Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme.
Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.
Brady
C P
CP
School of Biotechnology, Dublin City University, Dublin 9, Glasnevin, Ireland.
Brindley
P J
PJ
Dowd
A J
AJ
Dalton
J P
JP
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D018076:DNA, Complementary / Q000032:analysis; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008297:Male; D018510:RNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D018076:DNA, Complementary; D018510:RNA, Helminth; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L