VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 12, Number 11, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2011.0957
The 31-kDa Antigen of Angiostrongylus cantonensis
Comprises Distinct Antigenic Glycoproteins
Alessandra L. Morassutti,1 Keith Levert,2,3 Andrey Perelygin,3 Alexandre J. da Silva,3
Patricia Wilkins,3 and Carlos Graeff-Teixeira1
Abstract
Human angiostrongyliasis results from accidental infection with Angiostrongylus, an intra-arterial nematode.
Angiostrongylus cantonensis infections result in eosinophilic meningitis, and A. costaricensis infections cause eosinophilic enteritis. Immunological methodologies are critical to the diagnosis of both infections, since these
parasites cannot be isolated from fecal matter and are rarely found in cerebrospinal fluid samples. A. costaricensis
and A. cantonensis share common antigenic epitopes which elicit antibodies that recognize proteins present in
either species. Detection of antibodies to a 31-kDa A. cantonensis protein present in crude adult worm extracts is a
sensitive and specific method for immunodiagnosis of cerebral angiostrongyliasis. The objective of the present
work was to isolate and characterize the 31-kDa proteins using soluble protein extracts derived from adult
female worms using both one- (1DE) and two-dimensional (2DE) gel electrophoresis. Separated proteins were
blotted onto nitrocellulose and probed using sera from infected and non-infected controls. The 31-kDa band
present in 1DE gels and the 4 spots identified in 2DE gels were excised and analyzed by electrospray ionization
mass spectrometry. Using the highest scores obtained following Mascot analysis, amino acid sequences were
obtained that matched four unique proteins: tropomyosin, the 14-3-3 phosphoserine-binding protein, a protein
containing a nascent polypeptide-associated complex domain, and the putative epsilon subunit of coatomer
protein complex isoform 2. Oxidative cleavage of diols using sodium m-periodate demonstrated that carbohydrate moieties are essential for the antigenicity of all four spots of the 31-kDa antigen. In this article we describe
the identification of the 31-kDa antigen, and provide DNA sequencing of the targets. In conclusion, these data
suggest that reactivity to the 31-kDa proteins may represent antibody recognition of more than one protein, and
recombinant protein-based assays for cerebral angiostrongyliasis diagnosis may require eukaryotic expression
systems to maintain antigenicity.
Key Words: Abdominal angiostrongyliasis—Angiostrongylus—Eosinophilic meningitis—Immunodiagnosis—
31-kDa antigen.
Introduction
T
he nematode Angiostrongylus cantonensis is the
most common causative agent of eosinophilic meningoencephalitis (Graeff-Teixeira et al. 2009). Completion of its life
cycle requires two hosts: an intermediate mollusk host and a
definitive rodent host, typically Rattus norvegicus. The first
stage larva (L1) is released in rat feces and mollusks become
infected by ingesting organic debris contaminated with L1
larvae. Inside mollusk tissues, L1 larvae develop into the in-
fective third-stage L3 larvae. Rats may ingest L3 larvae that
penetrate the mucosa, invade blood vessels, and migrate to
the meninges. In the central nervous system (CNS) the larvae
mature into young adults (fifth-stage larvae) that complete
their maturation inside the pulmonary arteries and right
cardiac cavities. Humans can be accidentally infected by ingesting L3 larvae present in contaminated water, or food that
is raw or undercooked. In humans, L3 larvae are incapable of
completing the life cycle and die in the CNS, resulting in
disease.
1
Laboratório de Biologia Parasitária da Faculdade de Biociências e Laboratório de Parasitologia Molecular do Instituto de Pesquisas
Biomédicas da Pontifı́cia Universidade do Rio Grande do Sul (PUCRS), Porto Alegre RS, Brazil.
2
Department of Biology, Georgia State University, Atlanta, Georgia.
3
Centers for Disease Control and Prevention, Atlanta, Georgia.
961
962
Cerebral angiostrongyliasis has been reported in Southeast
Asia, Africa, Australia, America (Wang et al. 2008), and recently transmission foci have been identified in Brazil (Caldeira et al. 2007; Maldonado et al. 2010) and Ecuador (Pincay
et al. 2009). In addition, angiostrongyliasis is considered an
emerging public health problem in the United States (Diaz,
2008).
Confirmed diagnosis of cerebral angiostrongyliasis is seldom possible, since larvae are typically not found in cerebrospinal fluid (CSF; Yii, 1976). Several molecular targets
have been identified as potential antigens for angiostrongyliasis immunodiagnosis (Eamsobhana and Yong,
2009). However, these targets are not widely available for
independent evaluation or testing in either clinical or epidemiological investigations. Standardization of immunological
tests requires their validation using various geographical
isolates and sera collected from patients with different coinfections to rule out potentially cross-reactive responses.
Preparation of large quantities of the target antigens is a
complicated and laborious process. Molecular cloning and the
expression of recombinant proteins represent a reliable alternative for generating sufficient amounts of well-defined antigens for use in immunodiagnostic assays.
Immuoblotting studies have identified an immunoreactive
band with an estimated molecular weight of 31 kDa that has
been considered a target for a highly-sensitive and specific
antibody detection assay for A. cantonensis infections
(Nuamtanong, 1996; Kirsch et al. 2008). Eamsobhana and
associates demonstrated that the 31-kDa glycoprotein possessed sugar residues that did not affect antibody recognition
(Eamsobhana et al. 1998); furthermore, this protein was purified and employed in enzyme-linked immunosorbent (ELISA) and dot-blot assays, resulting in 100% sensitivity and
specificity (Eamsobhana et al. 2003; Eamsobhana and Yong,
2009). Nevertheless the identity of this 31-kDa antigen is unknown.
Heterologous antigens have been used in various immunodiagnostic assays, taking into account the various shared
epitopes present between different helminth species. This
approach has also been utilized in the diagnosis of angiostrongyliasis, since A. cantonensis and A. costaricensis possess
cross-reactive antigens that can be used to diagnose infections
with either pathogen (Dekumyoy et al. 2000; Ben et al. 2010).
Since A. cantonensis is more easily maintained in the laboratory, proteins from this nematode may be used to identify
antigenic targets with potential for use in the diagnosis of
infections with either pathogen.
In the present study we characterized the makeup of the
31-kDa A. cantonensis antigen complex using one- (1DE) and
two-dimensional (2DE) gel electrophoresis, which allowed
the identification of various targets that can be used in the
development of recombinant antigens for immunodiagnostic
purposes.
Materials and Methods
Biological materials
Worms. Adult A. cantonensis worms were recovered from
experimentally-infected rats. A. cantonensis worms were
originally obtained from the Department of Parasitology,
Akita Medical School, Akita City, Japan, and have been
maintained in our laboratory since 1997. Wistar rats served as
MORASSUTTI ET AL.
definitive hosts and Biomphalaria glabrata as intermediate
hosts. Rats were infected with 104 larvae by gavage inoculation, and 42 days post-infection the animals were sacrificed
and the worms collected.
Antigen preparation. Total extract (TE) was obtained
from harvested female worms that were macerated in liquid
nitrogen and homogenized in phosphate-buffered saline
(PBS; pH 7.4). The suspension was centrifuged at 12,000 g for
1 h at 4C, and the supernatants were used to derive the TE.
Protein concentrations were determined by the Bradford assay using bovine serum albumin as a standard.
Two-dimensional electrophoresis (2DE)
An aliquot of TE that contained 60 lg of total protein was
desalted using a 2-D Clean-Up Kit (GE Healthcare, Piscataway, NJ), followed by resolubilization in DeStreak Rehydration Solution (GE Healthcare), with 66 mM DTT and 0.5%
carrier ampholytes (v/v). The samples were in-gel rehydrated
on 11-cm pH 3–11 NL or 3–6 NL IPG strips (GE Healthcare),
and isoeletric focusing was performed using an IPGphor
Isoelectric Focusing System (GE Healthcare), with voltages
increasing stepwise as follows: 500 V for 500 V h, a linear
gradient from 500–8000 V for 6500 V h, followed by a hold at
6000 V for 22,000 V h.
After isoeletric focusing, the strips were soaked for 15 min
in fresh equilibration buffer (20% v/v glycerol, 6 M urea, 1%
DTT, and 2% SDS). IPG strips were run in the second dimension on 4–12% polyacrylamide Bis-Tris gels with sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; Bio-Rad, Hercules, CA). The gels were then stained
with colloidal Coomassie blue or mass spectrometrycompatible silver stain (Mortz et al. 2001), or transferred to
nitrocellulose membranes for immunological analyses.
Western blot analysis
Resolved proteins were electro-transferred onto nitrocellulose membranes using a semi-dry trans-blot apparatus (BioRad). The membrane was washed three times with PBS-T
(0.05% Tween), and blocked with 5% skim milk for 1 h at room
temperature. The membranes were then incubated for 2 h
with a pool of sera (1:200 dilution), prepared from either 20
patients histopathologically diagnosed with abdominal angiostrongyliasis, 20 patients positive for eosinophilic meningoencephalitis, or 20 pooled serum samples from uninfected
controls. After three washes, the membranes were probed
with a secondary peroxidase-conjugated anti-human IgG
(diluted 1:8000; Sigma-Aldrich, St. Louis, MO) for 1 h at room
temperature. Diaminobenzidine (DAB) (Sigma-Aldrich;
0.05% DAB and 0.015% H2O2 in PBS, pH 7.4) was added as
developer reagent.
Tandem mass spectrometry (MS/MS) analysis
Immunoreactive spots were manually excised from 2DE
gels and subjected to in-gel tryptic digestion (Promega, Madison, WI), and mass spectrometric analysis. Electrospray
ionization (ESI) mass spectrometric analysis was performed
using a Bruker model maXis ESI-Q-TOF instrument interfaced with an on-line nanospray source (Bruker Daltonics,
Billerica, MA), to perform LC-MS/MS using a U3000 HPLC
THE 31-KDA ANTIGEN OF Angiostrongylus cantonensis
configured for nanoliter-per-minute flows. The Dionex
U-3000 nanobore HPLC was configured with dual ternary
pumps with the flow output of one pump split using a calibrated 1:1000 splitter with an active flow control. This system
used a pulled-loop auto sampler configured with a 20-lL
sample loop. A desalting trap column (0.3 · 5 mm, 5 lm C18
PepMap 120 A; Dionex, Sunnyvale, CA), and C18 PepMap
(0.075 · 150 mm, 3 lm, 120 A; Dionex) were used. The solvents
used were 0.1% formic acid in water, and 80% acetonitrile/
0.1% formic acid. The gradient was 2–55% in 90 min. The eluent from the analytical column was introduced into the
maXis using the Bruker on-line nanospray source. The source
was operated at a spray voltage of 900 V with a drying gas of
nitrogen flowing at 6 L/min. The capillary temperature was
set to 150C. The mass spectrometer was set to acquire line
spectra of m/z 50–1900. MS/MS data were acquired in an
automated fashion using the three most intense ions from the
MS scan with precursor active exclusion for 90 sec after three
spectra were acquired for each parent ion. MS data were acquired at a scan speed of 3 Hz and MS/MS data were acquired
at a scan speed of 1–1.5 Hz, depending on the intensity of the
parent ion. MS internal calibration was achieved by the use of
a lock mass (HP-1222; Agilent Technologies, Santa Clara, CA).
Collected data were processed by Data Analysis (Bruker
Daltonics), to produce deconvoluted and internally-calibrated
data and saved as an xml peaklist, which was searched
against the NCBInr database with the Mascot on-line program
(http://www.matrixscience.com). The data were acquired in
data-dependent mode (DDA), and multiple-charged peptide
ions (+ 2 and + 3) were automatically mass selected and dissociated in MS/MS experiments. Mascot search parameters
allowed a maximum of one missed cleavage, carbamidomethylation of cysteine as fixed modifications, methionine
oxidation as a variable modification, peptide tolerance of
0.2 Da, and MS/MS tolerance of 0.2 Da. The significance
threshold was set at p < 0.05, and identification required that
each protein contain at least one peptide with an expected
value < 0.05.
Oxidation of the carbohydrates
Carbohydrate moieties were oxidized using sodium periodate to investigate their antigenicity. Proteins were electrotransferred onto nitrocellulose membranes, washed three
times with PBS-T, and incubated for 30 min with 100 mM
NaOAc (pH 5.0). The membranes were incubated with a sodium m-periodate solution (20 mM NaIO4 diluted in 100 mM
NaOAc), and kept at 37C for 1 h in the dark. After washing
with 100 mM NaOAc, the membranes were incubated with
50 mM NaBH4 in PBS-T for 30 min at room temperature, and
developed as described above (Western blot analysis).
Results
Immunodiagnostic targets were identified in crude female
worm extracts using 1DE gel electrophoresis. This analysis
identified a 31-kDa band (Fig. 1), which is consistent with
previously published data (Eamsobhana et al. 1998). However, 2DE resolved the 31-kDa band into four distinct antigenic spots in the acidic region that appeared elongated and
diffuse in shape. These spots were recognized by sera from
angiostrongyliasis patients, but not by sera from uninfected
controls (Fig. 2). A better separation of immunoreactive spots
963
FIG. 1. Identification of 31-kDa molecules on 1DE. Female
worm total protein extract (TE) was resolved in 1DE gel and
probed on Western Blot with: lane 1, pool of positive controls
for abdominal angiostrongyliasis; lane 2, pool of normal
human sera. The square represents the band excised from
the gel for MS analyses. (Color image available at www
.liebertpub.com/vbz).
distributed in the pH range 4–5 was obtained when a 3–6 pHNL strip was employed (Fig. 2). The sugar moieties of the 31kDa glycoprotein are essential to maintain antigenicity of the
protein, since carbohydrate oxidation revealed no recognition
by infected sera (Fig. 2f ).
The 31-kDa band detected on 1DE gels and the four antigenic spots identified on 2DE gels were excised and digested
with trypsin for further analysis using MS/MS ESI-Q-TOF. The
Mascot score was used to determine the probability that the
observed matches between the experimental data and the database sequences were not random. Analysis of mass spectrometry data for 1DE (Table 1), and three of the spots from
2DE (Table 2), identified amino acid sequences that matched
several unique proteins or protein domains in the database.
Since there are few Angiostrongylus sequence data available,
most protein identifications rely on homologous sequences
from closely related organisms within the database. No peptide
matches were obtained from spot 1. Amino acid sequences of
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MORASSUTTI ET AL.
FIG. 2. Identification of the 31-kDa protein complex on 2DE. (a) TE pH range 3–11, silver staining. (b) TE pH range 3–11
Western blot using sera derived from pooled A. costaricensis and A. cantonensis infection. (c) TE pH range 3–6 sera derived
from A. costaricensis infection. (d) TE pH range 3–6 sera derived from A. cantonensis infection. (e) Normal human sera. (f)
Carbohydrate oxidation. (g) Coomassie blue staining. The four spots of the 31-kDa proteins are indicated on the pH 3–11
strip. Circles represent the spots excised for MS analyses. (Color image available at www.liebertpub.com/vbz).
two proteins, the 14-3-3 protein and the NAC domain-containing protein, were obtained from all three of the spots in
which protein identifications were made. The highest Mascot
scores for the 14-3-3 protein were detected with database sequences derived from the 14-3-3 proteins of Ancylostoma caninum, and a 28-kDa protein of Meloidogyne incognita, a plant
nematode. Peptide sequences obtained from spot 4 matched
sequences from the A. cantonensis putative epsilon subunit of
the coatomer protein complex isoform 2 (33 kDa). Amino acid
sequences from spots 2 and 3 matched heat shock proteins of
Loa loa and Haemonchus contortus. Peptide sequences also matched sequences from two other A. cantonensis proteins (Table 2).
Discussion
The identified peptides present in the 1DE band did not
correspond to the peptides identified in the 2DE gel spots.
This is likely a result of the better separation obtained during
2DE, which allowed for improved isolation of the antigenic
protein. A comparison of the 2DE immunoblot and the total
protein stain of the 2DE gel demonstrates that most of the
proteins in the 31-kDa range are not immunogenic (Fig. 2).
Thus, mass spectrometry analysis following 2DE gel separation allowed for more specific identification of those proteins
with antigenic activity. One of the peptides obtained by 1DE
showed the highest Mascot score match to the 33-kDa
tropomyosin from Heligmosomoides polygyrus, a rodent
nematode.
Tropomyosin is a highly conserved muscle protein with
potent allergenic potential. This protein is known to induce
IgE production in parasitic nematode infections such as anisakiasis and onchocerciasis (Sereda et al. 2008), but due to
similarities between invertebrate tropomyosins, IgE antibodies cross-react with tropomyosins from other species, and
THE 31-KDA ANTIGEN OF Angiostrongylus cantonensis
965
Table 1. Proteins Identified Following One-Dimensional Gel Electrophoresis
Peptide sequence
R.ANTIEAQLK.E
R.LEDELVHEK.E
K.IVELEEELR.V
K.LAMVEADLER.A
K.EAQMLAEEADR.K
R.MTLLEEELER.A
K.VQEAEAEVAALNR.R
K.EVDRLEDELVHEK.E
K.AQEDLATATSQLEEK.D
R.ALQASCLAK.W
K.GILAADESTGSMEK.R
R.GAAQNIIPAATGAAK.A
R.VPTPDVSVVDLTCR.L
K.AGFAGDDAPR.A
K.DSYVGDEAQSK.R
K.QEYDESGPSIVHR.K
R.VAPEEHPVLLTEAPLNPK.A
K.ITETVLSYCYR.A
K.KPWALTFSYGR.A
K.EPDWVQSER.E
R.HLVGIADDNKDGK.L
R.DWIMPVGFDHAEAEAR.H
R.LLLEQMSQDPGAVR.E
K.LMEFQR.A
R.DYGVLKEDDGIAYR.G
R.LVQAFQFVDK.H
R.QITVNDLPVGR.S
Organism with
homologous target
Protein name (Da)
Scorea
Coverage %
Tropomyosin (33)
Heligmosomoides
polygyrus
262
36
Hypothetical protein
CBG15316 (39)
Glyceraldehyde-3-phosphate
dehydrogenase (36)
Actin-2 (41)
Caenorhabditis briggsae
172
9
Dictyocaulus viviparus
131
10
Ascaris suum
134
15
Aldolase (39)
Haemonchus contortus
88
6
CALUmenin (calcium-binding
protein) homolog family
member (36)
TPR domain (31)
Caenorhabditis elegans
87
12
Brugia malayi
78
7
Peroxiredoxin (21)
Ascaris suum
75
17
a
Mascot score is - 10 · log (P), where P is the probability that the observed match is a random event. Mass is molecular weight in
kilodaltons.
The band on the area of 31-kDa was excised from the gel and subjected to trypsin digestion, and then analyzed by mass spectrometry for
protein identification.
therefore tropomyosins are not useful for diagnostic purposes
(Sereda et al. 2008). However, specificity may be further tested
by epitope mapping of this protein.
The 14-3-3 proteins are dimeric phosphoserine-binding
proteins, which are members of a family of acidic regulatory
molecules that participate in signal transduction, transport,
and regulation, of several eukaryotic biochemical processes
(Obsilova et al. 2008; Mrowiec and Schwappach 2006). In
some parasites, such as Echinococcus multilocularis and Schistosoma mansoni, 14-3-3 proteins have been described to be
immunogenic, and therefore have been promoted as potential
vaccine targets (Schechtman et al. 2001; Siles-Lucas et al. 2008;
Wang et al. 2009). In addition, the 14-3-3 protein has been
identified as a prominent product in the S. mansoni female
worm reproductive system (Schechtman et al. 2001). This may
explain previous findings showing the female reproductive
system as the main source of antigenic targets useful for the
diagnosis of abdominal angiostrongyliasis caused by A. costarecensis (Bender, 2003). Moreover, these proteins might directly interact with immune system components, since these
interactions have been modulated by 14-3-3 proteins secreted
by Toxoplasma gondii and E. granulosus (Assossou et al. 2004;
Siles-Lucas et al. 2008).
Coatomer proteins (COP) form a coat protein complex that
mediates protein transport between the Golgi compartment
(COPI), endoplasmic reticulum (COPII), and the plasma
membrane (clathrin/adaptin; Lee and Goldberg, 2010). COPI
from rat liver peroxisomes contains stoichiometric amounts of
seven subunits, including alpha-COP (160 kDa), beta-COP
(107 kDa), beta-prime-COP (102 kDa), delta-COP (57 kDa),
epsilon-COP (36 kDa), gamma-COP (97 kDa), and zeta-COP
(20 kDa; Lay et al. 2006). To date, there is no evidence that
these proteins can induce immune responses. However, a
crystallographic analysis showed that the epsilon-COP and
alpha-COP complex were exposed on COPI vesicles, thereby
facilitating their extracellular targeting (Hsia and Hoelz,
2010), suggesting that the complex might be attached to the
Golgi membrane while transporting proteins that are eventually exposed to the host’s immune system.
The nascent polypeptide-associated complex (NAC) is associated with ribosomes and involved in nascent polypeptide
chain folding (Hayashi et al. 2011). NAC is also implicated in
the targeting of ribosomes to the ER membrane (Wiedmann
and Prehn, 1999).
Angiostrongylus 31-kDa antigen was first described as a
glycoprotein, and its antigenicity was considered independent of carbohydrate moieties (Eamsobhana et al. 1998). In the
present study m-periodate treatment eliminated the recognition by sera from infected individuals (Fig. 2f ), demonstrating
that carbohydrate moieties are essential for antibody
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MORASSUTTI ET AL.
Table 2. Identification of the 31-kDa Proteins Excised from A. cantonensis Two-Dimensional
Gel Electrophoresis Preparations
Spot no.
2
3
4
Peptide sequence
K.ADLVNNLGTIAK.S
K.EDQTEVLEER.R
R.ELISNSSDALDK.I
K.TLTIMDTGIGMTK.A
R.YQALTEPAELESGK.E
R.VLSSIEQK.T
K.DSTLIMQLLR.D
K.SQQSYQEAFDIAK.D
K.SPGSDTYIVFGEAK.I
K.NILFVINKPDVYK.S
K.AGIVFTGK.G
K.YMNQFTK.A
K.LEVGLFDTYR.C
R.YDDMAQSMK.K
K.DSTLIMQLLR.D
R.DICQDVLNLLDK.F
K.VTELGAELSNEER.N
K.SQQSYQEAFDIAK.D
K.MQPTHPIR.L
K.SPGSDTYIVFGEAK.I
K.NILFVINKPDVYK.S
K.EDQTEVLEER.R
R.ELISNSSDALDK.I
K.TLTIMDTGIGMTK.A
R.YQALTEPAELESGK.E
R.LAEYQNATDK.Q
K.ASLVLNEISER.T
K.AKENLFDELVAA
K.DAEALLHEAQLR.D
R.DINPNHPWVIDLK.A
R.VISSIEQK.T
K.DSTLIMQLLR.D
K.VTELGAELSNEER.N
K.SQQSYQEAFDIAK.D
R.VGPGIGEYIFDK.E
K.FLDEQVESIAEIAK.M
K.ASAANDPHMSDFLES
K.F
K.LAQIISQFER.A
R.ALTSVNSLIEGVVQK.
M
K.SPGSDTYIVFGEAK.I
Organism with
homologous target
Scorea
Coverage
(%)
Heat shock protein 90 (80)
Heat shock protein 90 (81)
Loa loa
Haemonchus contortus
201
144
7
5
14-3-3 product (29)
Meloidogyne incognita
111
14
NAC domain-containing
protein (24)
PCNA (proliferating cell
nuclear antigen) (29)
Brugia malayi
136
12
Caenorhabditis briggsae
98
11
14-3-3 protein isoform 2 (28)
Ancylostoma caninum
234
34
NAC domain containing
protein (24)
Heat shock protein 90 (80)
Brugia malayi
171
12
Loa loa
128
7
Putative epsilon subunit
of coatomer protein
complex isoform 2 (33)
Angiostrongylus
cantonensis
289
30
14-3-3b protein (28)
Meloidogyne incognita
277
29
Putative ferritin protein
2 (6.8)
Angiostrongylus
cantonensis
155
67
Putative uncoordinated
protein 23 (11)
Angiostrongylus
cantonensis
150
39
NAC domain containing
protein (24)
Brugia malayi
92
6
Protein identified (kDa)
The spots were excised from respective gels and subjected to trypsin digestion and then analyzed by mass spectrometry for protein
identification.
a
Mascot score is - 10 · log (P), where P is the probability that the observed match is a random event. Mass is the molecular weight in
kilodaltons.
recognition of the 31-kDa protein. The better separation of the
proteins by 2DE allowed us to distinguish the specific antigenic spots corresponding to the previously described 31-kDa
antigen, which was first detectable as a single band in 1DE
preparations. Another explanation for this conflicting result is
the amount of antigen loaded in the previous characterization
of the 31-kDa protein, for which 10 lg were applied in the gel,
while here only 3 lg in 2DE gels were loaded. The largest
amount of antigen in 1DE gels could contain unspecific sugars
being recognized by infected sera. This finding has strong
implications for the choice of appropriate vectors to express
such recombinant targets for the development of diagnostic
tests.
In order to achieve the complete DNA sequence of each
identified protein for further recombinant protein studies, we
sequenced the DNA in a random way using the parallel
THE 31-KDA ANTIGEN OF Angiostrongylus cantonensis
967
sequencing approach (Morassutti et al. in preparation). The
NAC domain was revealed to be composed of 185 amino
acids, while 14-3-3 protein had 249 amino acids. The sequences were published in Genebank under the numbers GI:
341864443 for NAC domain-containing protein, and GI:
341864441 for 14-3-3.
Analysis of the data presented in this report raises the
question of whether the reactivity observed with the native
parasite 31-kDa molecules is due to reactivity with one or
more of the putative proteins identified by MS/MS. Interestingly, the NAC domain-containing protein, epsilon-COPI,
and 14-3-3 protein, all play putative biological roles in protein
translocation. Therefore we hypothesize that they may form a
cell membrane complex, which may have led to co-isolation of
these proteins in the original TE preparation, and may ultimately explain how all three could be antigenic.
In conclusion, the set of proteins with an estimated
molecular weight of 31 kDa identified by 2DE consisted of
several potential antigens. Cloning of the corresponding
cDNAs and expression of these proteins is the next critical
step to further define their roles as diagnostic targets, and may
represent a tool to better understand host-Angiostrongylus
interactions.
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Acknowledgments
Financial support was provided by CNPq, CAPES, FAPERGS, and APHL-USA. is the recipient of a CNPq PQ 1D
fellowship, and of grants 300456/2007-7 and 477260/2007-1
(Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico do Brasil).
Author Disclosure Statement
No conflicting financial interests exist
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MORASSUTTI ET AL.
Address correspondence to:
Alessandra L. Morassutti
Laboratório de Biologia Parasitária da Faculdade de Biociências
e Laboratório de Parasitologia Molecular do Instituto de
Pesquisas Biomédicas da Pontifı́cia Universidade
do Rio Grande do Sul (PUCRS)
Av Ipiranga 6690
90690-900 Porto Alegre RS
Brasil
E-mail: almorassutti@gmail.com