From the Division of Parasitology and Tropical
Veterinary Medicine, Department of Infectious Diseases and Immunology,
Utrecht University, P.O. Box 80165, 3508 TD, Utrecht, The Netherlands
and the
Department of Biomolecular Mass Spectrometry, Bijvoet
Center for Biomolecular Research and Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands
Received for publication, December 6, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Haemonchus contortus is a
nematode that infects small ruminants. It releases a variety of
molecules, designated excretory/secretory products (ESP), into the
host. Although the composition of ESP is largely unknown, it is a
source of potential vaccine components because ESP are able to induce
up to 90% protection in sheep. We used proteomic tools to analyze ESP
proteins and determined the recognition of these individual proteins by
hyperimmune sera. Following two-dimensional electrophoresis of ESP,
matrix-assisted laser desorption ionization time-of-flight and
liquid chromatography-tandem mass spectrometry were used for protein
identification. Few sequences of H. contortus have
been determined. Therefore, the data base of expressed sequence tags
(dbEST) and a data base consisting of contigs from
Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like
Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were
identified from matches with H. contortus ESTs displaying
high similarity with proteins like cyclophilins, nucleoside diphosphate
kinase, OV39 antigen, and undescribed homologues of
Caenorhabditis elegans. Of special note is the finding of
microsomal peptidase H11, a vaccine candidate previously regarded as a
"hidden antigen" because it was not found in ESP. Extensive
sequence variation is present in the abundant Hc15 proteins. The Hc15
isoforms are differentially recognized by hyperimmune sera, pointing to
a possible specific role of Hc15 in the infectious process and/or in
immune evasion. This concept and the identification of multiple
novel immune-recognized components in ESP should assist future
vaccine development strategies.
Gastrointestinal (GI)1
nematodes are currently controlled by the use of chemicals
(anti-helminthics), but there is great interest in development of
vaccines, mainly because of the emergence of drug-resistant parasites.
Among the GI nematodes, Haemonchus contortus is economically
important because of its blood feeding characteristics in the abomasum;
it is able to cause severe losses in production of small ruminant
herds. The parasitic stages of the developmental cycle occur in a
single host, and during each phase the nematode releases a variety of
molecules into the host or in vitro culture environment.
These are usually referred to as excretory/secretory products
(ESP).
ESP are of practical value as a source of potential vaccine components.
Obtained after in vitro cultivation of adult worms in
serum-free medium, ESP or its partially purified fractions are able to
induce 65 to 90% protection against H. contortus in sheep
(1, 2), but protective properties have not conclusively been attributed
to individual proteins.
Regardless of its practical use in vaccination studies, there is hardly
any conclusive evidence on the biological function of ESP. For some
proteins, functional roles have been proposed on the basis of
similarity to other proteins in sequence data bases (3, 4), or roles in
immune evasion or in molting have been implied (5, 6). Furthermore, the
surface of the cuticle may be constituted of proteins derived from the
secretory system (7). The secretion of some proteins has been linked in
time to the transition of free-living stages to parasitism (8).
The identification of the proteins present in ESP of H. contortus using a proteomics approach will provide a basis for
studies on the following matters. (i) Identification of ES proteins as the products of specific genes, enabling bioinformatic analyses and
much more specific functional studies. (ii) The complexity of ES, which
has hardly been addressed by the almost exclusive use of
one-dimensional gel electrophoresis. (iii) The variability between batches of ESP, a topic highly relevant to vaccination studies.
(iv) The recognition of specific spots by immune sera, especially in
cases where multiple spots are derived from the expression of multigene
families or from variations in post-translational modifications. (v)
The cellular origin of ESP. Among others, ESP can contain proteins
secreted by the pharyngeal glands, the excretory system, epithelial
cells of the intestine (e.g. digestive enzymes), or rectal
and vaginal cells (7). Moreover, cytosolic components of decaying cells
(either by apoptosis or other causes of damage) may be present in
addition to epithelial membrane proteins that are cleaved off.
Here we have described 107 identifications in the ESP of adults of
H. contortus from gene sequence information deposited in GenBankTM nr as well as from H. contortus dbEST
tag sequences. A number of the identified proteins have
previously been associated with a protective immune response, although
in several cases their presence in ESP has not been reported. For many
proteins we have now shown that they appear in truncated forms or have
extensive sequence modifications, which may complicate the design of
vaccination strategies. Furthermore, many proteins have been detected
in ESP for the first time and will be discussed with regard to their potential function and relation to an immune response.
H. contortus ESP--
Standard procedures for harvesting ESP
have been used as described for H. contortus and other
nematodes (3, 9-13). All batches (A to D) were derived from
experimental infections initiated from different larval stocks
established over a period of 2 years from new generations of the same
isolate. H. contortus adult worms were harvested from the
abomasum of infected donor lambs, washed several times in PBS, and kept
in RPMI 1640 medium containing antibiotics (100 IU of penicillin, 0.1 mg/ml streptomycin, and 5 µg/ml gentamicin) at 37 °C under 5%
CO2. The parasites were first incubated for 4 h, after
which the medium was harvested and new medium containing 2% glucose
was added for overnight incubation. The supernatant was collected,
centrifuged, filter-sterilized (0.2 µm), concentrated, and desalted
(10 mM Tris, NaCl pH7.4) in 3-kDa filters (Centripep YM-3, Millipore).
Sample Preparation and Isoelectric Focusing--
Prior to
isoelectric focusing, the ESP were precipitated in a final
concentration of 10% trichloroacetic acid (dissolved in acetone) containing 10 mM dithiothreitol. The pellet was
washed one time in acetone with 10 mM dithiothreitol and
resuspended in rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS, 2% carrier ampholyte mixture, pH
3-10NL (IPG buffer) and 20 mM dithiothreitol). Isoelectric
focusing instrumentation, IPG gels, and related reagents were from
Amersham Biosciences unless otherwise indicated. Either 70 or 140 µg
of protein was loaded onto 13-cm IPG strips (pH 3-10NL) and
supplemented with protease inhibitors (Complete Protease Inhibitor mixture, Roche Molecular Biochemicals). The sample was rehydrated and
focused in an automated overnight run (IPGPhorTM) using
10-14 h of rehydration (30 V), followed by a step voltage focusing
procedure (1 h 500 V, 1 h 1000 V followed by 8000 V until a total
of 35-40 Kvh was reached).
Second Dimensional Electrophoresis--
The strips were
incubated in 10 ml of equilibration buffer (50 mM Tris, 6 M urea, 2% SDS, 30% glycerol, pH 8.8) containing 30 mM dithiothreitol for the first 15 min and replaced by
equilibration buffer with 135 mM iodoacetamide for another
15 min. Electrophoresis in second dimension gel SDS-gel was carried out
in a Hoefer SE600 system. Silver staining or Coomassie Brilliant Blue
R-250 was used to visualize proteins after second dimensional
electrophoresis. The images of the gels were acquired using LabScan
v3.0 software on an ImageScanner (Amersham Biosciences).
Immunoblotting--
ESP of H. contortus were
separated in 12.5% SDS-PAGE gels and transferred to polyvinylidene
difluoride membranes (ImmobilonP, Millipore) using a semi-dry system
(Novablot, Hoefer) in transfer buffer (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol) at 1.1 mA/cm2 for 1 h. The transfer efficiency was checked by
staining of the membranes with DB71 (14). The membranes were blocked
with 5% skimmed milk in PBS/0.05% Tween 20 (PBS-T, 1h, 37 °C) and
all the washing steps were done with PBS-T (1 × 15 min, 1 × 10 min, 2 × 5 min). Either a pool of sera from parasite-free
animals or a pool of polyclonal sera derived from five sheep
hyperimmune to H. contortus (1/500 in PBS-T/2% milk) was
incubated overnight at 22-24 °C, washed, and incubated with
anti-sheep total IgG coupled to horseradish peroxidase (1/75000 in
PBS-T/2% milk) for 1 h at room temperature. The chemiluminescent
development was performed with ECL Plus according to the
manufacturer's instructions (Amersham Biosciences). Films exposed to
the blots were densitometrically scanned using an ImageScanner, and
matching was done by comparing the films of the blots with the
DB71-stained membrane image and later with the master gel.
Mass Spectrometry--
Proteins were in-gel digested with
trypsin (Roche Molecular Biochemicals) in 50 mM ammonium
bicarbonate (Sigma). Before MALDI-TOF analysis, peptides were
concentrated using µC18-ZipTips (Millipore) and eluted directly on
the MALDI-target in 1 µl of a saturated solution of
Image Software Analysis--
The scanned images were analyzed
with Image Master two-dimensional v4.01 software (Amersham
Biosciences). The observed molecular weight and isoelectric points (pI)
of all the spots were calculated with protein markers (low molecular
weight calibration kit, Amersham Biosciences) and pI markers
(two-dimensional SDS-PAGE standards, Bio-Rad). Protein staining and
immunological recognition of individual spots were quantified by
calculation of spot volume after normalization of the image, using the
total spot volume normalization method multiplied by the total area of
all the spots (Image Master software). For the immunoblotting analysis,
the image of the 2-min exposure was used for the quantification procedures.
MS Fingerprint Searches against EST Clusters by
EMOWSE--
H. contortusEST sequences available from the
dbEST division of GenBankTM (4843 on September 30, 2002) were clustered by SeqMan (Lasergene sequence analysis
software, DNAstar) using a minimum match percentage of 90 and a minimum
overlap of 20 bp. The alignments of the 342 clusters containing three
or more ESTs were edited manually where necessary. The resulting 1876 clusters (of which 1253 contain a single EST) were annotated by running
locally a Blastx (NCBI, cut-off p > 100)
against the complete SwissProt and Trembl protein data bases. All EST
clusters and all separate ESTs were subjected to a six-frame
translation, and all peptide sequences longer than 60 amino acids were
used for making a local data base that was searched by EMOWSE (16)
(available from the EMBOSS package at www.hgmp.mrc.ac.uk/Software/EMBOSS). A tolerance of 0 was specified (effectively ± 0.5 dalton), and for cysteines modification by iodoacetamide was taken into account.
Identification of 107 Proteins in ESP by Mass
Spectrometry--
Under optimized conditions of sample preparation,
aiming at the prevention of proteolytic breakdown and limitation of
artificial modifications, 224 Coomassie Blue-stained spots were
detected in a 140-µg sample of H. contortus ESP (Fig.
1). This complexity substantially exceeds
previous estimations derived from one-dimensional SDS-PAGE and was even
more apparent after silver staining, resulting in the automated
detection of about 950 spots. Fig. 2
shows a comparison of silver-stained gels of four batches of ESP
obtained from different infections (see "Discussion"). Five
Coomassie Blue-stained gels holding samples of the same batch of ESP
were found by imaging software analysis to be nearly identical and were
used for the reported experiments.
130 spots were judged to contain sufficient material for fingerprinting
by MALDI-TOF mass spectrometry. Fingerprints were used for searching
the GenBankTM non-redundant protein data base with Mascot
software and allowed the identification of 61 spots of H. contortus origin. Because relatively few H. contortus
entries are present in GenBankTM nr, an attempt was made to
employ EST data for peptide mass fingerprint searching. H. contortus ESTs were clustered and searched by EMOWSE (as described
under "Experimental Procedures") for matching the fingerprints. The
top-score hits identified in 43 cases a contig matching one of the 61 spots identified by Mascot in GenBankTM nr. Additional
EMOWSE hits, scoring within the range observed for the 43 confirmed
Mascot hits, could represent significant identifications of proteins
that are currently only present in the EST-derived data. To check for
the reliability of these hits, selected spots were subjected to peptide
fragmentation by LC-MS/MS to obtain sequences that could be used for
searching both GenBankTM nr and EST data bases. In
addition, manual comparison of peptide fingerprints of 12 spots,
lacking LC-MS/MS data, revealed a very high similarity to the
fingerprints of adjoining spots (marked as "identified by fingerprint
similarity" in Supplementary Table S1 that includes all MW, PI, spot
volume, and scoring data), which in most cases was confirmed by a
matching EMOWSE top hit. These spots could represent
post-translationally modified forms of the same protein.
In this way, a total of 107 identifications were made from 102 spots,
of which 62 were confirmed as top hit by EMOWSE searches of the
EST-derived dataset (average score of 0.293, ranging from 0.132 to
0.556). In addition, for 14 spots confirmation by EMOWSE was obtained
by lower ranking hits. The fingerprints of 8 spots with high EMOWSE
scores (p > 0.250, average 0.345) lacked confirmation by other methods. Confidence in these hits was enhanced by a manual reinspection demonstrating that the fingerprints matched within 50 ppm
mass accuracy to the exact calculated MWs of the predicted peptides of
their respective EMOWSE hits (EMOWSE tolerates a less accurate MW range
of ± 0.5 dalton). They are taken up in the supplementary table as
preliminary identifications.
In Fig. 3 a colored scheme is used for
categorizing identified spots in functional groups. Immunoblotting
results are also incorporated.
Immunoblotting Identifies 193 Spots That Are Recognized by
Hyperimmune Serum--
Immunoblotting of two-dimensional gels in
combination with sensitive chemiluminescence detection permits
quantification of relative immunogenicity (included in Supplementary
Table S1) of individual spots by taking the ratio between the density
of a Coomassie Blue-stained spot and its chemiluminescence signal. Reproducible blots of ESP batch A were incubated with a pool of hyperimmune sera obtained from five sheep experimentally infected with
H. contortus several times. Spots on the blot and the gel were matched by imaging software; the final image is shown in Fig.
4. From 193 immune-recognized spots
detected by image analysis, 52 have been identified above by mass
spectrometry. Relative immunogenicity was observed to vary over a
2500-fold range (comparing spot 182 to spot 170) but is in fact larger
because some high-density spots display no immunological detection at
all, whereas several immunogenic spots cannot be detected by Coomassie
Blue staining. This also reflects the specificity of immune recognition
of the respective spots.
Candidate Vaccine Components Hc15, Hc24, and GA1 Are Members of
Protein Groups Displaying Differential Immune Recognition--
Table
I provides a list of protein
identifications; full data can be found in Supplementary Table S1.
Identifications that have exclusively been made by matches to
ESTs have been annotated on the basis of BLAST analysis.
Hc15, Hc24, and GA1 are the only proteins that have previously been
shown to be ESP constituents (17, 18) by N-terminal peptide sequencing.
These proteins will first be inspected in detail. Although for each a
single gene has been cloned, we identified multiple spots for each of
the three proteins (all of unknown function), hinting at sequence
modifications (post-translational or in primary sequence). This was
investigated in more detail for the spots identified as Hc15.
Mascot and EMOWSE searches of MALDI-TOF fingerprints in combination
with manual comparison of the fingerprints from this gel region
identified 21 potential Hc15 protein spots distributed over a wide
range of pI and MW (pI 5.53-7.04; 16.1-19.0 kDa) as indicated in Fig.
3. Tandem mass spectrometry was pursued on these spots until high
sequence coverage was obtained. All spots appear to be Hc15-related. In
Fig. 5 the single published sequence of Hc15 (AAC47713) is taken as a basis, and all sequenced parts of the
individual spots are shaded.
Amino acid substitutions, all of which can be explained by single
nucleotide changes, are observed in 10 of 21 spots. One to 5 residues
are substituted out of a set restricted to 6 positions (Fig. 5, shaded
in black) and occur in 9 different combinations. Four of 6 (T46A, R64P, T73A, and V90I) are also observed to be encoded by one or
more of the 19 ESTs matching Hc15 and are thus confirmed to be present
at the protein level in ESP. Two additional substitutions (H47R and
N72S), detected by de novo sequencing using the MS data,
were not found in ESTs.
Spectra of six spots contained a 1362-Da mass peak, the full sequence
(SGNQVMFENINK) of which does not correspond to a tryptic fragment and
therefore possibly represents the N terminus (Fig. 5). This N terminus
exactly matches the end of an 11-amino acid deletion (between Glu-20
and Gly-30) in three ESTs (BF059795, BF422966, BF423305). Both in these
three ESTs and in all other sequences, signal peptide cleavage is
predicted to occur after Gly-19 (SignalP). The predicted N-terminal
tryptic peptide of AAC47713 (ESQLNTK) and of the other 16 ESTs was not
observed in any of the spots, suggesting alternative cleavage at the N terminus at Gly-30. A total of 17 of the 19 available Hc15 ESTs contain
the stop codon and, as concluded from an alignment of these EST
sequences, the 35-amino acid C-terminal tryptic peptide seems to be the
most conserved part of the protein. Thus, spots 188 and 190 on the one
hand and spots 161, 162, 165, and 167 on the other hand are almost
fully sequenced by MS/MS and predicted to have an identical number of
amino acids but, nevertheless, display a ~2000-Da mass difference.
Possibly post-translational modifications are involved in such differences.
Collectively, the residue substitutions and alternative N-terminal
sequences result in considerable variation among Hc15 proteins. A
direct relationship between sequence variation and immune recognition, as measured by the relative immunogenicity described in Fig.
6, is far from obvious. Only 2 of the 10 spots in which substitutions have been found are recognized by
hyperimmune serum (Fig. 5, spots 177 and 189), and even spots in which
no sequence variation is detected by MS/MS display a 5-fold difference
in immunogenicity (e.g. spots 180 and 183) or are not
detected at all (e.g. prominent spots 175 and 193).
Variation in relative immunogenicity is even more pronounced within the
groups identified as Hc24-like (9 spots) and GA1-like (21 spots). For
the Hc24 group a 32-fold difference was observed (comparing spot 128 to
131), whereas one of nine was not recognized at all (spot 119). Similar
to the Hc15 group, spots are distributed over a wide pI and MW range
(4.80-6.71; 26.2-34.3 kDa). Spots of the GA1 group are located in a
more restricted area (see below) accommodating many other, sometimes
co-migrating, spots (more clearly demonstrated after silver staining).
This increases fingerprint complexity and might explain the low, but
significant, Mascot scores of a number of GA1 identifications (Table
S1). In addition, low scores may point at considerable sequence
modification between spots and the published GA1 sequence, a feature
that can only be clarified by an extensive MS/MS approach as used above
for Hc15. GA1 protein is a 92-kDa membrane-associated polyprotein from
which p46GA1 and p52GA1 subunits are released into solution after
cleavage by glycosylinositol-specific phospholipase C (18). The p52GA1
subunit has a glycosylphosphatidylinositol anchor, and only the
p46GA1 subunit was reported to be present in ESP. Here we have
identified 9 spots with similarity to p52GA1 (pI 5.76-6.79; 45.1-51.7
kDa) and 12 spots similar to p46GA1 (pI 5.84-5.96; 40.4-42.2 kDa). A
total of 18 of 21 GA1 spots are recognized within a broad range
(730-fold for p52GA1) of relative immunogenicity for both subunits.
However, the demonstrated co-localization of some proteins in this area
(e.g. spots 69, 76, and 96) emphasizes that even
two-dimensional separation cannot always definitely resolve which
component is actually immune-recognized.
General--
Control programs for GI nematodes in ruminants are
currently based on the use of anthelmintics. These interventions are
seriously hampered by the occurrence and rapid spread of drug-resistant parasites (e.g. Refs. 19-21). Part of the research
community has, for more than a decade, focused its efforts on the
development of vaccines with the ultimate aim of controlling helminth
infections and GI disease. This research was confronted with two basic
problems. First, the immunological toolbox to study and describe
host-parasite interactions in ruminants was limited, certainly when
compared with similar infections in humans or rodent models. Second,
identification and purification of promising unique antigens have been
difficult because of the complex life cycle of GI nematodes, the
absence of in vitro culture methods, and the inherent large
genetic heterogeneity of the population used. In this study we describe
a large scale, high accuracy mass spectrometric proteome analysis of
the ESP of H. contortus, a very important pathogenic GI
nematode of small ruminants. H. contortus was selected
because resistance to all classes of anthelmintics has been reported
for this species on all continents. In addition, several approaches
have been undertaken with the aim of developing a vaccine against this
parasite, some of which are based on ESP.
ESP of nematodes are produced by a standardized method of incubating
parasitic stages in protein-free medium (3, 9-13), resulting in a
protein pool of largely unresolved composition, origin, and function.
Considering composition, we revealed the pattern of 224 ES proteins of
H. contortus within a 3-10 pH range. Making use of
GenBankTM nr and dbEST data, we have assigned 107 identities from 102 spots. A major concern about ESP has been its
nematode-specific origin, especially with regard to bacterial and host
contamination. However, among the 130 most abundant protein spots, not
a single bacterial protein was detected. The identified host proteins
(serpins and complement factor C3) could be in ESP because of their
high binding affinity for nematode proteins (proteases and antigens,
respectively), thus revealing potential host-pathogen interactions. The
presence of a small number of intracellular enzymes of nematode origin (five glycolytic enzymes and one glutamate dehydrogenase) indicates protein leakage from cells. Serious cell damage or decay is not likely,
because no traces were found of abundant intracellular components like
ribosomal and cytoskeletal proteins, which probably do not exit their
cell through small leaks because of their association in high molecular
weight complexes. Factors discussed above can be considered as a
standard condition because a comparison of ESP batches obtained from
four different isolations displayed a high degree of similarity in
spots (Fig. 2).
This leaves us with a large set of specifically secreted products for
which a function, and possibly a purpose, should be resolved. The
purpose refers to potential applicability in vaccine development,
whereas function and applicability could find a common theme in the
diversity that we observed within a number of protein groups in
association with highly variable immunogenicity (Hc15 and -24 proteins). This variation may underlie functional diversity, improving
the adaptability of a parasite to fluctuating conditions, or may
represent antigenic diversity or variation counteracting host-immune
responses. Both options have hardly been addressed at all in metazoan
parasites. The outcome of our analyses has major implications for the
present set of vaccine candidates. These will be discussed in line with
future vaccine development. We will also discuss a set of newly
identified proteins in ESP.
Variation in and Validation of Vaccine Candidates Hc15, Hc24, and
GA1--
For three proteins (Hc15, Hc24, and GA1), a definite proof
for their ES origin had been previously obtained. The successful use of
these proteins in vaccination trials when in native partially purified
fractions (17, 22), followed by poor results with their corresponding
recombinant version (2), suggests that a single gene often
yields many products (e.g. Ref. 23). Sequence variations
among the 21 Hc15 spots, as established here by MS/MS, do not fully
explain the wide pI and MW ranges observed. Post-translational modifications in the C-terminal tryptic peptide (as nearly all other
peptides were resolved) or largely anomalous migration behavior can
thus also contribute to this diversity. N-linked
oligosaccharides of H. contortus have different
structures in comparison to the host (24) and have been shown to
contribute to immune recognition (25). In combination with the presence
of gene families, as has been demonstrated for an Hc15 homologue of the
nematode Cooperia punctata (26), this has clear implications
for vaccination trials.
Whereas the Hc15 family has no homologues in any organism outside the
Trichostrongyloidea superfamily of gastrointestinal nematodes, Hc24 and
Hc40 are related antigenic proteins (27, 28) and part of a venom
allergen antigen homologue/ associated secreted protein (VAH/ASP)
family present in vertebrates, yeast, plants, nematodes,
and hymenopteran venoms (8, 29-30). Only single Hc24 and Hc40
genes have been characterized in H. contortus, but it might
be a complex gene family with the clustering of ESTs in 22 different
contigs and 5-13% sequence variation between contigs (nema.cap.ed.ac.uk/nematodeESTs/Hemonchus/Hemonchus.html).
In C. punctata multiple Hc24 and Hc40 homologues have
recently been identified (31). This is the first direct identification of Hc40 in ESP, and the occurrence of multiple spots is clearly a
shared property of immunogenic proteins. GA1 proteins show similarity to bacterial TolB proteins, which are supposed to be involved, among
other functions, in outer membrane integrity and membrane transport of
colicins (32). Although only the 46PGA1 fragment has previously been
detected in ESP (18), we also consistently detected the 52PGA1 fragment
in different batches.
A large variation in immunogenicity is detected among members of all
three families. Some spots of the Hc24 group (spots 128 and 137) and
the GA1 group (spots 89 and 90) belong to the most immunogenic proteins
of ESP, but other members of the groups are not recognized at all. Only
six of the Hc15 spots are recognized. Hc15 has been cloned because of a
prominent immune reaction of the 15-kDa band in a one-dimensional
SDS-PAGE gel (17). After the identification of several other (novel)
immune reacting proteins in the same MW region, it has become clear
that previous interpretations correlating Hc15 to protection may have
been premature. The most immunogenic protein by far is spot 170, which matches a homologue of C. elegans Y105C5B.5. It
belongs to the transthyretin-like nematode-specific protein family (52 members in C. elegans) with weak similarity to
transthyretin, a thyroid hormone transport protein in vertebrates.
Interestingly, two other spots (179 and 184) match another EST cluster
that is also homologous to Y105C5B.5 but, distinct from spot 170, does
not contain a signal peptide and is not immune reactive. Another
identified transport protein is represented by spot 158, matching a
cuticular globin-like protein. Nematode globins have been isolated from
Nippostrongylus brasiliensis (33) and Trichostrongylus
colubriformis (34) and have been detected in H. contortus (35) and Ostertagia ostertagia (36). The
secreted form is present in the cuticle of the parasite and might be
involved in oxygen transport for muscular activity of the worm, because
nematode globins have higher affinity for oxygen than the host globins
(100 and 1000 times higher in N. brasiliensis and
Ascaris suum, respectively) (37, 38). Two spots represent homologues of OV39, a protein from the filarial nematode
Onchocerca volvulus (39) that is possibly involved in the
autoimmunity to ocular components because of its cross-reactivity with
a retinal auto antigen (hr44). Ov39 is supposed to be involved in the
autoimmune pattern of this parasitic disease that causes river
blindness in humans (40, 41). One spot matches C. elegans
F54D5.3 protein, which in H. contortus is represented by a
single contig containing 194 ESTs, indicating a high expression level.
A possible function has not been described.
ESP Contains Zinc Metalloproteases, Serine and Aspartic
Proteases--
Protease activity has been detected and partially
characterized by substrate specificity in ESP of H. contortus (42-44). Here we identified multiple spots matching
four different types of proteases belonging to three different protease
classes (metallo-, serine and aspartic proteases). Surprisingly, no
cysteine proteases were detected. Cathepsin-B-like proteases constitute
almost 5% of the 4843 ESTs presently analyzed and 17% of the ESTs
from gut tissue (45). All clusters contain a putative signal peptide, indicating their targeting for the secretory pathway (including lysosomal proteins). Purification of ESP, using recombinant H. contortus cystatin affinity chromatography, identified only a few
spots displaying high protease activity (not shown), indicating that
cysteine proteases may indeed be among the low-density spots in ESP,
not included for analysis by MS in the present study.
Four spots were identified as an amino-peptidase of the metalloprotease
class, previously identified as the antigenic protein H11 in microsomal
membrane fractions of H. contortus. H11 is taken as the most
effective H. contortus immunogen, inducing up to 93% protection (46-48). The presence of H11 in ESP was unexpected, because
it has been cloned from the insoluble gut membrane fraction (microvilli) of H. contortus and is classified as hidden
antigen, i.e. not presented to the host immune
system. Protection would be based on the binding of antibodies
to the gut of the worm, which would die from starvation. We have
consistently observed H11 in four different batches of ES.
In addition, three 85- to 95-kDa predicted endo-proteolytic zinc
metallopeptidases of H. contortus (mep1, mep1B, mep2) were identified by matching peptide mass fingerprints from ten spots of
40-46 kDa. Strikingly, all the MS fingerprints matched with either the
N-terminal or the C-terminal half of the protein. LC-MS/MS data
confirmed that the N-terminal half was represented by the three more
acidic (as predicted) and slightly smaller spots, whereas the other
seven spots matched the C-terminal domain. These observations suggest
that the protease is cleaved in two parts. None of the few studies on
metalloproteases from parasitic nematodes has described proteolytic
cleavage, although smaller sized bands have been implied as degradation
products (49, 50). Human metalloproteases are associated with a
membrane complex from which the active protease can be shed as an
inactive form by autocatalytic processing. A non-autocatalytic process,
taking place intracellularly, gives rise to the release of active
fragments (51). In H. contortus, the cellular location of
processing is unknown, and it remains to be determined whether the
cleavage products are proteolytically active.
A similar phenomenon was observed for seven spots matching either the
N- or the C-terminal part of a protein composed of a duplicated domain
strongly resembling serine proteases (as predicted from a cluster of 30 ESTs). In addition, the protein is predicted to be
glycosylphosphatidylinositol-anchored and the observed molecular mass of ~62 kDa (C-terminal half, spots 40, 41, 42,) and ~52
kDa (N-terminal half, spots 60, 63, 65, 69) fit to a predicted 127.6 kDa for the full-length protein (including signal peptide and anchor
peptide). Based on sequence comparisons, both halves may very well
encode an active serine protease, thereby classifying the full-length
protein as a polyprotein. C. elegans has three genes
encoding a serine protease double-domain version, apparently constituting a new type inside the family of serine carboxypeptidases (GenBankTM Accession nr NP_501598, NP_501599,
NP_507841).
Aspartic proteases in schistosomes (trematodes) and in H. contortus are involved in the processing of hemoglobin (52, 53). Localized in the gut lumen, cross-reaction with polyclonal antisera suggested the presence of aspartic proteases in ESP at a lower concentration compared with gut tissue (53). Our experiment suggests
the same with only one identification (spot 68).
ESP Contain Polypeptides Involved in Antioxidant Stress and
Extracellular Signaling--
Parasitic nematodes are sensitive to
oxidative stress generated by their own cellular metabolism or by the
host and therefore need an efficient oxidant defense system to ensure
survival inside the host (54, 55). We have identified antioxidant
enzymes such as superoxide dismutase (SOD) and glutathione
S-transferase in our study. Their secreted forms were found
in other nematodes (reviewed in Ref. 54), in trematodes (56), and were
indirectly shown to be present in ESP (with polyclonal antibodies) for
H. contortus (57).
Two types of proteins potentially involved in extracellular signaling
were identified. Cyclophilins (CYP) are peptidyl-prolyl cis-trans-isomerases acting both as catalysts and chaperones in protein
folding (58-60). For example, CYP-9 promotes the proper folding of
collagen in parasitic nematodes, with direct implications for cuticle
synthesis (61). CYPs have recently been classified as a pan-allergen
family after demonstration of high sensitization to Malassezia
furfur and Aspergillus fumigatus in human sera (62). The allergenic property of CYP has also been detected in the cestode Echinococcus granulosus (63). Secreted CYPs have
extracellular signaling functions, such as induction of chemotaxis and
adhesion of memory CD4 cells (64). Of 17 putative C.elegans
CYPs, only CYP-3 (matching 2 spots) is demonstrated to be present
exclusively in the single anterior mononuclear excretory cell (65).
CYP-5, with a match to spot 150, is the only C. elegans CYP
with a predicted signal peptide. Nucleoside diphosphate kinase (NDK)
(spot 157) is responsible for maintaining the levels of intracellular
nucleotide pools but is also involved in cell growth, differentiation,
and tumor metastasis (66). A secreted form has been found only in some
bacteria (67-69) and in the nematode Trichinella spiralis (70). A function is suggested by observed ATP-dependent
cytotoxicity for macrophages and mast cells and prevention of cells
from apoptosis (67, 71).
Independent Batches of ESP Have Similar Profiles--
Differential
immune recognition and the occurrence of multiple related spots,
representing the expression of multiple genes and/or variation between
individual worms within a population, present a challenge for the
comparison of ESP patterns and immune responses under variable
conditions. The effects of differences in worm isolates, host breed,
and immune status of the host should all be investigated. Four batches
of ESP, obtained over two years from experimental infections of the
same breed of sheep with the laboratory strain used in this study were
analyzed for variation. Larval stocks used were different because they
were continuously replaced by later generations produced for strain
maintenance. Thus the distribution of genetic variants within the
populations used may vary. Nevertheless, Fig. 2 shows very limited
variation of two-dimensional patterns between four batches.
Silver-stained gels of the same ESP batch (Fig. 2A)
and three other batches analyzed by MS demonstrate that the identified
proteins, including the Hc15, Hc24, and GA1 families, occur in similar
ratios in all gels. It is evident that even the majority of weakly
stained spots, not detectable by Coomassie Blue staining, are present
at comparable density in all four batches. Limited batch-to-batch
variation makes the use of two-dimensional ESP protein and immune
recognition patterns ideal tools for evaluating the parameters
mentioned above. Studies of ESP profiles of parasites propagated after
selection from (partially) immune hosts should allow evaluation of the
role of antigenic diversity or variation in immune evasion. A specific problem of helminth infections, which is also hard to address by this
technique, is discrimination between variants of a protein derived from
the same, but mutated, gene of different individuals and variants
derived from expression of different members of a multigene family
within an individual. Occurrence of the latter option has been
demonstrated by RT-PCR experiments on members of protein families
homologous to Hc15 and Hc24 in single C. punctata individuals (26, 31), but current protein detection methods (even by
specific monoclonals) preclude such experiments for single worms except
for a few of the most abundant proteins.
Despite the absence of high numbers of full-length protein
sequences from genomic and cDNA data, many of the identifications described above could be made on ESTs. Searching data bases of clustered and translated ESTs with MS fingerprints by EMOWSE was shown
to be an efficient tool for making a pre-selection of spots to be
analyzed further by LC-MS/MS. This tool would become even more useful
for a MS fingerprint screening of the large amount of minor spots
(>800 left), many of which may not be represented in the EST data,
making a pre-selection desirable. Because of the accuracy of MS
equipment, this approach would benefit from a more restricted tolerance
level than the current margin of ± 0.5 Da used by EMOWSE.
Finally, it may be instructive to compare our approach using
two-dimensional electrophoresis and the finding of a family of proteins
displaying extensive sequence variation to the trend in proteomics
toward LC-based methods (72). In the latter approach a complex protein
mixture (e.g. total ESP or a molecular weight fraction
thereof) is digested and peptides are separated and identified by
consecutive (multidimensional) LC and tandem mass spectrometry. Although many proteins can be identified this way, it will be extremely
difficult, if not impossible, to identify multiple isoforms of the same
proteins, such as Hc15. This study clearly shows that two-dimensional
electrophoresis is still the most powerful technique to resolve closely
related proteins, such as the many Hc15 and Hc24 isoforms observed in
this work.
The majority of abundant proteins have now been identified; the use of
two-dimensional electrophoresis will allow more detailed studies on the
triggers and kinetics of their secretion. In addition, it has become
clear that many of the major immune-recognized proteins result from
minor, as yet unidentified, spots. Intensified protein studies like
those reported here will be required to reveal their identity. Rational
approaches for design of possible vaccine components require a better
understanding of parasite biology. In describing here the first
proteome map from a parasitic nematode, we have provided an initial
step into a clearer understanding of the H. contortus
secretion profile.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
-cyanohydroxycinnamic acid in 50% acetonitrile. Peptides were
analyzed using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied
Biosystems) operated in reflectron mode at 20 kV accelerating voltage. Tandem MS measurements were performed on an electrospray ionization (ESI) quadrupole time-of-flight instrument (Q-Tof; Micromass
Ltd., Manchester, UK) operating in positive ion mode and equipped with
a Z-spray nano-ESI source. Nano-ESI needles were prepared from
borosilicate glass capillaries (Kwik-FilTM, World Precision
Instruments Inc., Sarasota, FL) on a P-97 puller (Sutter Instrument
Co., Novato, CA). The needles were coated with a gold layer using an
Edwards Scancoat sputter-coater 501 (at 40 mV, 1 kV, for 200 s).
The capillary voltage was set at 1500 V; the cone voltage was 40 V. For
the characterization of Hc15, the collision energy was optimized for
individual peptides for optimal fragmentation. In all other cases,
instead of nano-ESI needles a nano-LC system was coupled to the Q-TOF
essentially as described in Ref. 15. Peptide mixtures were delivered to the system using a Famos autosampler (LCPackings, Amsterdam, The Netherlands) at 3 µl/min and trapped on an AquaTM C18RP
column (Phenomenex, Torrance, CA; column dimensions 1 cm × 100 µm inner diameter). After flow splitting down to 150-200 nl/min,
peptides were transferred to the analytical column (PepMap; LC
Packings, Amsterdam, The Netherlands; column dimensions 25 cm × 50 µm inner diameter) in a gradient of acetonitrile (1% per min). Fragmentation of eluting peptides was performed in
data-dependent mode, and mass spectra were acquired in
full-scan mode. For protein identification, Mascot software
(www.matrixscience.com) was used for data base searches both for
peptide mass fingerprinting and peptide sequence tagging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
View larger version (74K):
[in a new window]
Fig. 1.
Two-dimensional-SDS-PAGE gel of H. contortus ESP. ES proteins (140 µg) were
separated by isoelectric focusing on a non-linear immobilized pH
gradient from 3-10 and second dimension electrophoresis on a 12.5%
gel as described under "Experimental Procedures." Identified spots
are marked by their number; non-identified spots are
indicated with a line. Molecular mass markers are indicated
on the right (in kDa).
View larger version (140K):
[in a new window]
Fig. 2.
Silver-stained two-dimensional-SDS-PAGE gels
of H. contortus ESP. Four independent batches (70 µg) were run on a 12.5% two-dimensional gel (pI 3-10NL)
as described under "Experimental Procedures." A, batch
used for the mass spectrometry procedures (in Fig. 1). B,
C, and D, three other independent batches. For
comparison, some of the identified spots are marked in all four gels.
Molecular mass markers are indicated in kDa.
View larger version (81K):
[in a new window]
Fig. 3.
Categorized view of the identified ES
proteins. Colored representation of different categories of
proteins in the gel. All identified spots are surrounded by
circles, and analyzed spots without identification by
squares. For all spots a solid line indicates
immune recognition, and a dashed line stands for not
observed immune recognition. Double-colored spot means
double identification for a single spot. Markers are indicated on the
right (in kDa).
View larger version (97K):
[in a new window]
Fig. 4.
Immunoblotting of H. contortus
ESP. ESP was probed with a pool of sera from five animals
protected from H. contortus after several
infections. The image is composed of two exposures
(A is 30 s, and B is 5 min) to prevent
overexposure of the upper part. Some of the spot ID numbers are
indicated in the figure (some non-recognized spots are in
circles). Markers are indicated on the right (in
kDa).
ES proteins of H. contortus identified
, Only EST sequences available from H. contortus yet.
, No sequence available from H. contortus.
, Not detected previously in H. contortus
ESP.
View larger version (53K):
[in a new window]
Fig. 5.
Peptide sequences of Hc15 spots.
Alignment of the Hc15 protein (AAC47713) and other homologous ESTs with
the identified peptide sequences (shaded in gray) of the 21 spots based on the template AAC47713. The predicted signal peptide
cleavage site is indicated by an arrow.
View larger version (11K):
[in a new window]
Fig. 6.
Comparison of immune recognition of the Hc15
spots. Volumes of the Hc15 spots present in the Coomassie
Blue-stained gel and on the immunoblot (normalized as described under
"Experimental Procedures"). The spot with the highest normalized
volume in the gel or on the blot were taken as 100%; the relative
volume of all other spots is expressed in percentages on the
y-axis. Spots are ordered by increasing pI on the
x-axis. Open bars indicate the relative volumes
in the Coomassie Blue-stained gel and solid bars the volumes
on the immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank the collaborators at the laboratories of Dr. D. Knox, Scotland, and Prof. J. Vercruysse, Belgium. We thank Nicole Bakker for technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by European Union Project QLK2-CT-1999-00565.
** Supported by the Center for Biomedical Genetics and the Dutch Organization for Scientific Research (NWO).
To whom correspondence should be addressed. Tel.:
31-30-253-2582; Fax: 31-30-254-0784; E-mail: e.vries@vet.uu.nl.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212453200
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ABBREVIATIONS |
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The abbreviations used are: GI, gastrointestinal; MW, molecular weight; pI, isoelectric point; ES, excretory/secretory; ESP, ES products; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Cyp, cyclophilins; EST, expressed sequenced tag; dbEST, data base of EST; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LC-MS/MS, liquid chromatography-tandem mass spectrometry.
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