Schistosoma mansoni: a Cu/Zn superoxide dismutase is glycosylated when expressed in mammalian cells and localizes to a subtegumental region in adult schistosomes.
We previously isolated a gene from Schistosoma mansoni with the capacity to encode a 20-kDa polypeptide that had homology to Cu/Zn superoxide dismutase from 19 other species. The predicted protein sequence contained a hydrophobic N-terminus as well as a site for N-linked glycosylation, suggesting that the protein was a secreted or membrane-associated form of the enzyme. To study this protein further, we first expressed it in a prokaryotic system and used the gene product to make both monoclonal and polyclonal antibodies. We then expressed the protein in CMT3 cells, a monkey kidney cell line, to investigate possible post-translational modifications. Our results demonstrated that the schistosomal protein expressed in CMT3 cells migrated on an SDS-polyacrylamide gel as multiple glycosylated species with molecular masses of 20, 22, and 23 kDa. Glycosylation was inhibited by tunicamycin, resulting in the expression of an unglycosylated product which migrated with a molecular mass of 18 kDa. The CMT3-cell expressed protein eluted from a gel filtration column with a molecular mass larger than 200 kDa, suggesting that it was membrane-associated or bound to a high-molecular-weight component. The product could not be detected in the medium of the CMT3 cell culture and apparently was not secreted. Comparison between the protein expressed in CMT3 cells and that found in adult schistosomes showed that the parasite-derived gene product was also heterogeneous but had different molecular masses (20, 23, and 25 kDa). The protein was localized in frozen sections of adult worms to the subtegumental area as detected by indirect immunofluorescence using monoclonal antibodies. Since the protein was glycosylated but not secreted we suggest it be called signal peptide-containing superoxide dismutase.
Hong
Z
Z
Department of Biochemistry, State University of New York, Buffalo 14214.
LoVerde
P T
PT
Thakur
A
A
Hammarskjöld
M L
ML
Rekosh
D
D
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017238:Genes, Helminth; D006031:Glycosylation; D007438:Introns; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D021382:Protein Sorting Signals; D014415:Tunicamycin; D012313:RNA; D013482:Superoxide Dismutase