INFECTION AND IMMUNITY, Mar. 2006, p. 1989–1993
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 3
Identification of Secreted Cysteine Proteases from the Parasitic Nematode
Haemonchus contortus Detected by Biotinylated Inhibitors
Ana P. Yatsuda,1,2Nicole Bakker,1Jeroen Krijgsveld,3David P. Knox,4
Albert J. R. Heck,3and Erik de Vries1*
Departamento de Ana ´lises Clı ´nicas, Toxicolo ´gicas e Bromatolo ´gicas, Faculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o Preto,
Universidade de Sa ˜o Paulo, Ribeira ˜o Preto, Brazil2; Division of Infection Biology, Department of Infectious Diseases and
Immunology,1and Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and
Utrecht Institute for Pharmaceutical Sciences,3Utrecht University, Utrecht, The Netherlands;
and Moredun Research Institute, International Research Centre,
Pentlands Science Park, Penicuik, United Kingdom4
Received 12 October 2005/Returned for modification 22 November 2005/Accepted 18 December 2005
Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-
secretory products of Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors
were employed to localize active CBLs in 2-D protein gels. Mass spectrometry provided the identification of
AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered expressed sequence tags.
Cysteine proteases are prime targets for vaccine develop-
ment against parasitic nematodes (11, 14, 19). In Haemonchus
contortus, a highly pathogenic parasite of ruminants, cathepsin
B-like cysteine proteases (CBLs) are encoded by a family of at
least 22 genes (7, 13, 21) and are abundantly expressed, rep-
resenting 4% of all adult worm expressed sequence tags
(ESTs) (6, 12). No function for any of the CBLs, nor their
potential functional diversity or redundancy, has been re-
solved. Such knowledge is key to the design and evaluation of
vaccination experiments, and it will be necessary to trace back
immune protection to uniquely identified proteins. Specific
inhibitor profiles of H. contortus CBL activity have mainly been
determined in crude extracts of whole worms and gut tissue.
These contain a mixture of CBL gene products (4, 8, 9, 10, 15),
of which only a few have directly been identified by N-terminal
amino acid sequencing (20).
Cysteine proteases are the most active proteases of the ex-
cretory-secretory products (ES) of H. contortus (12) and are
likely to be involved in induction of protective immunity (2).
Proteomic analysis of 102 prominent spots present on a two-
dimensional (2-D) protein gel of ES identified only members
of three other protease classes (28). Therefore, the ability of
CBLs to separate and migrate into 2-D protein gels was inves-
tigated by gel activity assay (zymogram), as commonly per-
formed after 1-D electrophoresis in gelatin containing sodium
dodecyl sulfate (SDS)-polyacrylamide gels. ES (200 ?g) was
submitted to isoelectric focusing on 13-cm immobilized pH
gradient strips (pH 3 to 10 nonlinear [NL]) as described pre-
viously (28), but alkylation by iodoacetamide was omitted. Sep-
aration in the second dimension was performed on a SDS–
12.5% polyacrylamide gel containing 0.1% gelatin in the
absence of dithiothreitol (DTT). Under conditions favoring
cysteine protease activity (18 h at 37°C in 10 mM Tris, 20 mM
NaCl, 10 mM DTT, pH 5.0), abundant proteolytic activity was
found in a region between 30 and 35 kDa (Fig. 1) correspond-
ing to the position of cysteine proteases in 1-D zymograms of
H. contortus ES (8). No proteolytic activity was detected at the
higher molecular weight (MW) range, where serine proteases,
metalloproteases, and aspartic proteases are expected to be
located (28), possibly due to unfavorable experimental condi-
The observed wide pI range corresponds to the range of
predicted pI values for individual CBLs (Table 1). However,
reduced resolution due to the presence of gelatin and the
absence of DTT prohibits precise colocalization with spots
present in a silver-stained 2-D gel run in parallel. Therefore,
spots representing putative CBLs were localized by affinity
labeling with a biotinylated irreversible dipeptide inhibitor spe-
cific for cysteine proteases (20). ES (200 ?g) was incubated
with 5 ?M biotin-phenylalanine-alanine-fluoromethylketone
(Bt-FA-FMK) (Enzyme Systems Products) for 15 min at 37°C.
* Corresponding author. Mailing address: Division of Infection Bi-
ology, Department of Infectious Diseases and Immunology, Utrecht
University, Yalelaan, 1, 3584CL, Utrecht, The Netherlands. Phone:
31-30-2532582. Fax: 31-30-2540784. E-mail: email@example.com.
FIG. 1. Two-dimensional zymography (13-cm strips; pH 3 to 10 NL).
Proteolysis is visualized as a clear area where gelatin has been digested
against a blue background stained with Coomassie blue R250. Molecular
masses were estimated from a standard molecular mass marker.
After 2-D gel electrophoresis (immobilized pH gradient strips,
pH 3 to 10 NL; 12.5% polyacrylamide gel electrophoresis ),
proteins were blotted onto polyvinylidene difluoride membranes
and blocked overnight with 5% nonfat dry milk in PBS–0.05%
Tween (PBS-T). Membranes were incubated with streptavidin-
horseradish peroxidase conjugate (GE Healthcare) diluted
1:500 in 2% nonfat dry milk in PBS-T, followed by detection by
chemiluminescence (ECL plus; GE Healthcare). The non-
charged and low-MW inhibitor Bt-FA-FMK binds covalently
to a cysteine in the active site of the protease and is unlikely to
cause changes in pI and MW in comparison to the silverstained
gel, in which cysteines are blocked by alkylation with iodoac-
etamide. As on the zymogram, spots were detected between 30
and 35 kDa within a pI range of 5.0 to 8.3, a number of which
could be colocalized with spots on a silver-stained gel (Fig. 2)
using Phoretix Software (NonLinear). Mass spectrometry (ms)
analysis by liquid chromatography (LC)/MS/MS allowed the
identification of seven CBLs, an aspartic protease, and a met-
alloprotease by searching the GenBank protein database and a
database of 21,791 clustered H. contortus ESTs with the ob-
tained fragmentation spectra. CBL identifications are summa-
rized in Table 1, and LC/MS/MS-derived peptide sequences
are indicated in the alignment of Fig. 3. We obtained peptide
sequences encoded by the cDNA sequences of four known
CBLs (AC-4, spots 4 and 5; HMCP1, spot 8; HMCP2, spot 6;
GCP7, spot 7) as well as the corresponding homologous EST
clusters. However, the EST clusters most similar to HMCP1
and HMCP2 display a few remarkable sequence variations
(Fig. 3). The gene clusters may encode allelic variants, but
spots 8 and 6 could also derive from paralogous genes (repre-
sented by the EST clusters) most similar to HMCP1 and
FIG. 2. (A) Silver-stained two-dimensional SDS-PAGE gel of H.
contortus ES (13-cm strips; pH 3 to 10 NL). (B) Detection and local-
ization of ES cysteine proteases of H. contortus by the biotinylated
peptide inhibitor Bt-FA-FMK. C) Zoom of the region in panels A (gel
on the left) and B (membrane on the right) marked with a square.
Specificity is exemplified by two circled spots showing no binding of
inhibitor (right) despite intense silver staining (left). The estimation of
the molecular mass of stained proteins was done using a biotinylated
marker (GE Healthcare).
TABLE 1. Cathepsin B-like cysteine protease spots identified from H. contortus ESa
pI of spot
4 and 5
1 and 2
6.34 and 6.75
5.05 and 5.34
32.5 and 32.4
aThe predicted isoelectric points (pI) and molecular masses (in kDa) are indicated without signal peptide (?SP) and without the propeptide region (?PR). The
observed pI and mass (in kDa) of the spots are also indicated.
bNA, not applicable.
Three novel CBLs, designated HMCP7 (spot 3), HMCP8
(spot 1 and 2), and HMCP9 (spot 8), are encoded by EST
clusters with little similarity at the DNA level to any known
CBL, thus excluding the possibility that they represent allelic
variants of known CBLs.
A full-length protein sequence was obtained for HMCP7 by
conceptual translation from the EST clusters. Sequence compar-
ison (Fig. 3) reveals 71% amino acid identity to HMCP4. Re-
markably, HMCP7 carries a glutamic acid at position 338,
whereas most H. contortus CBLs have a hydrophobic residue at
this position. Amino acids at the homologous position, lining the
S2 substrate pocket (depicted in Fig. 3), in cathepsin B-like pro-
teases of other organisms determine substrate specificity. Site-
residue at this position supports the typical cathepsin B-like ac-
AMC (RR), whereas a hydrophobic residue results in cathepsin
L-like specificity leading to hydrolysis of only FR. Thus, the po-
tential presence of abundantly expressed HMCP7 may well ex-
plain the previous observation that H. contortus intestinal extracts
containing CBL activity seemed hardly more efficient in hydroly-
of individual CBLs, in ES as well as in intestinal extracts, is clearly
important in determination of their function. With the identifica-
tion and mapping of specific CBLs, predicted specificities can be
tested by further fractionation from native extracts or character-
ization of recombinant proteins.
HMCP8 displays 57% identity to a Trichuris suis cysteine pro-
tease (accession no. AAC78691) and is represented by a single
of this protease. A replacement of tyrosine by phenylalanine in
the putative hemoglobinase domain (Fig. 3) is likely to modify
substrate specificity. This domain is strictly conserved within most
CBLs from blood feeding helminths (1), and detection of CBLs
FIG. 3. Alignment of the CBL sequences enclosing the identifications of CBLs made from ES of H. contortus. GenBank accession numbers of
the displayed sequences have been indicated in Table 1. Positions having four or more identical residues have been shaded. The peptide sequences
obtained from each spot by LC/MS/MS (as described in reference 28) are boxed in the alignment. The hemoglobinase motif and S2 substrate
binding site described in the text are shown, and the propeptide region is indicated by a black bar.
VOL. 74, 2006NOTES 1991
with and without this motif in the excretory/secretory products of
diversity of the secreted CBLs.
CBLs are translated as preproteins harboring an N-terminal
propeptide that blocks access to the active site. Activation
results from autocleavage triggered by a drop in pH (25). All
peptide sequences obtained by LC/MS/MS are localized after
the cleavage site (Fig. 3), with the exception of the peptide
mapping to the predicted propeptide (13, 20) region of
HCMP9 (spot 8, colocalizing with HCMP1), indicating that it
may be secreted as a nonactive protease. HCMP9 is repre-
sented by three ESTs and is 55% identical to GCP7. The
observed MW and pI of AC 4 (spots 4 and 5) and HMCP7
match better with the values predicted for their precursor
proteins still containing the propeptide (Table 1).
In addition to HCMP8, spots 1 and 2 provided simultaneous
identifications with the aspartic protease PEP2 (CAE12199;
spots 1 and 2) and the metalloprotease MEP3 (AAC31568;
spot 2). Both are components of a galactose-containing glyco-
protein complex (H-gal-GP) with immunoprotective proper-
ties located on the luminal surface of the intestine (22–24).
PEP2 is cleaved into N- and C-terminal domains that are held
together by disulfide bonds which are broken under the reduc-
ing conditions used for gel electrophoresis in the second di-
mension (28). The estimated molecular masses of spots 1 and
2 (32.4 and 32.5 kDa, respectively) correspond to the 31-kDa
size reported for the C-terminal domain (22). Similarly, MEP3
(with a predicted size of 95.5 kDa) was shown to resolve in N-
and C-terminal domains of 41 and 47 kDa under reducing
conditions (24), and the size observed for MEP3 in spot 2 (32.5
kDa) indicates further processing. Previous proteomic analysis
of H. contortus ES identified the presence of the N- and C-
terminal domains obtained after cleavage of other metallopro-
teases (MEP1, MEP1B, and MEP2) and serine proteases (28).
As for PEP2 and MEP3, the fragments may be kept together
by disulfide bonds, possibly giving rise to proteolytically active
complexes under native conditions. Colocalization with
HCMP8 is considered to be coincidental, and labeling with
Bt-FA-FMK is probably exclusively due to binding of this sub-
strate to HCMP8.
Several CBLs induce protective immune responses (14), but
a function has not been identified. A proposed (27) and par-
tially reconstituted proteolytic cascade for the metabolism of
hemoglobin, the major food source of blood-feeding parasites,
encompasses the sequential cleavage by aspartic proteases,
cysteine proteases, metalloproteases, and exopeptidases (26).
Aspartic and metalloproteases are also involved in the activa-
tion of procathepsin B to cathepsin B (5). Immunohistology
has demonstrated localization of many proteases at the surface
of the microvilli (26). Their presence in ES suggests that he-
moglobin digestion may take place not only at the cell surface
but also in the lumen of the gut, thus greatly enhancing the rate
of digestion. Proteases in ES may perform other essential func-
tions, outside of the worm, in penetration of mucus layers,
gaining access to blood vessels and intervention with host pro-
cesses like blood-clotting and immune responses. The molec-
ular identification of proteases in ES provides specific tools to
explore these options.
The protective properties against infection induced by im-
munization with ES of H. contortus have inspired attempts to
determine the molecular components involved (17, 18). A
global analysis (28) of the most abundant proteins in ES al-
ready identified several known vaccine candidates (H11 and
GA1) and demonstrated the complexity and variability of other
immunologically relevant molecules (Hc-ASP1, Hc-ASP2, and
Hc15). The identification in ES of new CBLs and proteases
with previously demonstrated protective properties further ex-
pands this group of proteins, thus contributing to the challeng-
ing search for the minimal set of proteins required for induc-
tion of a protective immune response.
This work was supported by the European Union (Project QRLT-
PL-1999-00565) and The Netherlands Proteomics Center.
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Editor: W. A. Petri, Jr.
VOL. 74, 2006NOTES1993