Download Proteolytic Degradation of Hemoglobin in the Intestine of the Human

MAJOR ARTICLE
Proteolytic Degradation of Hemoglobin in the
Intestine of the Human Hookworm
Necator americanus
Najju Ranjit,1,3 Bin Zhan,5 Brett Hamilton,2 Deborah Stenzel,3 Jonathan Lowther,4 Mark Pearson,1 Jeffrey Gorman,2
Peter Hotez,5 and Alex Loukas1
1
Helminth Biology Laboratory and 2Protein Discovery Centre, Division of Infectious Diseases, Queensland Institute of Medical Research, and
School of Life Sciences, Queensland University of Technology, Brisbane, and 4Institute for the Biotechnology of Infectious Diseases, University of
Technology, Sydney, Australia; 5Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, DC
3
Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme
cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in
catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in
baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult
N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not
cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered
process of hemoglobinolysis. Seventy-four cleavage sites within Hb ␣- and ␤-chains were characterized after
digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb;
noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for NaAPR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a
network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which
some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection.
More than 700 million persons in developing countries
are infected with the human hookworms Necator americanus and Ancylostoma duodenale [1]. The main pathology associated with hookworm infection stems from intestinal blood loss, which can lead to iron deficiency
anemia in heavy infections. Hookworms are voracious
blood feeders, causing an estimated loss of up to 9 mL of
blood per day in heavily infected subjects [2]. Although
Received 17 June 2008; accepted 26 September 2008; electronically published
3 February 2009.
Potential conflicts of interest: none reported.
Financial support: National Health and Medical Research Council (NHMRC),
Australia (Senior Research Fellowship 442906 and program grant 496600 to A.L.
and project grant 389813 to A.L. and P.H.); Sabin Vaccine Institute and Bill and
Melinda Gates Foundation (grant to P.H. and A.L.); Australian Research Council/
NHMRC Research Network for Parasitology (travel award to N.R.); Queensland
University of Technology (QUTBLU award to N.R.).
Reprints or correspondence: Dr. Alex Loukas, Helminth Biology Laboratory, Div.
of Infectious Diseases, Queensland Institute of Medical Research, 300 Herston
Rd., QLD 4006, Australia ([email protected]).
The Journal of Infectious Diseases 2009; 199:904 –12
© 2009 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2009/19906-0019$15.00
DOI: 10.1086/597048
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anthelmintic chemotherapies are generally effective at
removing adult worms from the gut, reinfection occurs
rapidly, leading to concerns about the long-term viability of such practices [3]. Unlike other human helminthiases, clear-cut protective immunity does not occur in
most individuals exposed to hookworms [4]. This has
culminated in efforts to develop a prophylactic vaccine
against hookworm infection as a sustainable solution for
long-term control [5, 6].
Hematophagous parasites depend on the catabolism of
hemoglobin (Hb) for survival. Schistosome blood flukes
and the malarial parasite Plasmodium falciparum digest Hb
by means of cooperative proteolytic cascades. In schistosomes, the Hb tetramer is cleaved initially by aspartic proteases [7, 8] and to a lesser degree by papainlike cysteine
proteases; the cysteine proteases are thought to exert most
of their activity downstream of the aspartic protease by further catabolizing globin fragments [8]. On the other hand,
treatment of schistosomes with double-stranded RNA for
the gastrodermal cathepsin B cysteine protease SmCB1 interrupted the ability of worms to digest serum albumin [8],
suggesting that the order in which the different proteases act differs
for each distinct protein substrate.
There are degrees of redundancy in hemoglobinolysis in
blood-feeding parasites. The roles and hierarchical positions of
various Schistosoma and Plasmodium proteases in the hemoglobinolytic processes have been debated, but the application of
gene knockout (Plasmodium) and gene silencing (Schistosoma)
technologies have resolved some of these issues. For example,
hemoglobinase knockout and double-knockout P. falciparum
have been used to show that Hb degradation is partially redundant and relies on dual protease families with overlapping functions [9]. For Schistosoma mansoni, silencing of the mRNAs for
different hemoglobinases has shown an ordered and somewhat
less redundant pathway of Hb degradation [8, 10].
Gene silencing techniques are available for schistosomes and
plasmodia but have not yet been successfully used with hookworms; indeed, some have questioned whether parasitic nematodes have the required enzymes to process double-stranded
RNA [11, 12]. We have therefore taken a different approach to
explore hemoglobinolysis in blood-feeding hookworms. We
previously identified proteases involved in the digestion of Hb in
the canine hookworm Ancylostoma caninum and used recombinant enzymes to reveal a semiordered cascade of proteolysis in
vitro [13]. Two of the enzymes involved in this cascade were then
shown to confer protection as recombinant proteins against
hookworm infection in dogs [14, 15].
To characterize the hemoglobinolysis pathways used by human hookworms, we isolated intestinal tissue from adult N.
americanus and identified the most highly expressed mRNAs
[16], including homologues of some of the A. caninum hemoglobinases. Here, we describe the recombinant expression of intestinal aspartic proteases, cysteine proteases, and metalloproteases from the gut of N. americanus. We provide evidence for an
ordered cascade of hemoglobinolysis whereby aspartic proteases
(Na-APR-1), cysteine proteases (Na-CP-3), and metalloproteases (Na-MEP-1), in that order, digest Hb and subsequent
globin fragments, and provide a map of the cleavage sites. Our
findings shed light on the molecular mechanisms used by hookworms to obtain nutrition from human blood and further support the pursuit of hemoglobinases as targets for the development of new drugs and vaccines against blood-feeding parasites
of humans and livestock.
METHODS
cDNA cloning. Na-apr-1 was reported earlier [17] and assigned GenBank accession number CAC00543. Na-cp-3 was reported recently [18] and assigned GenBank accession number
ABL85237. The Na-mep-1 cDNA was initially identified as an
expressed sequence tag (EST; BG467946) by using Ac-mep-1
as the query in a BLAST search of the Parasite Genomes
Database (http://www.ebi.ac.uk/blast2/parasites.html). Gene-
specific primers were designed for 5' and 3' rapid amplification
of cDNA ends (RACE) using adult N. americanus RNA (GeneRacer; Invitrogen), as described elsewhere [19]. The full-length
cDNA sequence of Na-mep-1 was obtained by creating a consensus consisting of the 5' and 3' RACE products and the BG467946
EST sequence. The cDNA sequence of Na-mep-1 has been deposited in GenBank with accession number EU523699.
Protein expression and purification. Na-APR-1 was expressed in the yeast Pichia pastoris, as described for Ac-APR-1
[14]. Expression of Na-CP-3 in P. pastoris was reported by us
recently [20]. Attempts to express Na-MEP-1 in yeast were unsuccessful (data not shown), so we expressed it in baculovirus.
The entire open reading frame (ORF) minus the predicted signal
peptide was expressed as a secreted protein with an N-terminal
melittin signal peptide and a C-terminal 6⫻His tag, and recombinant protein was expressed and purified by nickel–nitrilotriacetic acid (Ni-NTA) chromatography using methods described
elsewhere [21]. Protein concentration was determined using a
bicinchoninic acid assay kit (Pierce). Na-MEP-1 was also expressed in Escherichia coli to obtain sufficient protein for antibody production. The entire ORF minus the signal peptide was
ligated into pBAD/Thio-TOPO (Invitrogen), and E. coli strain
BL21-DE2 (Invitrogen) was transformed with ligation product.
Subsequent induction of recombinant protein was performed as
described in the manufacturer’s instructions (Invitrogen). The
cell pellet was collected 4 – 6 h after induction by centrifugation
at 25,000 g for 20 min and resuspended in 20 mmol/L NaH2PO4
and 500 mmol/L NaCl. The cells were subjected to 2 cycles of
disruption in a French press (SLM Instruments). Lysates were
centrifuged at 20,000 g for 15 min, supernatant was retained, and
pellets were incubated in 6 mol/L guanidine HCl, 50 mmol/L
Tris, and 10 mmol/L imidazole for 2 h at room temperature (RT)
and then centrifuged again. Solubilized pellet was purified under
denaturing conditions on a Ni-NTA agarose column, in accordance with the manufacturer’s instructions (Novagen). Purified
protein was electrophoresed through SDS-PAGE gels to assess
purity, and the identity of the purified protein was confirmed by
Western blot analysis using an anti-6⫻His antibody (Invitrogen).
Catalytic activity of recombinant hemoglobinases. Recombinant Na-MEP-1, expressed in both E. coli and baculovirus, was
electrophoresed on precast gelatin zymogen gels (Invitrogen), in
accordance with the manufacturer’s instructions with slight
modifications: 10 mmol/L CaCl2 or 10 mmol/L ZnCl2 were
added to the zymogram developing buffer, and the pH of the
developing buffer was titrated in single pH unit increments from
3 to 7. Excretory/secretory proteins of adult A. caninum were
used as a positive control for the zymogram, and 1,10phenanthroline (final concentration, 10 ␮mol/L) was included
in some reactions to inhibit metalloprotease activity. Zymograms were incubated overnight at 37°C and stained with Coomassie brilliant blue followed by destaining. Catalytic activity of
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Table 1.
Orthologues of Necator americanus proteases involved in hemoglobin degradation by other hematophagous parasites.
Protease type
N. americanus
Ancylostoma caninum
Aspartic
Cysteine
Na-APR-1 (CAC00543.1); family A1
Na-CP-3 (ABL85237); family C1A
Ac-APR-1 (AAB06575.1)
Not determineda
Metalloproteases
Na-MEP-1 (ACB13196); family M13
Ac-MEP-1 (AAK67057)
NOTE.
Plasmodium falciparum
Schistosoma mansoni
Plasmepsin 1 (AAW71448)
catD (AAB63442)
Falcipain 2 (AAF97809)
Falcilysin (AAF06062)
SmCB1 (CAC85211)
Noneb
Orthology is determined by sequence identity and hemoglobinolytic activity where known. GenBank accession nos. are in parentheses.
a
Ac-CP-2 is a hemoglobinase from A. caninum that digests intact hemoglobin tetramer, whereas Na-CP-3 digests only globin peptides; these proteases are
therefore not considered orthologous.
b
The complete genome sequence is available, but an obvious orthologue has not been identified.
Na-APR-1 toward the fluorogenic substrate 7-methoxycoumarin4-acetyl-GKPILF2FRLK(DNP)-D-Arg-amide (MoCAc-GKPILFFRLK; Sigma) (2 represents the site where the peptide is cleaved)
[22] was assayed in a FLUOstar Optima microplate reader (BMG
Labtech) at 37°C. Rates of hydrolysis were recorded by monitoring
the increase in fluorescence at excitation and emission wavelengths
of 330 and 390 nm, respectively. The optimum pH for enzyme (1.0
␮g) activity was determined by assaying in 50 mmol/L sodium acetate buffers at half-unit pH increments from 2 to 6. For determining kinetic parameters, the final substrate concentration was 1.0
␮mol/L, and the final volume of each reaction was 100 ␮L. Enzyme
(0.105 nmol/L) efficiency was assessed at pH 3.5 by measuring initial rates over a range of substrate concentrations (0.2–25 ␮mol/L).
The catalytic constants kcat, Km, and kcat/Km were determined from
the resulting Michaelis-Menten plot. The catalytic activity of NaCP-3 against Z-Phe-Arg-aminomethylcoumarin has been described by us elsewhere [20].
Antibody production and immunolocalization. All animal
work was conducted with the approval of the Queensland Institute of Medical Research Animal Ethics Committee. In female
BALB/c mice, antibodies were raised against Na-MEP-1 expressed in E. coli, using methods described elsewhere [20]. Production of anti-Na-CP-3 serum has been reported elsewhere
[20]. A rabbit antiserum to recombinant Na-APR-1 was produced as described elsewhere for Ac-APR-1 [14]. Immunolocalization with fixed adult N. americanus worms was performed as
described elsewhere [23], with minor modifications. Briefly, sections were rehydrated in PBS and blocked with 5% fetal calf
serum/PBS for 1 h at RT. Sections were incubated with antisera
(dilutions of 1:100 to 1:1000) in 1% bovine serum albumin
(BSA) for 1 h at RT. Anti–mouse IgG Alexa Fluor 555 (Invitrogen) was used at a dilution of 1:500 in 1% BSA/PBS/0.05%
Tween 20 (PBST); sections were incubated for 1 h at RT. Sections
were washed in PBST and air dried, and cover slips were applied
with mounting medium (Sigma). Slides were viewed and photographed using a Leica IM 100 fluorescent microscope.
Proteolysis of Hb by Na-APR-1, Na-CP-3, and Na-MEP-1.
Human Hb was prepared as described elsewhere [7]. Hb was
incubated with individual recombinant proteases at molar ratios
of 72:1 Hb to Na-APR-1, 5:1 and 50:1 Hb to Na-CP-3, and 14:1
and 140:1 Hb to Na-MEP-1, in 0.1 mol/L sodium acetate, 0.5
mmol/L dithiothreitol, and 10 mmol/L CaCl2 at half-unit pH
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increments from 3 to 7 for up to 18 h at 37°C. Hb was also
incubated with various combinations of the recombinant proteases at 37°C for 6 –18 h. Each sample was electrophoresed on a
15% SDS-PAGE gel under native conditions to observe the
amount of intact Hb that remained.
Analysis of Hb hydrolysates by liquid chromatography
(LC)–mass spectrometry (MS) and tandem MS (MS/MS). Hb
samples that had been incubated with various combinations of
hookworm proteases were subjected to reverse-phase highperformance LC, and the peptides were identified by LC-MS.
LC-MS analysis was performed using an UltiMate 3000 Nano LC
system (Dionex) with a capillary LC flow splitter and a variablewavelength UV-visible detector scanning at 214 nm, coupled to
a quadrupole time-of-flight mass spectrometer (MicrOTOF-Q;
Bruker Daltonics) operated with a low-flow electrospray needle.
The column used was a Vydac monomeric C18 column (300 Å,
3 ␮m, 150 ␮m ⫻ 150 mm; flow rate, 1.2 ␮L/min). The mobilephase buffers used for the gradient program were water with
0.1% formic acid (buffer A) and acetonitrile-water (4:1) with
0.1% formic acid (buffer B). The gradient program consisted of
5% buffer B for 5 min, linear ramping to 55% buffer B over 29
min, linear ramping to 90% buffer B over 1 min, holding at 90%
buffer B for 9 min, ramping back to 5% buffer B over 1 min, and
holding at 5% buffer B for 20 min. The mass spectrometer
scanned 50 –3000 m/z (mass-to-charge ratio) and acquired data
for 50 min of each analysis.
Data acquisition was facilitated with HyStar software (version
3.2; Bruker) and processed with Data Analysis software (version
3.2; Bruker). The mass spectrometer used an AutoMSn feature
that collected MS/MS spectra for the 2 most intense ions in each
full-scan spectrum. The scan time was set to 0.5 s for the survey
scan, and the MS/MS spectra recorded were the result of 2 microscans, giving an overall duty cycle of 2.5 s. The data were
analyzed using Data Analysis and BioTools software (version
2.2; Bruker). Mascot searches were performed using the SwissProt database with a 20-ppm tolerance on the precursor, 0.2-Da
tolerance on the product ions, methionine oxidation as a variable modification, and charge states 1, 2, and 3. Searches were
performed using no enzyme, so that novel protease cleavage sites
generated by the hookworm hemoglobinases could be detected.
Data were entirely dependent on mascot identity, and an individual ion score of ⬎30 was used as a threshold to elucidate false
Figure 1. Expression and purification of Necator americanus hemoglobinases. A, Na-APR-1 expressed in the yeast Pichia pastoris X33. Lane 1, culture
supernatant; lane 2, flowthrough from nickel–nitrilotriacetic acid (Ni-NTA) column; lane 3, 20 mmol/L imidazole wash; lane 4, 60 mmol/L imidazole wash;
lane 5, 1 mol/L imidazole eluate. B, Na-MEP-1 expressed in baculovirus (BEVS). Lane 1, Culture supernatant; lane 2, flowthrough from Ni-NTA column;
lane 3, 40 mmol/L imidazole wash; lane 4, 60 mmol/L imidazole wash; lane 5, 250 mmol/L imidazole eluate. C, Na-MEP-1 expressed in insoluble form
in Escherichia coli. Lane 1, Urea solubilized pellet; lane 2, flowthrough from Ni-NTA column under denaturing conditions (6 mol/L guanidine HCl [GuHCl]);
lane 3, 40 mmol/L imidazole and 6 mol/L GuHCl wash; lane 4, 60 mmol/L imidazole and 6 mol/L GuHCl wash; lane 5, 250 mmol/L imidazole and 6 mol/L
GuHCl eluate. Arrows denote purified recombinant proteins.
identities. The relative entropy between the observed and background distributions of each amino acid at the P4 –P4' subsites
for each enzyme was calculated using the WebLogo program
[24].
RESULTS
cDNAs encoding N. americanus hemoglobinases. Cloning of
Na-apr-1 [17] and Na-cp-3 [18] has been reported elsewhere.
Na-MEP-1 ORF contained 846 aa (excluding the signal peptide
of 26 residues) with a predicted molecular mass of 96.77 kDa.
Na-MEP-1 belonged to the M13 peptidase family. The closest
homologues included predicted M13 proteases from other
hookworms (Ancylostoma species) and the free-living nematode
Caenorhabditis elegans. Na-MEP-1 was 56% identical at the
amino acid level to Ac-MEP-1 from A. caninum (data not
shown). Table 1 lists orthologues of each of these 3 N. americanus hemoglobinases from 3 other blood-feeding parasites: the
dog hookworm A. caninum, the blood fluke S. mansoni, and the
malaria parasite P. falciparum.
Expression and purification of Na-APR-1 and Na-MEP-1.
Na-APR-1 was expressed in yeast and secreted as a zymogen into
culture medium. The purified recombinant protein was produced at an approximate yield of 2.0 mg/L (figure 1A). Na-CP-3
was expressed in yeast and purified as described elsewhere [20].
Na-MEP-1 was expressed in baculovirus/Hi5 cells, secreted into
culture medium, and purified at a yield of 0.2 mg/L (figure 1B).
The identities of the 3 recombinant proteins were confirmed by
Western blot analysis using anti-6⫻His antibodies (data not
shown). Na-MEP-1 was also expressed in E. coli but required
denaturing conditions to solubilize. The protein was purified
under denaturing conditions and verified by Western blot anal-
Figure 2. Catalytic activity of recombinant Na-APR-1, expressed in yeast, and Na-MEP-1, expressed in baculovirus. A, Catalytic activity of Na-APR-1,
determined using the fluorogenic peptide MoCAc-GKPILFFRLK in 50 mmol/L sodium acetate. A range of concentrations of peptide was hydrolyzed by
0.105 nmol/L recombinant Na-APR-1 at pH 3.5. B, Gelatin zymogram showing catalytic activity of purified recombinant Na-MEP-1 produced in
baculovirus. Lane 1, 10 ng of Na-MEP-1 and CaCl2; lane 2, 5 ng of Na-MEP-1 and CaCl2; lane 3, 10 ng of Na-MEP-1 and 10 ␮mol/L 1,10-phenanthroline.
FU, fluorescence units.
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Figure 3. Immunolocalization of Na-APR-1, Na-CP-3, and Na-MEP-1 in
transverse (A, C, D) and longitudinal (B) sections of adult Necator
americanus. Arrows indicate the gut. A, Mouse anti–Na-APR-1. B, Mouse
anti–Na-CP-3. C, Mouse anti–Na-MEP-1. D, Normal mouse serum. The
scale bar denotes 50 ␮m. int., intestine.
ysis using anti-6⫻His antibody (figure 1C). The yield of purified, denatured protein was 1.5 mg/L. Attempts to refold denatured Na-MEP-1 expressed in E. coli were unsuccessful (data not
shown).
Catalytic activity assays. Na-APR-1 was secreted from P.
pastoris in its pro form and underwent autoactivation at acid pH.
Optimal cleavage of MoCAc-GKPILFFRLK was at pH 3.5 (data
not shown). MoCAc-GKPILFFRLK was hydrolyzed by 0.105
nmol/L recombinant Na-APR-1 at pH 3.5, with K m ⫽ 15.4 ⫾ 0.7
␮mol/L, k cat ⫽ 32.2 ⫾ 0.7, and k cat/K m ⫽ 2,090,909 L/(mol·s)
(figure 2A). As little as 5.0 ng of recombinant Na-MEP-1 displayed gelatinolytic activity in the presence of CaCl2 (figure 2B);
catalytic activity was not observed when CaCl2 was replaced with
ZnCl2 (data not shown). Activity was observed at pH 4.5– 6.5
(data not shown), and addition of 1,10-phenanthroline completely inhibited catalytic activity.
Immunolocalization. Immunofluorescence experiments were
conducted with tissue sections of adult N. americanus that had
been removed from euthanized hamsters. Antibodies to NaAPR-1, Na-CP-3, and Na-MEP-1 all localized to the brush border of the intestine of adult worms, confirming earlier reports of
intestinal localization for Na-APR-1 [17] and Na-CP-3 [20] (figure 3). Prevaccination control serum did not bind to any structures.
Hb degradation and LC-MS analysis of Hb hydrolysates.
Human Hb was readily degraded by Na-APR-1 within 5 min at
pH 3.5 and 4.5; digestion was slower at pH 5.5 but still occurred
(figure 4A). In contrast, Na-CP-3 and Na-MEP-1 did not digest
intact Hb (figure 4B), even after 24-h incubation (data not
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shown), but they did act downstream of Na-APR-1 in the hemoglobinolysis pathway, digesting globin fragments after initial digestion of Hb with Na-APR-1 (figure 5A). Nineteen cleavage
sites were detected in the ␣-chains and another 18 in the
␤-chains of Hb after digestion with Na-APR-1 (figure 5B). Neither Na-CP-3 nor Na-MEP-1 digested Hb when assessed by LCMS/MS (data not shown). However, when Na-APR-1 and NaCP-3 were added to Hb together, 4 additional cleavage sites were
detected in Hb-␣ and 9 in Hb-␤. Na-MEP-1 alone did not digest
Hb, but when added to Hb in the presence of Na-APR-1 and
Na-CP-3, another 16 cleavage sites in the ␣-chain and 8 in the
␤-chain were detected (figure 5). None of the proteases displayed a distinct preference for defined residues at the P4 –P4'
subsites. However, Na-APR-1 showed a preference for aromatic
and hydrophobic residues at P1, cleaving at 8 sites with P1 Phe,
10 with P1 Leu, and 5 with P1 Ala. Na-APR-1 preferred hydrophobic P1' residues, notably Leu and Ala, but nonhydrophobic
residues were also accommodated. Na-CP-3 was also promiscuous in its preferred P1 residues, again mostly cleaving after hydrophobic and neutral residues. Na-MEP-1 preferred P1 hydrophobic residues including Ala (5 sites) but also readily cleaved
sites with P1 Lys, Ser, and Asp (figure 6).
DISCUSSION
To liberate Hb into the gut lumen, hookworms must first lyse the
ingested erythrocytes via pore formation [25]. Hookworms
then, like other blood-feeding parasites, digest Hb via a cooperative and often redundant network of proteases [26]. Here we
show that the human hookworm N. americanus uses a pathway
of distinct proteases (hemoglobinases) to digest Hb and the sub-
Figure 4. Hemoglobin (Hb) digestion with recombinant Na-APR-1, NaCP-3, and Na-MEP-1. A, Human Hb incubated for 1 h with recombinant
Na-APR-1 at a molar ratio of 72:1 (Hb to protease), at pH 3.5 (lane 1), pH
4.5 (lane 2), and pH 5.5 (lane 3). B, Human Hb incubated with various
recombinant proteases at pH 4.5, at molar ratios of 72:1 (Hb to Na-APR1), 5:1 (Hb to Na-CP-3), and 14:1 (Na-MEP-1). Lane 1, Hb; lane 2, Hb and
Na-APR-1; lane 3, Hb and Na-CP-3; lane 4, Hb and Na-MEP-1.
Figure 5. Cleavage of hemoglobin (Hb) by various Necator americanus recombinant proteases for 18 h at 37°C and pH 4.5. In panel A, colored lines
represent the UV trace (214 nm) of Hb, and black lines represent the total ion count (i, overlapping UV trace of all 4 samples; ii, Hb only; iii, Hb and
Na-APR-1; iv, Hb, Na-APR-1, and Na-CP-3; v, Hb, Na-APR-1, Na-CP-3, and Na-MEP-1). Panel B shows a map of Hb ␣- and ␤-chains highlighting the
cleavages made by each recombinant hemoglobinase when all 3 enzymes were coincubated with Hb at pH 4.5. Black arrows indicate Na-APR-1 cleavage
sites, green arrows indicate Na-CP-3 cleavage sites, red arrows indicate Na-MEP-1 cleavage sites, and stars indicate hinge regions. AU, absorbance
units; Intens., mean signal intensity.
sequent globin peptides. The pathway is similar to that used by P.
falciparum [27] in that it consists of at least 3 distinct classes of
endopeptidases—aspartic, cysteine, and metalloenzymes—and
is a major target for the development of new therapies.
Plasmodia are no more closely related to nematodes than they
are to humans, which implies that convergent evolution has resulted in phylogenetically distant pathogens adopting similar
proteolytic cascades of Hb hydrolysis. P. falciparum makes initial
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Figure 6. P4 –P4' subsite specificities of Na-APR-1 (A), Na-CP-3 (B), and Na-MEP-1 (C). Tandem mass spectrometry data compiled from Mascot
searches were used to create an alignment of peptide cleavage sites, which was submitted to the WebLogo program to generate the image shown.
Plots depict relative entropy between the observed and background distributions of amino acids at each subsite. The larger the letter denoting each
amino acid, the more frequently that residue occurred. Hydrophobic residues are black, polar residues are green, basic residues are blue, and acidic
residues are red.
cleavages of Hb with aspartic proteases, called “plasmepsins.”
Four distinct plasmepsins cleave Hb in a semiordered fashion
[28], but P. falciparum clones with plasmepsin gene deletions
remained viable despite prolonged doubling times, suggesting
that at least the early phases of the hemoglobinolysis process are
redundant [9, 27]. This is in contrast to the P. falciparum cysteine hemoglobinases, termed “falcipains” (falcipain-1, -2, -2',
and -3); falcipain-3 is essential for Hb digestion and parasite
growth, and falcipain-2 knockout parasites were shown to be
⬎1000-fold more sensitive to the aspartic protease inhibitor,
pepstatin, than wild-type controls, highlighting the cooperative
action of cysteine and aspartic hemoglobinases in this parasite
[29]. A combination of pepstatin and E64, inhibitors of plasmepsins (aspartic proteases) and falcipains (C1 family cysteine
proteases), respectively, inhibit 98% of globin proteolysis by P.
falciparum digestive vacuole extract [30]. Although the hemoglobinolysis process in this species is redundant to some extent,
these 2 major groups of proteases initiate digestion of the blood
meal.
In contrast to Plasmodium species, hemoglobinolysis is less
redundant in the hematophagous flatworms [31]. Current data
suggest that only 1 aspartic protease and 3 clan CA cysteine proteases are present in the intestine of schistosomes, and only the
aspartic and 2 of the cysteine proteases (SmCB1 and SmCL1)
have been shown to digest the intact Hb tetramer [7, 32, 33].
Further evidence of an ordered cascade in schistosomes comes
from studies using RNA interference [8]. Silencing of the S.
mansoni mRNA encoding cathepsin D aspartic hemoglobinase
made larval worms unable to digest Hb in vitro, and none of the
cathepsin D dsRNA–treated worms matured to adulthood when
injected into mice [10]. Suppression of mRNA encoding the cathepsin B cysteine protease SmCB1 did not overtly affect Hb
degradation but slowed parasite growth considerably [34].
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The degree of redundancy, in terms of both numbers of proteases and overlapping functions/substrate preferences, has not
been thoroughly explored in hookworms or other parasitic nematodes. Progress in this area has been hampered by a number of
roadblocks, including the lack of a hookworm genome sequence
and, until recently, a substantive number of ESTs. Additionally,
neither robust RNA interference nor transgenesis technologies
have been developed for hookworms and most other parasitic
nematodes. As a result, studies addressing the functions of parasitic nematode proteases has been restricted to in vitro observations only.
Na-APR-1 is not the only hemoglobinase that cleaves intact
Hb in N. americanus. A pepsinlike aspartic protease, Na-APR-2,
also cleaves Hb and does so at distinct sites to Na-APR-1 [35].
Moreover, redundancy exists among the hookworm gut cysteine
proteases. Na-CP-3 did not cleave Hb but did cleave globin peptides; in contrast, A. caninum Ac-CP-2 cleaved canine Hb [13].
We recently reported a family of 5 cathepsin B cysteine proteases
from the gut of N. americanus [20], most of which were identified from just 480 ESTs. Only 1 of these enzymes, Na-CP-3, was
expressed in active form, precluding analyses of their hemoglobinolytic properties. However, Na-CP-3 but not the other N.
americanus intestinal cathepsins B had a C-terminal insertion in
the same region as the falcipain-2 “Hb-interacting motif” [36],
suggesting that not all of the N. americanus intestinal cathepsins
B are involved in Hb digestion [29]. Indeed, adult hookworms
can be kept alive for numerous months in medium containing
just serum in the absence of Hb or blood (D. Smyth, A.L., unpublished data), implying that Hb is a major food source required by hookworms for survival but not the only one, in vitro
at least.
N. americanus intestinal cells produce an array of proteolytic
enzymes [16], and our study has now attributed a hemoglobinolytic function to several of these. This is by no means the com-
plete set of enzymes involved in the pathway, but our findings
serve to highlight the complexity of the process and show that at
least some level of order exists. The pathway is similar to that
described for the canine hookworm A. caninum, but several key
differences exist. Na-CP-3 was incapable of cleaving intact Hb
and instead cleaved globin peptides generated by hydrolysis with
Na-APR-1; however, Ac-CP-2 is the only cysteine hemoglobinase from A. caninum that cleaves intact Hb identified to date.
Numerous cysteine proteases are present in the gut of N. americanus [16], so we cannot yet discount the presence of an intestinal cathepsin B that digests intact Hb.
In addition to the proteases mentioned above, which have
mainly endopeptidase and oligopeptidase activities, it has been
suggested that aminopeptidases and other proteases with exopeptidase functions play a vital role in the blood digestion process in hematophagous parasites. They are thought to exert their
activities downstream of the endopeptidases, removing terminal
amino acids and dipeptides for transport into the intestinal cells
(helminths) or in the cytosol (Plasmodium species). 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 the
endopeptidases [37]. S. mansoni and Schistosoma japonicum express an intracellular leucine aminopeptidase in the gastrodermal cells, which has been proposed to function in removing terminal amino acids from internalized globin peptides [38].
Hookworm ESTs encoding aminopeptidases and carboxypeptidases are present in public databases, and we are currently exploring their roles in the Hb-digestion process (T. Don, A.L.,
unpublished data).
Another blood-feeding nematode, Haemonchus contortus, is
highly pathogenic to sheep [39]; its gut contains a plethora of
mechanistically distinct endo- and exopeptidases [26, 40, 41],
but most have not been unequivocally assigned hemoglobinolytic functions. Complexes that are enriched for these intestinal
proteases are highly efficacious vaccines [41, 42], further supporting the case for dissecting the molecular basis of hemoglobinolysis in blood-feeding nematodes. Like these Haemonchus enzymes, the hookworm hemoglobinases described in
the present study are expressed in the gut lumen of the parasite and are therefore exposed to host antibodies when the
worm ingests blood. Antibodies to Ac-APR-1 and Ac-CP-2 bind
to the gut of feeding hookworms, neutralize the catalytic activity of the target enzymes, and damage the epithelial surface
[14, 15]. Given the involvement in hemoglobinolysis of the
N. americanus proteases described in the present study, they
are potential targets for vaccines that interrupt hematophagy.
Because of the potential redundancy in the hemoglobinolysis
cascade, we are now considering multivalent vaccines consisting
of fragments or epitopes, which are the targets of neutralizing
antibodies from multiple hemoglobinases, such as those described here.
Acknowledgments
We thank Tristan Wallis, Jason Mulvenna, Darren Pickering, Danielle
Smyth, Michael Smout, Gaddam Goud, and Vehid Deumic for technical
assistance, provision of materials, and advice.
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