Hemoglobin-degrading, aspartic proteases of blood-feeding parasites - Substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria par asites, employ aspartic proteases to make initial or early cleavages in ing ested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic protease s of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to th e inhibitor pepstatin as the template. The catalytic subsites S5 through S4 ' were determined for the modeled parasite proteases. Subsequently, the cry stal structure of mouse renin complexed with the nonapeptidyl inhibitor t-b utyl-CO-His-Pro-Phe-His-Leu [CHOHCH2]Leu-Tyr-Tyr-Ser-NH2 (CH-66) was used t o build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included repre sentative sites in hemoglobin known to be cleaved by both Schistosoma japon icum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues t hat would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-paras itic agents.