A Multienzyme Network Functions in Intestinal Protein Digestion by a Platyhelminth Parasite

Abstract

Proteases frequently function not only as individual enzymes but also in cascades or networks. A notable evolutionary switch occurred in one such protease network that is involved in protein digestion in the intestine. In vertebrates, this is largely the work of trypsin family serine proteases, whereas in invertebrates, cysteine proteases of the papain family and aspartic proteases assume the role. Utilizing a combination of protease class-specific inhibitors and RNA interference, we deconvoluted such a network of major endopeptidases functioning in invertebrate intestinal protein digestion, using the parasitic helminth, Schistosoma mansoni as an experimental model. We show that initial degradation of host blood proteins is ordered, occasionally redundant, and substrate-specific. Although inhibition of parasite cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cathepsin B predominated in albumin degradation. Nevertheless, in both cases, inhibitor combinations were synergistic. An asparaginyl endopeptidase (legumain) also synergized with cathepsin B and L in protein digestion, either by zymogen activation or facilitating substrate cleavage. This protease network operates optimally in acidic pH compartments either in the gut lumen or in vacuoles of the intestinal lining cells. Defining the role of each of these major enzymes now provides a clearer understanding of the function of a complex protease network that is conserved throughout invertebrate evolution. It also provides insights into which of these proteases are logical targets for development of chemotherapy for schistosomiasis, a major global health problem.

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade

“…First, SmCD 1.2 kb-dsRNA was employed for treatment of 3-hour-old schistosomules that were cultured for the next 14 days. dsRNA of this length was employed because earlier studies with cathepsin B1 of S. mansoni had shown that dsRNA of at least one kb in length induced strong knockdown and phenotypic effects against a proteolytic enzyme, which like cathepsin D, is gut-localized and participates in hemoglobin proteolysis [6,18]. Second, a SmCD dsRNA of 0.5 kb in length was prepared for use with seven, nine and 11 day old somules that were cultured for seven days after dsRNA treatment.…”

“…Second, a SmCD dsRNA of 0.5 kb in length was prepared for use with seven, nine and 11 day old somules that were cultured for seven days after dsRNA treatment. We tested the shorter dsRNA probe because it had become apparent that shorter dsRNAs of 300 to 700 bp, and indeed very short 21 bp short interfering RNAs (siRNAs), triggered profound gene suppression of gut-associated proteases of S. mansoni [6,15], and because there exists the possibility that residual dsRNAs might interfere with downstream analysis of gene-silenced cells, specifically by serving as spurious templates in RT-PCRs employed to investigate levels of target gene transcripts [24].…”

“…Cysteine and aspartic proteases have been described in secretions and/or worm extracts of schistosomules and adult worms [2], and previous studies have shown that inhibitors of either cysteine proteases [3] or aspartic proteases [4] block hemoglobin degradation and arrest schistosome development and egg production. A proteolytic cascade or network involving aspartic and cysteine proteases has been proposed as catalyzing hemoglobin degradation in schistosomes [5][6][7]. Aspartic proteases also play central roles in the degradation of ingested blood and hemoglobin in other blood-feeding parasitic worms, and sites of cleavage within hemoglobins by both hookworm and schistosome aspartic proteases have been mapped [1,5,8].…”

“…Aspartic proteases also play central roles in the degradation of ingested blood and hemoglobin in other blood-feeding parasitic worms, and sites of cleavage within hemoglobins by both hookworm and schistosome aspartic proteases have been mapped [1,5,8]. Moreover, from an evolutionary perspective, McKerrow and colleagues have hypothesized, based on findings with parasitic helminths, that early metazoans evolved a successful digestive network of proteases comprising cathepsins D, B, L, C and legumain that predated the evolution of the pancreas and the subsequent primacy of serine proteases as digestive enzymes in vertebrates [6].…”

“…Due to its physiological significance for schistosomes, deeper studies on its molecular and biochemical properties, gene structure and phylogeny have been undertaken [6,11,12]. Although cathepsin D-like protease (clan AA) has been involved in digestion of hemoglobin within the adult stages of S. mansoni, it remains unclear whether this enzyme is expressed in and functions in the schistosomula stage early in mammalian infection.…”