
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D018509:DNA, Helminth / Q000032:analysis; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D008842:Microfilariae / Q000201:enzymology / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D012727:Sex Characteristics; D013482:Superoxide Dismutase / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D018509:DNA, Helminth; D012333:RNA, Messenger; D013482:Superoxide Dismutase