Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin

Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrie nts utilized by these parasites. We have shown previously that the predomin ant cysteine proteinase activity in soluble extracts and excretory/secretor y (ES) products of adults of Schistosoma mansoni and S. japonicum is cathep sin L-like in its substrate specificity. However, biochemical analysis of t he cathepsin I, activity in extracts and ES products of schistosomes has be en complicated by the presence of at least two distinct forms of schistosom e cathepsin L, termed SmCL1 and SmCL2. We now report the purification and e nzyme characteristics of active, recombinant SmCL1 which was obtained by tr ansforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1, Recombinant SmCL1 was secreted by the transformed y east into the culture media from which it was purified by gel filtration an d ion-exchange chromatography. The purified enzyme exhibited substrate spec ificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; k(cat)/K-m = 17.25 and 6.24 mM(-1) s(-1), respectively ) and against gelatin and hemoglobin, characteristic of cathepsin L. Immuno blot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extract s of S. mansoni. Using this antiserum and thin tissue sections, we localize d the native SmCL1 to the gastrodermis and to the tegument of adult schisto somes, 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 it s catalysis by cathepsin L and other gut-associated proteinases, Together, these results support a role for SmCL1 in the degradation of host hemoglobi n within the gut of the schistosome.