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Digestive proteases of blood-feeding nematodes

Angela L. Williamson1, Paul J. Brindley2, David P. Knox3, Peter J. Hotez1 and Alex Loukas1,4
Department of Microbiology and Tropical Medicine, George Washington University Medical Center, 2300 Eye St NW, Washington DC, 20037, USA 2 Department of Tropical Medicine, Tulane University, 1430 Tulane Avenue SL-17, New Orleans, LA 70112, USA 3 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 OPZ, UK 4 Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Room CF. 42, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Queensland 4006, Australia
1

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major bloodfeeding nematodes hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus but also compares and contrasts these proteases with recent ndings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the ne specicity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets. Proteases encompass a broad class of hydrolytic enzymes that play essential roles in cellular, developmental and digestive processes, blood coagulation, inammation, wound healing and hormone processing. Parasite proteases, some of which are in the excretory secretory (ES) products, facilitate the invasion of host tissues, aid in the digestion of host proteins, help parasites evade the host immune response and mediate molting in parasitic nematodes. As a result, parasite proteases are considered to be potential targets for the development of novel immunotherapeutic, chemotherapeutic and serodiagnostic agents for the next generation of antiparasite interventions [1,2]. The diversity of the enzymes secreted by haematophagous parasites suggests that they are involved in a range of functions related to the host parasite relationship, including the catabolism of ingested host blood and tissues (Fig. 1). Blood-feeding parasites probably use proteases for digestion. Direct evidence for the intestinal lumen as the site of haemoglobin (Hb) digestion in blood-feeding
Corresponding author: Alex Loukas (alexL@qimr.edu.au).

helminths is lacking; however, most of the proteases described in this review are expressed in the intestine and many of them do not appear in the ES products, implying that they act locally in the intestine. Moreover, the acidic pH optima of many of these proteases is below that of host tissues, further implying that their sites of activity are in the acidic environment of the parasite intestine. Proteins are the most abundant nutrients of the blood and, therefore, the major digestive enzymes in haematophagous parasites and blood-sucking insects are thought to be proteases. The blocking of proteolysis of host Hb with protease inhibitors results in signicant antiparasitic and antipathology effects in schistosomiasis [3,4]. However, the identity and roles of the schistosome enzymes that participate in the degradation of host Hb have yet to be fully elucidated [5]. Until recently, not much was known about the proteases of nematodes and their roles in Hb proteolysis and nutrient acquisition. Over the past decade, complementary DNAs (cDNAs) encoding proteases of various mechanistic classes have been cloned from blood-feeding

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Fig. 1. Histological section through an adult Ancylostoma caninum attached to the intestinal mucosa of a dog. Note the unlysed red blood cells (arrows) containing haemoglobin in the buccal capsule. Scale 0.1 mm.

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nematodes that infect humans, ruminants and domestic companion animals. Biochemical analyses, expression of the recombinant proteases and localization of their anatomical sites of expression have shed light on the proteases used by haematophagous nematodes and their putative roles in a cascade of Hb digestion. Both hookworms and Haemonchus contortus are liberal blood-feeders and have an enormous impact on infected hosts. An estimated 730 million people are infected with the human hookworms Ancylostoma duodenale and/or Necator americanus, and many hookworm-infected people, particularly women and children, develop subclinical or clinical hookworm disease including anaemia and impaired physical and cognitive development [6]. Related hookworms infect the majority of dogs in the warmer latitudes, and some of these zoonotic species can also infect humans, often resulting in diverse pathological sequelae [7]. Haemonchus contortus is a highly pathogenic gastrointestinal nematode of sheep and goats, and blood loss caused by feeding of the adult parasite can result in severe anaemia, weight loss and death. Haemonchus contortus is a major constraint on agricultural production in many subtropical and tropical regions of the world, including Australasia, Southern Africa and South America. Proteases Proteases are classied according to the natural substrates upon which they act, historical ndings and/or known physiological function. Distinct classes of proteases are now assigned according to their respective catalytic mechanisms and are named after their active catalytic centre residues (aspartic, serine and cysteine proteases) or after their dependence on co-factors for activity (metalloproteases) [8]. A proteolytic cascade In mammals, gastric digestion of proteins derived from food involves a cascade of mechanistically distinct proteolytic enzymes including pepsin, trypsin and gastricsin. Similarly, the major food source of blood-feeding parasites, Hb, is degraded by a cascade of proteases in the intestine that cleave the intact Hb tetramer into successively smaller fragments; these are used to provide nutrients for growth and maturation. In Plasmodium, an ordered pathway of Hb digestion has been revealed and includes members of at least three different mechanistic classes of enzyme. It has been postulated that host Hb is attacked initially by aspartic proteases and degraded to smaller peptides by cysteine proteases and then metalloproteases. Finally, parasite exopeptidases complete the digestion to constituent amino acids. Recent evidence shows that homologous proteases line the intestinal brush borders of haematophagous nematodes, suggesting that Hb is degraded by a multi-protease cascade in the alimentary canals of these parasites (Fig. 2; Table 1). Aspartic proteases General acid-base catalysis is thought to mediate peptide hydrolysis by aspartic proteases, instead of the formation of covalent enzymesubstrate intermediates. Aspartic proteases belonging to clan AA (see http://merops.sanger.ac. uk/index.htm) have two catalytic aspartic acid residues at
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Haemolysis (e.g. undefined membrane proteins in A. caninum and H. contortus) Haemoglobin Aspartic proteases (e.g. hookworm APR-1, APR-2, H. contortus PEPI) Cysteine proteases (e.g. hookworm necpain,AcCP2, H. contortus cathepsins B) Metalloproteases (e.g. hookworm Ac-MEP-1, H. contortus MEPs) Exopeptidases? (e.g. dipeptidyl peptidase, aminopeptidase, cathepsin B, H11)

Free amino acids or small peptides absorbed across the gut lumen
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Fig. 2. The putative proteolytic cascade responsible for haemoglobin degradation in the intestines of blood-feeding nematodes.

their active site clefts. Aspartic proteases are considered to be the most conserved group of the four classes of proteases (consisting of aspartic, cysteine, serine and metalloproteases) and the most well known aspartic proteases belong to the A1 family of enzymes typied by the mammalian gastric enzymes pepsin and gastricsin. The A1 family also includes the lysosomal processing enzyme cathepsin D, and digestive enzymes from blood-feeding parasites including Plasmodium falciparum plasmepsins [9] and schistosome cathepsins D [5]. In both of these parasites, aspartic proteases are thought to be responsible for the rst step in degradation of host Hb and, in Plasmodium at least, this step can be inhibited by addition of the aspartic protease inhibitor, pepstatin [10]. However, there is controversy over the roles of Plasmodium aspartic and cysteine proteases and where each enzyme ts into the cascade of Hb proteolysis in vivo. Schistosoma cathepsin D is expressed in the gastrodermis of adult parasites, is overexpressed in female worms (presumably to accommodate their higher metabolic requirements for egg production) and digests Hb at acidic pH in vitro [5]. Extracts of the human hookworm, N. americanus, digest Hb and brinogen, and activity is inhibited by the aspartic protease inhibitor, pepstatin A [11]. Several cDNAs encoding aspartic proteases have been identied in nematodes, including cathepsin D-like enzymes from canine and human hookworms [1214]. The cathepsin D-like protease from the canine hookworm Ancylostoma caninum (Ac-APR-1) and the orthologous protease from N. americanus (Na-APR-1) were expressed in the intestinal microvillar border of blood-feeding adult hookworms [13] (Fig. 3). Both proteases cleaved Hb at numerous distinct sites, showed different substrate preferences, and cleaved Hb at the hinge region [13], a step that would facilitate unravelling of the Hb tetramer and facilitate its proteolysis by other aspartic proteases and degradative enzymes. A second family of aspartic proteases has been identied from the intestines of blood-feeding strongyle

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Table 1. Proteases of blood-feeding nematodes with known/putative roles in digesting the bloodmeal
Enzyme Na-APR-1b Ac-APR-1b Na-APR-2 AcCP-1 AcCP-2 Ac-MEP-1 AC-1 AC-5 MEP1 MEP 4 HMCP-1, HMCP -4, HMCP -6 Pep1 H11
a b c

Class Aspartic cathepsin D

Species Necator americanus Ancylostoma caninum N. americanus A. caninum A. caninum A. caninum Haemonchus contortus H. contortus H. contortus H. contortus H. contortus

Knowna/Putative Function Digestion of Hb, serum and connective tissue proteinsa

Anatomical location Gut, cephalic and excretory glands

Refs [13]

Aspartic nemepsin Cysteine cathepsin B Cysteine cathepsin B Metallo-neprilysin Cysteine cathepsin B Metallo-neprilysin Cysteine cathepsin B Aspartic nemepsin Amino-peptidase

Digestion of Hb and serum proteinsa Tissue degradation Digestion of Hba,c Digestion of Hb Digestion of blood Blood-feeding Blood-feeding Digestion of Hb Digestion of bloodmeal

Gut, cephalic and excretory glands Oesophagus, cephalic and excretory glands Gut microvillic Gut microvilli Intestine Gut microvilli Gut microvilli Gut microvilli Gut microvilli

[12] [33] [33] [41] [27,28,34,63] [43] -d [29,35] [15] [64]

Recombinant protein shown to cleave host-derived substrates are listed here. Abbreviation: Hb, heamoglobin. Known to also be expressed in 3rd stage larvae. A. Loukas and A.L. Williamson, unpublished. d GenBank accession numbers for H. contortus MEP-2, MEP-3 and MEP-4 are AF080117, AF080172, AF132519, respectively.

nematodes, the nemepsins [12]. These aspartic proteases more closely resemble mammalian pepsin than cathepsin D, and this group includes Na-APR-2 from adult N. americanus [12], Haemonchus Pep1 [15] and a similar protease from infective larvae of Strongyloides stercoralis [16]. Haemonchus contortus Pep1 is a component of the highly host-protective integral membrane protein complex H-gal-GP, isolated from the intestinal brush border of adult worms [17]. The native enzyme has a high afnity for Hb as a substrate and is expressed almost exclusively in the parasitic, blood-feeding stages of H. contortus, implying a function in digestion of the host bloodmeal [18]. Na-APR-2 was localized primarily to the intestinal microvillar surface in adult worms, and the recombinant enzyme readily cleaved human Hb and other serum proteins [12]. Differences in the pH proles (and cleavage sites within Hb) between Na-APR-1 and Na-APR-2 also suggests an ordered pathway for Hb degradation in the hookworm intestine; perhaps Na-APR-1 makes the initial cleavage (at the Hb hinge region, among other sites [13]) and, after unravelling of the Hb molecule Na-APR-2, is responsible for additional degradation at acidic pH. Based on this evidence, it is proposed that at least one of the nematode aspartic proteases catalyses a dened scission, thereby exposing other sites to digestion by a cascade of proteases. This concept is further supported by the numerous differences between the catalytic residues (and subsequent Hb cleavage sites) of Na-APR-2 and Na-APR-1 [12,13]. This ordered pathway of Hb degradation is thought to occur in schistosomes [19] and has been shown to take place in P. falciparum, where an aspartic protease located in the acidic digestive vacuole cleaves the a-chain of Hb at the hinge region, after which other proteolytic enzymes digest the Hb fragments into smaller peptides and dipeptides [20]. Cysteine proteases The most widely reported class of protease from parasitic nematodes is the cysteine proteases belonging to clan CA. These enzymes are secreted by larval and adult parasites,
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and are probably involved in tissue penetration, feeding and defence against effector mechanisms of the host immune response [1]. Cysteine proteases possess an essential cysteine residue that forms a covalent intermediate complex with substrates. A major cysteine protease grouping is the papain superfamily (family C1), which has a catalytic triad comprising Cys, His and Asn residues. Cathepsin B [21] and cathepsin L-like [22] proteases of adult Schistosoma mansoni are expressed in the gastrodermis [23]; moreover, cathepsin B degrades Hb in vitro. In addition, cysteine protease inhibitors have been found to interrupt Hb digestion of S. mansoni schistosomula [3]. Plasmodium falciparum expresses at least three cysteine proteases, the falcipains, which are thought to function in Hb proteolysis in the digestive vacuole [24 26]. Falcipain-2 can initiate cleavage of native Hb in the P. falciparum food vacuole and rapidly hydrolyses subsequent Hb fragments [25]. Falcipain-3 undergoes efcient

(a)

(b)

(c)

u i

i c r

c r

i
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Fig. 3. Aspartic proteases are expressed in the intestines of adult, blood-feeding hookworms. Fluorescence microscopic analysis showing immunolocalization of the aspartic proteases Ac-APR-1 (a) and Na-APR-2 (b) to the intestinal brush border of longitudinal sections of adult hookworms. (c) A section of an adult Ancylostoma caninum probed with normal mouse serum. Abbreviations: c, cuticle; i, intestinal lumen; r, reproductive organs; u, uterus containing eggs. Fig. 3b reproduced, with permission, from Ref. [12].

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processing to an active form at acidic pH, and is particularly suited for the hydrolysis of native Hb in the acidic food vacuole [26]. Papain-like enzymes are common in nematodes, and a family of cathepsins B has been cloned from H. contortus [27 29], some of which are expressed in the intestine of adult worms [29,30]. Cysteine proteases in H. contortus extracts are capable of digesting Hb, brinogen, collagen and immunoglobulin G [31]. Similarly, cysteine proteases in ES products of N. americanus exhibit lytic activity against Hb, brinogen [11] and antibodies [32]. At least two cathepsin B-encoding messenger RNAs (mRNAs) have been cloned from adult A. caninum [33]. AcCP1 is detected in ES products of adult parasites and is expressed in the secretory glands and oesophagus [33], but not in the intestine (A. Williamson and A. Loukas, unpublished), suggesting that it does not degrade Hb in vivo. Preliminary ndings show that AcCP2 is produced in the intestine, and that the recombinant protein is haemoglobinolytic in vitro; native Hb was found to be digested, but optimal activity was seen after initial cleavage of Hb by the aspartic protease Ac-APR-1 (A. Loukas and A. Williamson, unpublished). Cathepsin B-like protease genes constitute large multigene families in parasitic and non-parasitic nematodes. One of the best-characterized families of cathepsins B is that from intestinal tissues of adult H. contortus [30,34,35], all of which play potential roles in the digestion of Hb and other nutrients. Recently, a specic sequence alignment program was used to identify a haemoglobinase motif that was present, with one exception, only in the cathepsin B-like proteases of helminth blood-feeders [36]. The absence/infancy of gene knockout and RNA silencing techniques for parasitic helminths has made it difcult unequivocally to attribute haemoglobinolytic roles for these proteases. However, the tissue localization of cysteine proteases, activity of recombinant enzymes and, in some cases, overexpression in female worms strongly suggests a role in degradation of substrates for nutrient uptake. Unlike haematophagous trematodes, cathepsins L are less abundant in nematodes [1,37] and appear to be involved in embryogenesis and molting [38,39], rather than digestion of nutrients. Metalloendopeptidases Metalloproteases, which can be endoproteases or exoproteases, coordinate essential metal ions (typically zinc) at their active site centres. This enables the polarization of the target scissile peptide bond before nucleophilic attack and subsequent cleavage. Metalloendopeptidase activity was rst described from the ES products of adult A. caninum in 1983 [40]. This protease was shown to cleave brinogen and plasminogen, and was thought to inhibit clot formation at the site of attachment. Jones and Hotez have recently described a cDNA, Ac-mep-1, which probably encodes the same protease [41]. Ac-MEP-1 is a neprilysin-like (clan MA, family M13), zinc-dependent enzyme expressed in the intestinal lumen of blood-feeding adult parasites (Fig. 4). Falcilysin is a metalloendopeptidase, expressed in the digestive vacuole of P. falciparum, that digests Hb fragments
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(a)

(b)

re in in

re ex

(c)

(d)

in

E74 H77 H73

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Fig. 4. Metalloproteases are expressed in the intestines of adult, blood-feeding hookworms. Immunolocalization of Ac-MEP-1 to the intestinal brush border of a longitudinal section (a) and a transverse section (b) of adult Ancylostoma caninum hookworms. (c) The absence of reactivity is indicated here when sections were probed with rabbit serum before immunization with recombinant MEP-1. (d) Molecular model of the C-terminal domain of Ac-MEP-1 based on the crystal structure of human neprilysin. Side chains are shown for the active site histidine and glutamate residues that coordinate the catalytic zinc ion. The model was constructed using Swiss Model and drawn with Swiss Pdb Viewer. Abbreviations: ex, excretory glands; in, intestine; re, reproductive organs.

only after initial digestion with the plasmepsins and falcipains [42]. Ac-MEP-1, similar to falcilysin, contains the hallmark features of metallopeptidases including the catalytic His and Glu residues, and, given its anatomical location, it is predicted that it is involved in the enzyme cascade of Hb hydrolysis in the hookworm gastrointestinal tract. Work is ongoing to investigate whether Ac-MEP-1 is involved in Hb digestion, and, if so, to determine the point at which it acts in the haemoglobinolytic cascade. Preliminary data suggests that Ac-MEP-1 does not degrade intact Hb, but does further digest Hb that has previously been cleaved by other mechanistic classes of hookworm proteases (A. Williamson and A. Loukas, unpublished). Ac-MEP-1 exhibits signicant similarity to a developmentally regulated metalloendopeptidase, MEP1, from H. contortus [43]. MEP1 was identied as part of the protease-rich, H-gal-GP complex, which is expressed exclusively on the microvillar surface of the intestinal cells and provides signicant protection against challenge infections in sheep. The complex also contains three additional metalloproteases, MEP-2, MEP-3 and MEP-4 (see Ref. [18]). In addition to a haemoglobinolytic role, metalloproteases of adult hookworms appear to play a role in immune evasion by cleaving eotaxin, thereby inhibiting recruitment of eosinophils to the site of attachment [44]. Exopeptidases Considerably less is known on the roles of exopeptidases (proteases that cleave residues from the termini of peptide

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substrates) in Hb degradation by parasites. Dipeptidyl peptidases remove dipeptides from the N- or C-termini of peptide substrates, whereas amino- or carboxy-peptidases remove single, terminal amino acids. Exopeptidases belong to numerous mechanistic classes, but are often metalloproteases. Some cathepsin B cysteine proteases display both endopeptidases and exopeptidase activity [45], and this class of enzyme is highly expressed in blood-feeding nematodes. Aminopeptidase activity was recently described in P. falciparum trophozoites, suggesting that this activity was responsible for generating free amino acids from Hb peptides after prior digestion with plasmepsins, falcipains and falcilysin [46]. Moreover, inhibition of Plasmodium aminopeptidases with specic inhibitors resulted in potent blocking of parasite growth in vitro [47]. H11, a type II membrane glycoprotein with aminopeptidase A and M activities, is expressed exclusively in the intestinal microvilli of H. contortus; a role has not been attributed to this peptidase, although it could be involved in the terminal degradation of Hb peptides into free amino acids before uptake across the intestine, thus representing a promising vaccine antigen against haemonchosis [48,49]. Aminopeptidase activity is detected in the intestine of N. americanus [50], and a cDNA encoding an aminopeptidase from A. caninum has been cloned (T. Don and A. Loukas, unpublished). However, an H11 orthologue has not been recovered so far from hookworms. Targets for therapy Anthelmintic resistance in nematode populations and concerns about the effects of drug residues on consumer health and the environment have focused attention on developing effective anti-nematode vaccines [49]. The use of hidden or cryptic antigens (i.e. those not recognized by the host during the course of a natural infection) as vaccines against blood-feeding helminths warrants attention. Gut-derived molecules have been effectively employed as vaccine antigens against ectoparasites of domestic animals [51,52], and protection against haemonchosis in sheep by vaccinating with H11 and the H-gal-GP complex implies that this strategy is also efcacious against helminths [53]. Much effort has been directed towards the development of a vaccine against Haemonchus. The most promising candidates are the gut membrane antigens H11 and H-gal-GP [49,54]. H-gal-GP is a co-migrating, multiprotease complex comprising cysteine, aspartic and metalloproteases. Immunization of sheep with H-gal-GP induces protective and, importantly, neutralizing antibodies that can be passively transferred in serum [55]. Most of these perceived protective proteases are located on the surface of the parasites gut and are hidden from the immune system during natural infection. However, they are accessible to host antibodies ingested by the parasite. Thus, gut antigen-based vaccination takes advantage of Haemonchus being an obligate blood-feeder [29]. Vaccination trials with individual components of the complex in recombinant form are eagerly awaited and, if successful, will be the rst recombinant protein vaccine available against a nematode parasite.
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A major hookworm vaccine initiative is also underway at The George Washington University (Washington DC, USA). Preliminary trials have shown a modest reduction in the adult hookworm burden from the small intestine of dogs immunized with recombinant Ac-APR-1 expressed in baculovirus [56]. Of interest, an associated increase was noted in the number of worms recovered from the large intestine, a site not normally occupied by hookworms, suggesting that antibodies raised to Ac-APR-1 forced adult hookworms to exit the small intestine and enter into the colon. One possible explanation for this observed phenomenon is that adult hookworms are less able to feed on host blood that is spiked with anti-Ac-APR-1 antibodies. Moreover, female worms were more likely selectively to enter the colon, and because female hookworms are more voracious blood feeders than males, they would be exposed to more anti-APR-1 immunoglobulin [56]. Antibodies to hookworm aspartic proteases also interfere with larval migration; by incubating N. americanus L3 with mouse antibodies to Na-APR-2, 50% of L3 did not penetrate hamster skin in vitro when compared with controls [12] and, moreover, antibodies to aspartic protease inhibited hydrolysis of a synthetic peptide. Proteases that line the intestinal lumen of blood-feeding helminths are viable candidate antigens for controlling these parasites. The importance of hookworms in humans and Haemonchus in the agricultural industry render it imperative that vaccine projects aimed at controlling these nematodes are supported and continue to receive the appropriate funding. Proteases of infectious agents are the focus of inhibitor drug design programmes for viruses (HIV), pathogenic fungi and parasitic protozoa (for reviews, see Refs [57,58]). However, the development of peptidomimetic protease inhibitors to combat helminth infections is less relevant for numerous reasons. The people who have the greatest need for protection against helminth parasites are those who are least likely to afford expensive drugs. Moreover, helminth infection is rapidly reacquired after treatment with anthelmintics, and existing drugs (e.g. albendazole, praziquantel) are cheap and effective. Concluding remarks An emerging body of evidence suggests that not only host blood, but also Hb in particular is an important driving force in the evolution of invertebrate parasite enzymes. It is impressive that four phylogenetically unrelated parasite genera, namely Plasmodium spp., Schistosoma spp., Haemonchus spp. and Ancylostoma/Necator spp. independently evolved to select Hb as an important nutrient source of amino acids. Moreover, host Hb was targeted by relying on the concerted action of similar cascades of enzymes. The observation suggests that the study of the sequence and structural organization of the active site of parasite-derived haemoglobinases provide a basis for which host parasite specicities will be elucidated at the molecular level (Box 1). In the coming decade, the chemical biology of the haemoglobinase active site is likely to prove to be a gold mine for developing new targets for drug and vaccine development.

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Box 1. A contribution to host specicity and host species range

Brinkworth et al. hypothesized that the specicity and afnity of haemoglobin (Hb)-degrading enzymes of blood-feeding parasites have evolved in response to the mutations in Hbs of their mammalian hosts, and that a key contributor to the evolution of the host species range of hookworms and other haematophagous parasites is an implication of the nely tuned interplay between the Hb-degrading proteases and their natural substrates, mammalian Hbs [59]. The hypothesis predicts that a Hb-degrading enzyme from parasite species A will process Hb from host species X more efciently than will the orthologous enzyme from blood-feeding parasite species B, where A and B are closely related phylogenetically, but where host species X is a permissive host for parasite species A but not for B. In a similar fashion, the reverse situation is also expected where host species Y is a permissive host for parasite species B, but not A. Underpinning the hypothesis is a small number of discrete, yet inuential, mutations in the molecular structures of the orthologous enzymes from parasite species A and B, and also in the peptide sequence of Hbs from host species X when compared with Y. For example, the hypothesis would predict that the cathepsin D aspartic protease of Schistosoma japonicum involved in Hb degradation in the schistosome gut [5] would digest bovine Hb more efciently than would the cathepsin D from Schistosoma mansoni because cattle are permissive hosts for S. japonicum but not for S. mansoni. Recently, studies on orthologous hookworm aspartic proteases supports the Brinkworth et al. hypothesis [59]. Ac-APR-1 and Na-APR-1 are cathepsin D-like proteases expressed in the gut of adult Ancylostoma caninum and Necator americanus, respectively [13]. The denitive host of A. caninum is the dog, and although larvae can infect humans, worms do not attain sexual maturity in humans, despite sometimes reaching the intestine and attempting to blood-feed [60]. Conversely, humans are the denitive host for N. americanus and, although larvae can infect dogs, they do not mature sexually in that host [61]. Both Ac-APR-1 and Na-APR-1 digested Hb from their permissive hosts between twofold and sixfold more efciently than Hb from closely related non-permissive hosts, despite the two proteases having identical residues lining their active site clefts (i.e. Na-APR-1 was more efcient at cleaving human Hb than dog Hb and vice versa for Ac-APR-1 [13]). A similar, host-specic phenomenon was observed with the nemepsin Na-APR-2 in relation to canine and human Hb and serum proteins [12] (Fig. I). As such, the elevated species-specic afnity of hookworm aspartic proteases for human or canine Hb provides molecular evidence for the co-evolution of parasite enzymes and their host protein substrate. Furthermore, with respect to the evolution of host species range of parasites, the complementarity (or precision of t) between the key catalytic residues of proteases and their target Hbs can be generalized to most, if not all, parasite enzyme host molecular substrate processes and, indeed, to many receptor ligand interactions

(a) kDa 29 21 30 5

Incubation time (mins) 15 30 5 15 30

(b) 0.6 Free amino groups (nM) 0.5 0.4 0.3 0.2 0.1 0 0

Buffer only

Dog Hb

Human Hb

10

15

20

25

30

35

Time (mins)
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Fig. I. Na-APR-2 aspartic protease from Necator americanus preferentially cleaves haemoglobin from its permissive human host. Recombinant Na-APR-2 generated more hydrolytic fragments from human haemoglobin (Hb) than from dog Hb after 15 and 30 min incubation (a). Quantication of free amino groups from dog (red diamonds) and human (green squares) Hb released after hydrolysis by recombinant Na-APR-2 at different time points using ninhydrin (b). Results are expressed as linear regressions of substrate cleavage over time. When the inhibitor pepstatin A was included in reactions, there was no detectable increase in free amino groups above background levels. Fig. I reproduced, with permission, from Ref. [12].

[59]. Moreover, we have observed evidence for this hypothesis with serum proteins, including brinogen and serum albumin [62]. Once ecological and ethological requirements are met, a whole spectrum of molecular interactions is likely to govern the host species range of a given parasite. One suggestion is that interactions between a parasite protease and its host substrate are just one of what is clearly a panoply of molecular relationships that govern parasitism.

Acknowledgements
The work described here was supported by grants from the National Health and Medical Research Council of Australia, Australian Research Council, National Institutes of Health (AI-32726) and The Human Hookworm Vaccine Initiative of the Bill and Melinda Gates Foundation. We thank Maria Elena Bottazzi, Mina Owlia and Mohamad Lazkani for technical assistance with immunolocalization studies.

References
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6 Hotez, P.J. et al. (1995) Molecular pathobiology of hookworm infection. Infect. Agents Dis. 4, 71 75 7 Loukas, A. and Prociv, P. (2001) Immune responses in hookworm infections. Clin. Microbiol. Rev. 14, 689 703 8 Berger, A. and Schechter, I. (1970) Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos. Trans. R. Soc. Lond. B Biol. Sci. 257, 249 264 9 Banerjee, R. et al. (2002) Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proc. Natl. Acad. Sci. U. S. A. 99, 990 995 10 Francis, S.E. et al. (1997) Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu. Rev. Microbiol. 51, 97 123 11 Brown, A. et al. (1995) An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus. Parasitology 110, 555 563 12 Williamson, A.L. et al. (2003) Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specic fashion. J. Infect. Dis. 187, 484 494 13 Williamson, A.L. et al. (2002) Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its contribution to host-specicity. FASEB J. 16, 1458 1460 14 Harrop, S.A. et al. (1996) Acasp, a gene encoding a cathepsin D-like

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