Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Abstract

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested 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 proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4 were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH 2 ]Leu-Tyr-Tyr-Ser-NH 2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum 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 that 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-parasitic agents.Blood flukes, hookworms, and the malaria parasites are among the most important pathogens of humans in terms of both numbers of people infected and the consequent morbidity and mortality (1). Although phylogenetically unrelated, these parasites all share the same food source; they are obligate blood feeders, or hematophages. Hb from ingested or parasitized erythrocytes is their major source of exogenous amino acids for growth, development, and reproduction; the Hb, a ϳ64-kDa tetrameric polypeptide, is comprehensively catabolized by parasite enzymes to free amino acids or small peptides. Intriguingly, all these parasites appear to employ cathepsin D-like aspartic proteases to make initial or early cleavages in the Hb substrate (2-4).The vertebrate endopeptidase, cathepsin D (EC 3.4.23.5), is a member of the aspartic protease category of hydrolases, which also includes renin, pepsin, chymosin, cathepsin E, HIV 1 protease, and several other enzymes (5, 6). Cathepsin D is expressed in a diverse range of mammalian cells and tissues and is located predominantly in lysosomes (6). The molecule comprises two rather similar lobes, each incorporating a homologous Asp-Thr-Gly catalytic site motif, with the substrate binding groove located between these lobes. In aspartic proteases generally, the nucleophile that attacks...

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

“…These enzymes play a key role in the digestion of haemoglobin by schistosomes (Brinkworth et al 2001, Koehler et al 2007, Plasmodium falciparum (Francis et al 1997, Banerjee et al 2002, Necator americanus (Brown et al 1995(Brown et al , 1999 and A. caninum (Williamson et al 2003). Interestingly, cysteine protease activity was not detected under the experimental conditions tested.…”

Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase

“…Several cysteine peptidases and an aspartic endopeptidase are secreted into the gut of S. mansoni and each of these has been proposed to facilitate degradation of ingested host proteins including hemoglobin [2,4,13,18,24]. Whether a single peptidase is the major digestive enzyme or a number act in concert is unknown.…”

Blood-feeding in the young adult filarial wormsLitomosoides sigmodontis

“…is considered a favourable adaptation because haemoglobin has a high nutritional value and is frequently associated with completion of the reproductive cycle. This feeding adaptation requires involvement of specific enzymatic pathways to prevent clotting (Urata, Shojo & Kaneko, 2003) and to permit haemoglobin digestion (Brinkworth et al 2001 ;Williamson et al 2003). In L. sigmodontis, only young adults ingest blood.…”