Affinity isolation and characterization of the cathepsin B-like proteinase Sj31 from Schistosoma japonicum.
The major cathepsin B-like proteinase of adult Schistosoma japonicum has been isolated for the first time. Affinity chromatography with the mammalian cathepsin B inhibitor glycyl-phenylalanyl-glycine-semicarbazone purified a protein that was identified by N-terminal sequencing as Sj31. Sensitivity of Sj31 to PNGase F demonstrated the presence of asparagine-linked N-glycan. Marked resistance to the action of Endo-beta-glycosidase H indicated that most of the N-glycan chains are of the complex type. Binding of horseradish peroxidase-conjugated lectins demonstrated the presence of N-mannose, N-acetylglucosamine, and N-acetyllactosamine type 2 in the N-glycan. Fucose was not detected, and the presence of sialic acid remained questionable. Sj31 degraded the fluorogenic substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec with an optimum between pH 5.0 and 6.0. The specific activity was 18-21-fold higher with the Phe-Arg substrate compared with the Arg-Arg substrate, whereas this value was 4-6-fold for bovine spleen cathepsin B, thus suggesting differences in the S2 subsite between parasite and host proteinases. Quantitative purification of Sj31 led to the conclusion that cathepsin B-like activity predominates over cathepsin L-like activity in S. japonicum. Because Sj31 degraded hemoglobin in vitro and was localized in the parasite gut, the proteinase may degrade ingested proteins in vivo.
Caffrey
C R
CR
Institute of Tropical Hygiene, University of Heidelberg, Germany.
Ruppel
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D006031:Glycosylation; D015801:Helminth Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D008297:Male; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D009928:Organ Specificity; D012549:Schistosoma japonicum / Q000201:enzymology*; D013379:Substrate Specificity
D015801:Helminth Proteins; D006454:Hemoglobins; D009842:Oligopeptides; D002401:Cathepsin B