title: Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin.
abstract: Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of Schistosoma mansoni and S. japonicum is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; kcat/Km = 17.25 and 6.24 mM-1 s-1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S. mansoni. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.
authors.affiliation: School of Biological Sciences, Dublin City University, Ireland.
authors.forename: C P
authors.initials: CP
authors.lastname: Brady
authors.forename: A J
authors.initials: AJ
authors.lastname: Dowd
authors.forename: P J
authors.initials: PJ
authors.lastname: Brindley
authors.forename: T
authors.initials: T
authors.lastname: Ryan
authors.forename: S R
authors.initials: SR
authors.lastname: Day
authors.forename: J P
authors.initials: JP
authors.lastname: Dalton
mesh_terms: D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006454:Hemoglobins / Q000378:metabolism*; D006790:Host-Parasite Interactions; D008297:Male; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D012555:Schistosomiasis mansoni / Q000097:blood* / Q000201:enzymology / Q000469:parasitology; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D006454:Hemoglobins; D010455:Peptides; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L