Hookworm Aspartic Protease,Na‐APR‐2, Cleaves Human Hemoglobin and Serum Proteins in a Host‐Specific Fashion

Abstract

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.Hookworms are highly host specific, obligate bloodfeeders. Infective, third-stage larvae (L3) in soil penetrate the skin and migrate (and mature) through the

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

“…Plasmepsin II is capable of digesting native Hb but is more active against denatured or fragmented globin such as that produced by the action of plasmepsin I (34). In addition to Na-APR-1, we identified a second aspartic protease from the human hookworm, N. americanus, Na-APR-2; APR-2 was most similar to a family of nematodespecific aspartic proteases, termed nemepsins (19). APR-2 digested Hb but at very different sites than APR-1, suggesting that in N. americanus, at least, multiple aspartic proteases are responsible for digesting Hb in the intestine of adult worms.…”

“…Immunohistochemistry-Adult A. caninum were prepared for immunostaining as described previously (19). Nonspecific binding sites were blocked with 5% fetal bovine serum in phosphate-buffered saline, 0.05% Tween 20 (PBS-T) for 1 h. Dog antisera were produced against each protease formulated with the adjuvant AS03 as described for CP-2 (21); animals were bled after the third vaccination but before parasite challenge.…”

“…Antisera (diluted 1:100, 1:500, 1:1000 in phosphate-buffered saline) were applied to each tissue section and incubated for 2 h at room temperature in a humidity chamber. Subsequent steps were as described elsewhere (19).…”

“…Until now, only the aspartic proteases, APR-1 and APR-2, had been shown to digest Hb in vitro (19,20). Moreover, these hookworm proteases were more efficient at digesting Hb from permissive host species than from nonpermissive hosts, despite sharing identical active sites residues; this highlights the exquisite co-evolution of these enzymes with host substrates (19,20,27). We have suggested that hookworms likely employ a cascade of hemoglobinolysis utilizing multiple proteases of distinct mechanistic classes (18).…”

“…Moreover, the intestinal contents from the related blood-feeding helminth, Schistosoma mansoni, are acidic pH (11). mRNAs encoding hookworm proteases of different mechanistic classes, including aspartic (19,20,23), cysteine (24), and metalloproteases (25,26), have been cloned, and the expression sites of some have been localized to the intestinal brush border of the blood-feeding adult stage (for a review, see Ref. 18).…”