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Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity¹

ANGELA L. WILLIAMSON^*,, PAUL J. BRINDLEY, GIOVANNI ABBENANTE, PAUL PROCIV, COLIN BERRY^||, KAREN GIRDWOOD^||, DAVID I. PRITCHARD^¶, DAVID P. FAIRLIE, PETER J. HOTEZ^#, JOHN P. DALTON and ALEX LOUKAS^*,#2

^* Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, QLD, Australia;
Department of Microbiology and Parasitology and
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia;
Department of Tropical Medicine, Tulane University, New Orleans, Louisiana, USA;
^|| School of Biosciences, Cardiff University, Cardiff, UK;
^¶ The Boots Science Institute, University of Nottingham, Nottingham, UK;
^# Department of Microbiology and Tropical Medicine, George Washington University Medical Centre, Washington, D.C., USA; and
School of Biotechnology, Faculty of Health and Science, Dublin City University, Dublin, Ireland

²Correspondence: Department of Microbiology and Tropical Medicine, Ross Hall, Room 726, George Washington University Medical Centre, 2300 Eye St. NW, Washington DC, 20037, USA. E-mail: mtmacl{at}gwumc.edu

SPECIFIC AIM

Human and canine hookworms are blood-feeding nematode parasites that reach the gut of nonpermissive mammalian hosts but fail to successfully feed, develop, and reproduce, presumably as a consequence of intimate coevolution between the parasite and its normal definitive (permissive) host. To identify molecular examples of host specificity in blood-feeding pathogens, we hypothesized that hookworm digestive proteases were more efficient at cleaving hemoglobin substrates from permissive than nonpermissive host species.

PRINCIPAL FINDINGS

We cloned and expressed cathepsin D-like aspartic proteases from canine and human hookworms and showed that a cathepsin D-like protease from the canine hookworm Ancylostoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) were expressed in the gut, and both cleaved human and dog hemoglobin in vitro. Each protease digested hemoglobin from its permissive host between two- (whole molecule) and six- (synthetic peptides) fold more efficiently than hemoglobin from the nonpermissive host even though the two proteases have identical residues lining their active site clefts. Both proteases cleaved hemoglobin at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor of host-parasite compatibility and host species range.

1. Hookworm cathepsin D-like aspartic proteases
The predicted Ac-APR-1 and Na-APR-1 proteins exhibited 85% sequence identity at the amino acid level and shared 49% identity with human cathepsin D. It is notable that the residues that constitute the active site clefts of the two enzymes were identical in the hookworm proteases yet exhibited differences with human cathepsin D and homologues from other helminths. Phylogenetic analysis of the hookworm cathepsins D and other aspartic proteases showed that Ac- and Na-APR-1 formed a robust clade within a larger, strongly supported group that contained cathepsins D from other nematodes.

2. Hookworm cathepsins D are expressed in the gut and amphidial glands
Ac- and Na-APR-1 were expressed as secreted proenzymes in Sf9 lepidopteran cells, activated by low pH, and purified using pepstatin agarose. Purified proteases were used to immunize mice for antibody production and antisera were used to localize expression of the proteases to the gut, excretory, and amphidial glands of adult hookworms (Fig. 1 ).

View larger version (201K): [in this window] [in a new window]   Figure 1. Hookworm cathepsins D are expressed in the gut and amphidial glands of adult parasites. Antiserum raised against recombinant Ac-APR-1 bound to the intestine (in), esophagus (oe), excretory glands (ex), and amphidial glands (am) of A. caninum (B, D) whereas preimmunization serum did not (A, C).

3. Hookworm recombinant cathepsins D cleave fluorogenic substrates
Both enzymes were proteolytically active against o-aminobenzoyl-IEF-nFRL-NH₂ with pH optima of 5.5 and were completely inhibited by pepstatin A. Mouse IgG purified from anti-protease sera completely inhibited cleavage of the peptide substrate by its homologous protease. Incubation of each protease with heterologous antisera led to significant reductions (60–80%) in cleavage of the peptide by the proteases.

4. Hookworm cathepsins D preferentially cleave hemoglobin from permissive hosts
Both enzymes cleaved dog and human Hb, but at different rates (Fig. 2 ). Ac-APR-1 degraded dog Hb almost twice as efficiently as it did human Hb; conversely, Na-APR-1 cleaved human Hb with greater efficiency than dog Hb. The host-specific differences in cleavage efficiencies were remarkable considering that the residues lining the active site clefts of both hookworm proteases were identical in sequence.

View larger version (60K): [in this window] [in a new window]   Figure 2. Hookworm cathepsins D preferentially cleave hemoglobin (Hb) from permissive definitive hosts. Ac-APR-1 (dog hookworm cathepsin D) generates more hydrolytic fragments from dog Hb than human Hb, whereas Na-APR-1 (human hookworm cathepsin D) generates more hydrolytic fragments from human Hb than dog Hb as determined by SDS-PAGE (A) and quantitation of free amino groups over time (B).

5. Mapping of the hemoglobin cleavage sites for hookworm cathepsins D
To further investigate the apparent species specificity of the hemoglobinolysis, cleavage sites were determined by Edman degradation and RP-HPLC separation of hydrolysates. Despite some conserved sites, many cleavage events were unique to just one or the other protease. Though residues that constituted the S1 pocket of both proteases were identical at the sequence level, the P1 Hb- chain substrate residues differed substantially between these proteases: Ac-APR-1 showed a preference for bulky, aromatic residues (Trp, Phe) whereas Na-APR-1 preferred other hydrophobic residues (Leu, Ala, and Val). In contrast, both proteases accommodated Ser or Ala in the P3 position of Hb, but Na-APR-1 was exclusive in its affinity for substrates with His at P3. Indeed, Ac-APR-1 failed to cleave any Hb sites with a P3 His, indicating that the S3 pocket in Na-APR-1 might be more accommodating for large, aromatic residues whereas the equivalent pocket in Ac-APR-1 is less accessible to residues with bulky side chains. Both proteases cleaved at sites with a P1' Pro. Mammalian aspartic proteases are not thought to cleave at such sites, although two other parasitic cathepsin D-like proteases have been observed to process peptide bonds with Pro at P1': HIV-1 protease and schistosome cathepsin D. By Edman sequencing, Ac-APR-1 favored Trp14-Asp-15 and Gly51-Ser52 in both Hb species whereas Na-APR-1 cut human and dog chains at different sites but showed a preference for P1 Asp residues in dog Hb. Ac-APR-1 and Na-APR-1 cut both species of Hb ß chains at different sites; both sites cleaved by Ac-APR-1 in human ß Hb had aromatic P1 residues, one of which represented an exopeptidic site (Val1-His2).

6. Host-specific cleavage of synthetic hemoglobin peptides
To observe whether the species-specific cleavage of Hb was evident using synthetic peptides representing known Hb cleavage sites for both enzymes, we synthesized the octapeptides LDKFLASV (where indicates the cleavage site) of the human Hb chain and the equivalent peptide in canine Hb (LDKFFAAV) and determined kinetic measurements. Ac-APR-1 cleaved the dog Hb peptide (LDKFFAAV) sixfold more efficiently than did Na-APR-1 (k_cat/K_m 59 M^-1.s^-1 vs. 10 M^-1.s^-1) whereas Na-APR-1 cleaved the human Hb peptide (LDKFLASV) fourfold more efficiently than did Ac-APR-1 (k_cat/K_m 41 M^-1.s^-1 vs. 10 M^-1.s^-1).

CONCLUSIONS

Every species of infectious organism exhibits a varying degree of host specificity, a nebulous characteristic that essentially defines the species range of its potential hosts. Once a parasite makes intimate contact with its prospective host, physiological compatibility becomes the most critical, but still a poorly understood, determinant of the success of parasitism and thus of host specificity. Among factors in the host that define physiological compatibility are the availability of appropriate and sufficient nutrients and suitable physical, chemical, and immunological conditions that allow the parasite to develop and reproduce.

Here we have shown that hookworm cathepsin D-like aspartic proteases are expressed in the gut and in excretory and amphidial glands of adult parasites where, at least in the former site, they likely cleave host Hb in the gut lumen. Moreover, each enzyme showed a marked proficiency for cleaving Hb from its true definitive host, providing molecular documentation of a contributing factor in host specificity of these parasites.

Whereas aspartic proteases play a key role in the digestive process, blood-feeding parasites are thought to use a proteolytic cascade to process Hb through an acidification process. Despite the apparent complexity, however, inactivation of just one class of enzyme in the cascade can significantly impair or completely disrupt the ability of parasites to feed on Hb. We predict that similar host-specific cleavage events occur between other mechanistic classes of parasite digestive proteases and food substrate proteins of their hosts.

Hookworms, like many other multicellular pathogens, negotiate a circuitous pathway through the host en route to their predilection site in the small intestine. When L3 infect a less favorable host, physical and molecular barriers limit the number of parasites that reach the small intestine. Nonetheless, those worms that do successfully establish within the host gut have to feed, mature, and reproduce. We suggest we have now identified a contributing factor to host compatibility: comparative proficiency for digesting proteinaceous food substrates. In this case, complementarity between different host Hb sequences and the Hb-degrading proteases of the parasites reflects the coevolution of host and parasite and contributes to determining in which hosts a given hookworm species can survive and reproduce. Indeed, Hb is probably not the only proteinaceous food source of hookworms, and we predict that host-specific cleavage will be seen with other physiological substrates of these and other parasite proteases.

Clearly, a panoply of ligand–receptor and enzyme–substrate interactions have been finely tuned to allow a parasite to develop in a given host. Such selective pressure likely manifests at most, if not all, points throughout the hookworm’s migratory route in the host. Nonetheless, some parasites do reach the gut of nonpermissive hosts and unsuccessfully attempt to feed. The complementarity we describe here between a putative digestive protease of a parasite and its substrate protein highlights some key issues: 1) molecular examples of compatibility between helminth parasites and their hosts are poorly documented and even less well understood; 2) subtle differences in fold result in distinct substrate cleavage preferences; 3) the coevolution of hookworms and their mammalian hosts has resulted in a finely tuned molecular relationship between aspartic proteases and Hb substrates that has allowed each hookworm to be intimately suited to survival in just one or few closely related host species. The phenomenon of matching a parasite protease to a specific host ligand clearly is not restricted to hookworms; direct parallels might be drawn with other blood-feeding parasites such as Plasmodium and schistosomes. The compatibility between digestive proteases of parasites and the nature of available food substrates might also affect the acceptable host range for non-blood-feeding pathogens. The fine molecular specificity between parasite protease and host substrate shown here suggests a very real prospect for the development of inhibitor therapies that specifically target parasite enzymes yet have no effect on host homologues.

View larger version (40K): [in this window] [in a new window]   Figure 3. Like other parasites in hostile environments, hookworms have adapted to survive in a limited number of host species. To avoid immune destruction, a given hookworm species has coevolved with its specific mammalian host. The result is a finely tuned molecular relationship (ligand–receptor interactions) permitting survival in a limited range of hosts, e.g., compatibility between cathepsin D digestive proteases and host hemoglobins.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0181fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.02-0181fje

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