Toxocara canis is a parasitic ascarid nematode that
infects large numbers of dogs worldwide. Adult worms infest the gut and
lay large numbers of eggs that, on excretion, embryonate and lie
dormant in the soil. Upon ingestion by non-canid paratenic hosts,
tissue-invasive infective stage larvae are released(1) . In
humans these cause visceral and ocular disease
syndromes(2, 3, 4) . Infective larvae have
been shown to persist up to 9 years(5) , demonstrating their
remarkable facility for evading host immune responses.
T. canis infective larvae are developmentally arrested, and large numbers
may easily be cultured in vitro for long periods(6) .
For this reason this organism has been used as a laboratory model for
characterizing the nematode surface in order to understand the
mechanisms of escape from immune attack(7) . Cultured larvae
release five major glycoprotein excretory/secretory (TES) (
)antigens(8, 9, 10) , which are
well characterized
biochemically(8, 10, 11, 12, 13, 14) ,
as well as a number of minor components that have been little studied.
Infective larvae also possess a glycocalyx-like surface coat that
overlies the cuticle, comprised largely of a 120-kDa mucin-like
glycoprotein, TES-120(15, 16) . (
)Monoclonal antibodies reactive to TES-120 bind to
secretory glands within the body, from which it is thought that coat
components are ducted to the surface via the buccal cavity and
secretory pore(18) .
The mRNA for TES-120 is hyperabundant,
representing approximately 10% of a cDNA library, and is trans-spliced with the nematode 22-nucleotide spliced leader
(SL) sequence.
cDNA amplified from mRNA by PCR using SL and
oligo(dT) primers reveals the TES-120 hyperabundant band, together with
a less abundant mRNA species. We report here the isolation of cDNA
corresponding to this second SL-bearing message (TcSL-2), demonstrate
its stage-specific expression in infective larvae as the secreted
antigen TES-26, and describe the primary structure of the protein.
Comparisons to previously known sequences reveal that TcSL-2 belongs to
a recently identified family of genes from a range of organisms, some
of which bind phosphatidylethanolamine with high specificity.
MATERIALS AND METHODS
Parasites
Live adult T. canis were
obtained from the intestinal tract from post-mortem dogs. Male worms
were snap frozen and stored at -70 °C prior to use. Eggs were
obtained from uteri excised from gravid females. Hatching of eggs and in vitro culture of infective larvae was as described
previously(19) .
Preparation of RNA and DNA
Approximately 1,000
infective larvae that had been in culture for at least 3 days were
suspended in a drop of RPMI 1640 medium and snap frozen in liquid
nitrogen. This was ground to a fine powder in a pre-cooled mortar with
a pestle, and RNA was extracted using the acid guanidinium thiocyanate
method (20) and resuspended in 10 µl of H
O. DNA
was prepared from 10 adult males by grinding to a fine powder under
liquid nitrogen with a pestle and mortar and resuspending in 5 ml of
lysis buffer (100 mM NaCl, 100 mM EDTA, and 10 mM Tris/HCl, pH 7.5, supplemented with 1% SDS and 1 mg/ml proteinase
K). This was incubated at 50 °C for 3 h with gentle agitation, then
extracted with an equal volume of warm phenol (37 °C), and spun at
20,000
g for 10 min at 20 °C. After two
extractions with phenol/chloroform and one with chloroform, RNase was
added to the aqueous phase to a concentration of 250 µg/ml, and the
sample was incubated for 20 min at room temperature with gentle
agitation. After another chloroform extraction DNA was ethanol
precipitated from the aqueous phase.
Genomic DNA Library Construction
Aliquots of 5
µg of T. canis DNA were partially digested using RsaI, HaeIII, and AluI. These were pooled
and protected from EcoRI restriction using EcoRI
methylase. EcoRI linkers were then ligated on and digested
with EcoRI. The inserts were then ligated into the EcoRI site of the expression vector
gt11. After packaging
(Amersham packaging extract) a titer of 10
plaque-forming
units/ml was obtained, with 98% of clones containing genomic DNA
inserts.
Antibody Screening of Expression Library
Anti-TES
polyclonal sera were raised in CBA/Ca and BALB/c mice. These sera were
found to bind to all components of TES in Western blots. For screening,
sera were first preabsorbed against sonicated Escherichia coli and then used at a dilution of 1:25.
cDNA Preparation
To prepare cDNA, 1 µl of
total RNA solution was used with a GeneAmp RNA PCR kit (Perkin-Elmer)
with Pfu polymerase (Stratagene Ltd., United Kingdom) substituting for
Amplitaq in the PCR. RNA PCR has been described
previously(21, 22) . The cDNA was amplified at 94
°C for 1 min, 60 °C for 3 min, and 72 °C for 15 min for 35
cycles. For first strand cDNA synthesis, primer DGDT containing a 5` BamHI restriction site
(5`-AATTCGGATCCCCCGGG(T)
-3`) was used. Three
second primers were added for PCR: DGSL1
(5`-GGGCGGCCGCGGTTCAATTACCCAAGTTGGAG-3`), DGSL2A
(5`-GGGCGGCCGCGGGTTTAATTACCCAAGTTGGAG-3`), and DGSL3
(5`-GGGCGGCCGCGGGGTTTAATTACCCAAGTTTGAG-3`). These primers
contained a NotI site positioned so that each primer would
result in PCR products being in a different frame relative to the lacZ gene after ligation into the plasmid pBluescript
SK(+). Sequence changes were introduced into the SL1 sequence of
DGSL1 and DGSL2A to remove termination codons.
PCR of TcSL-2 cDNA
Total cDNA prepared as
described above was used as a template. Two primer pairs were used:
DG9a (5`-GCGCGGCGGCCGCGTCAATTAACCTCTGCCAGAAC-3`), containing 21
bases of the sense genomic sequence determined for clone TcG9 and a 5` NotI site with the oligo(dT) primer DGDT, and DG9c
(5`-GCGCGGCGGCCGCGTTCTGGCAGAGGTTAATTGA-3`), containing 21 bases
of the antisense TcG9 sequence and a 5` NotI site with
DGSL-2A. Amplification was at 94 °C for 1 min, 60 °C for 2 min,
and 72 °C for 3 min for 35 cycles.
Southern Blot Analysis
DNA was electrophoresed on
0.8% agarose gels and transferred to Hybond-N membrane (Amersham
Corp.). DNA probes were labeled with digoxygenin by random priming and
detected after hybridization using a digoxygenin DNA Labeling and
Detection kit (Boehringer Mannheim). The chemiluminescent substrate
AMPPD was used as a substrate for the anti-digoxygenin
hapten-conjugated alkaline phosphatase. Hybridization and washing was
carried out at high stringency as described in the Boehringer kit
protocol.
DNA Sequencing
The nucleotide sequences of the
cDNAs were determined using the Sanger dideoxy chain termination method
with double-stranded DNA (23) using
S-dATP and a
T7 sequencing kit (Pharmacia Biotech Inc.). The sequences of both
strands were determined from two copies of each of the two portions of
the TcSL-2 cDNA. Where sequence differences were seen a third copy was
sequenced across the region in question.
Expression of TcSL-2 (TES-26) Protein
Two
expression systems were used to prepare TcSL-2 (TES-26) protein by
inserting a PCR-amplified fragment extending from Gln-21 to the
C-terminal Ala-263, thereby omitting the putative signal peptide. For
pET15b (Novagen), the 5` primer contained a restriction site for NdeI (AGTGGACATATGCAACAGTGTATGGACAGT), and the 3`
primer contained a BamHI site
(TACGGATCCTTAGGCCTGCGATCGATAGAA). Cloning of this insert into a
site under control of the T7 promoter was accomplished in E. coli TB-1 cells, which lack the T7 RNA polymerase gene; expression was
achieved in BL21 cells carrying the DE3 bacteriophage, which carries
T7. The recombinant TcSL-2 was found in the soluble fraction of a
bacterial lysate and was purified on a NiSO
-charged
chelating Sepharose column by elution at pH 3.8. A second expression
system, pMAL (New England BioLabs Inc.) produced a form of TcSL-2,
fused with maltose-binding protein (MBP), which performed better in
enzyme-linked immunosorbent assays (see below). For this vector, the
same 3` PCR primer containing a BamHI site was combined with a
5` primer utilizing a BglI restriction sequence.
Immunological Assays
Western blot analysis was
performed on TES antigens as described previously(18) .
Briefly, 5 µg of TES/track was electrophoresed on a 5-25%
polyacrylamide gradient gel, transferred onto nitrocellulose paper,
blocked in 5% fetal calf serum, and incubated for 2 h with 1/400
dilution of sera from normal or TES- or TcSL-2-immunized mice. Blots
were then probed with 1/2500 alkaline phosphatase-conjugated anti-mouse
Ig and finally incubated in substrate, nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma). For
enzyme-linked immunosorbent assays, MBP-TES-26 or control MBP from
unrecombined pMAL were coated onto plates at 1 µg/ml overnight, and
sera were tested at 1/200, incubating for 2 h at 37 °C. Wells
subsequently received peroxidase-conjugated anti-human IgG, followed by
2,2`-di-(3-ethylbenzthiazoline sulfonic acid) substrate (Kirkegaard and
Perry Laboratories, Inc.). Absorbances for each serum on MBP and
MBP-TES-26 were read at 405 nm, and the net reactivity to TES-26 was
calculated by subtraction.
Lipid Binding Assay
A binding assay for
phospholipids was adapted from that described for other members of the
PEBP family(24, 25) . Lipidex-1000 beads (Packard
catalog number 6008301) were first washed in 10 volumes of 10 mM potassium phosphate buffer, pH 7.4, and then poured into 2-ml
columns in siliconized glass. Radiolabeled
[
C]phosphatidylethanolamine (DuPont NEN catalog
number NEC-783) was mixed with either recombinant 26-kDa MBP fusion
protein or MBP alone; all mixtures contained 500 pmol of PE and
0-10 µg of protein in a total volume of 300 µl of 10
mM potassium phosphate buffer, pH 7. Samples were incubated in
siliconized glass for 15 min at 37 °C and then cooled on ice before
loading onto parallel Lipidex columns. Columns were washed with 5 ml of
phosphate buffer, and the flow-through fraction was collected; this
fraction contains lipids sequestered by proteins. Free lipids bound to
the column were then eluted with 5 ml of methanol at room temperature.
Aliquots from each fraction were assayed by liquid scintillation
counting, and the proportion of protein-bound PE was calculated as a
percentage of total counts recovered. Additional mixtures were set up
in which proteins were preincubated with 5 nmol of unlabeled PE or
phosphatidylcholine for 15 min at 37 °C.
Computer Analysis of Sequences
Nucleic acid
sequences were analyzed using MacMolly (Softgene) and the University of
Wisconsin Sequence Analysis (UWGCG) package at the SEQNET facility
(Daresbury, UK). Data base searches for amino acid sequence
similarities were performed using the Lipman and Pearson algorithm (26) FASTA on the SwissProt data bank, TFASTA on a translated
data base of DNA sequences (GenEMBL), and by the BLAST E-mail server (27) to search a collection of data bases including
GenBank
, SwissProt, and dbest.
RESULTS
Preparation of cDNA from T. canis Adult Males and
Infective Larvae
cDNA was synthesized from unfractionated RNA
prepared from T. canis infective larvae and from adult males.
For first strand cDNA synthesis a
poly(A)
-complementary primer carrying a 5` EcoRI restriction enzyme site (DGDT) was used. A second primer
containing the conserved nematode 5` spliced leader (SL1) sequence and
a 5` NotI site was added, and PCR was carried out. It has been
demonstrated in the closely related nematode Ascaris lumbricoides that most mRNA species carry a 5` SL1 sequence(28) ; the
occurrence of spliced leaders in T. canis was previously
unknown. The cDNA preparations were separated by agarose gel
electrophoresis (Fig. 1A). The cDNA profiles from adult
male and infective larvae were similar apart from two distinct bands
visible only in infective larval cDNA. The upper band was approximately
1.1 kb in size; analysis of the lower 0.73-kb band is described
elsewhere.
Figure 1:
RNA PCR
reveals hyperabundant stage-specific transcripts among total
SL
cDNA. A, ethidium bromide-stained agarose
gel with cDNA prepared by RNA PCR using as template unfractionated RNA
from infective larvae (lane 2) and adult males (lane
3). Primers used were complementary to the 5` spliced leader (SL1)
and the 3` poly(A)
tail. Lane 1 contains 1-kb
ladder size standards (Life Technologies Inc.). B, Southern
blot of the same gel probed with digoxygenin-labeled DNA from a genomic
excretory/secretory antigen clone, TcG9.
Hybridization of TcG9 to cDNA and genomic
DNA
In a parallel experiment, a 1.3-kb genomic
gt11 clone,
TcG9, was isolated by immunoreactivity to an anti-TES polyclonal serum
raised in mice. To determine if mRNA from this antigen was represented
in the SLcDNA, TcG9 was used to probe a Southern blot of adult and
infective larva-derived cDNA (Fig. 1B). It was found
that hybridization occurred to a single band approximately 1.1 kb in
size in the infective larval cDNA. No hybridization to the adult male
cDNA was observed. Digoxygenin-labeled TcG9 was hybridized to a
Southern blot of T. canis genomic DNA digested with EcoRI at high stringency. A single band of approximately 3 kb
was seen (data not shown), indicating the presence of a single copy of
the gene.
PCR Amplification of the 1.1-kb cDNA Using
TcG9-complementary Primers
273 bp of the 5` end of TcG9 were
sequenced, and upstream (DG9a) and downstream (DG9c) primers were
designed from the 5` 21 bp. PCR was carried out on template cDNA from
adults or infective larvae. Using infective larval cDNA and DG9a with
DGDT, an 850-bp product was obtained (Fig. 2). This was not
obtained when adult cDNA was used as a template. The upstream primer
(DG9c) was used in PCRs with three SL1-complementary primers. When
infective larval cDNA was used as a template, a 235-bp product was
obtained in each case (Fig. 2). Where adult cDNA template was
used, no such product was seen. Both fragments were ligated into the
plasmid pBluescript SK(+). A plasmid containing the 850-bp
fragment was digoxygenin-labeled and used to probe TcG9 on a Southern
blot, and hybridization occurred (data not shown). These results
confirm the correspondence of the cloned 1.1-kb cDNA with the TcG9
genomic sequence and indicate that expression of the gene from which
the 1.1-kb mRNA is derived is stage-specific.
Figure 2:
Reverse transcriptase PCR amplification of
cDNA using primers designed from TcG9 sequence. Lane 1, size
standards (1-kb ladder, Life Technologies, Inc.); lanes 2 and 3, reverse transcriptase PCR using as template cDNA prepared
from infective larvae and as primers DGDT and DGK9a (lane 2)
and DGSL1 and DGK9c (lane 3); lanes 4 and 5,
reverse transcriptase PCR using adult cDNA with the primers DGDT and
DGK9a (lane 4) and DGSL1 and DGK9c (lane
5).
DNA Sequence Analysis
Using double-stranded
sequencing methods two copies of each of the 5` and 3` PCR products
were sequenced along the full length of both strands (Fig. 3). A
group of overlapping clones was assembled of 1,056 bp, including SL1
and excluding the 3` poly(A)
tail. The sequence
contained a single open reading frame encoding a protein of 263 amino
acid residues. The start codon was located 9 bases downstream of the
end of the spliced leader sequence. Analysis based on a weight matrix
consensus (29) indicated that the first 20 residues comprised a
signal peptide for targeting secretion. The predicted protein molecular
mass was 28,034, but it was 26,007 after removal of the signal peptide
with a pI of 8.5 in each case. The most abundant residues in TcSL-2 are
alanine (11.0%), asparagine (8.8%), valine (8.8%), threonine (7.6%),
and serine (7.2%).
Figure 3:
DNA
and protein sequence of abundant 1.1-kb infective larval cDNA. The
amino acid residues of the putative N-terminal hydrophobic secretory
signal peptide are shown in italics. The 36-residue NC6 motif
repeats are highlighted by open boxes. The potential
transmembrane sequence is marked with solid boxes, and the
flanking pairs of charged residues are circled. The putative
phosphatidylethanolamine-binding site identified in rat and bovine
homologues is shown in the shaded
box.
A hydropathy plot shows the protein to be largely
hydrophilic apart from the N-terminal signal peptide and a 20-amino
acid region at residues 94-113 (data not shown). The presence of
charged residues on either side of this region suggests that this may
be a transmembrane domain, although no direct evidence for membrane
association exists for this protein. The N-terminal domain also
contains an NPT triplet at amino acids 78-80, but the presence of
proline generally prohibits N-glycosylation because the
asparagine and hydroxyamino acid side chains cannot be juxtaposed.
Antibody Reactivity of Recombinant and Native
Proteins
Recombinant TcSL-2 was expressed as a full-length
protein from Gln-21 to the C-terminal Ala-263 in two vectors, pET15b
and pMAL. The former was used to prepare recombinant protein for
immunization of mice, and sera collected after secondary challenge were
used to probe a Western blot of TES antigens. The TcSL-2-specific
antiserum reacted with a 26-kDa band distinct from the major TES-32
glycoprotein, which was designated TES-26 (Fig. 4). The 26-kDa
antigen showed no shift in mobility when anti-TcSL2/TES-26 was used to
probe N-glycanase-treated TES (data not shown). In testing for
naturally elicited antibodies in human infection with T.
canis, it was found that the pMAL-expressed protein fused to
maltose-binding protein performed better than the pET product. Sera
from a total of 118 patients with toxocariasis (as confirmed by A >0.5 by enzyme-linked immunosorbent assay on TES) were
screened, together with 38 low titer Toxocara-reactive sera
and 8 normal controls. Only 18 individuals (11.5%) were seropositive
against TES-26, but one normal control was also reactive (data not
shown). The relatively low frequency and nonspecific reactivity to
TES-26 excludes it from consideration as a diagnostic antigen. The same
antibodies have been tested in immunofluorescence assays in which
strong staining of the anterior surface of the larvae was observed
(data not shown). Because the nematode cuticle and surface coats are
extracellular structures, this pattern supports the contention that
TES-26 follows a secretory pathway.
Figure 4:
Western blot reactivity of TES antigens
with normal mouse serum (NMS), antibody to pET-expressed
TcSL-2 protein (Anti-TcSL2), and antibody to TES (Anti-TES). The major TES glycoproteins, TES-32 and TES-120,
are indicated, together with the TES-26 band reactive with anti-TcSL-2
antibodies.
Sequence Data Base Searches
Seven protein
sequences showing a high degree of similarity to the 1.1-kb cDNA were
identified by data base searching: the filarial nematode Onchocerca
volvulus Ag16 antigen(30, 31) , the 21-kDa bovine
brain cytosol PEBP(24, 32, 33) , the 23-kDa
rat sperm plasma membrane PEBP(25) , identical to the 23-kDa
morphine binding protein found in brain and liver cytosol(34) ,
a macaque epididymal homologue(35) , a homologous sequence from
human HepG3 cDNA(36) , an antennal cDNA from Drosophila(37) , and the Saccharomyces cerevisiae 24-kDa TFS1 protein(38) . Similarities within this gene
family are summarized in Fig. 5. The Ag16 cDNA sequence from O. volvulus(30) shows a frame shift due to a 1-bp
deletion relative to the other members of the family at nucleotide 454
(residue 136, Fig. 5). A strong residual similarity can be found
between the -1 frame and the rest of the family almost to the end
of the protein(37) , indicating that this deletion is likely to
be a recent event.
Figure 5:
Sequence alignment of TcSL-2 and seven
homologues. Residues identical to TcSL-2 in at least 5 of the 7
homologues are marked with solid boxes. The region in the
TcSL-2 sequence with similarity to von Willebrand's factor (amino
acids 107-114) is denoted in the shaded box; identical
residues within this region are indicated with asterisks. Oncho, Ag16, a cuticular and secreted antigen from the
filarial nematode parasite O.
volvulus(30, 31) ; Drome, an antennal
protein from Drosophila melanogaster(37) ; Human: a protein expressed by HepG3 hepatoma
cells(36) ; Macaca, an epididymal protein from Macaca fasciculatis(35) ; Bovine, Bov21, a
bovine cytosolic phosphatidylethanolamine-binding protein(33) ; Rat, a 23-kDa epididymal, brain, and liver protein also
identified as a morphine-binding protein(25, 34) ; Yeast, S. cerevisiae TFS1, 24-kDa suppressor of CDC25
mutation, also termed DKA1 and
NSP1(38) .
The sequence alignment reveals several blocks of
highly conserved sequence, especially between residues 240 and 253. The
run of hydrophobic residues YV(W/F)LVY at 245-250 (Fig. 3)
was previously noted as a putative PE-binding sequence (33) and
has a similar composition to a known binding site for
phosphatidylcholine, VFMYYF (39) . In all seven homologues this
hydrophobic stretch is bracketed by pairs of charged residues (H,R and
E/K/R,Q), possibly reflecting hydrophilic boundaries of a short
hydrophobic functional region.
Both the 21-kDa bovine protein (33) and the 23-kDa rat protein (25) have been shown to
bind PE. For this reason the 1.1-kb cDNA putative protein was compared
with three other families of lipid-associated proteins: (a)
the 14-16-kDa fatty acid-binding proteins, which include surface
proteins of Schistosoma mansoni(40) and Fasciola
hepatica(41) ; (b) the mammalian lipid-binding
proteins, e.g. human adipocyte lipid-binding protein (42) and rat nonspecific lipid transfer protein(43) ;
and (c) the plant phospholipid transfer proteins, e.g. barley phospholipid transfer protein(44) . No significant
similarity was detected between the PEBP family and the other three
lipid associated protein families (data not shown). A short stretch of
similarity (7 out of 8 residues identical) was also found between
residues 54-61 of TcSL-2 and residues 107-114 of von
Willebrand's factor(45) , a mammalian serum protein
involved in platelet adhesion and blood coagulation (Fig. 5).
TES-26 Is a Functional Phosphatidylethanolamine-binding
Protein
To test whether the sequence homology reflected a
functional conservation, recombinant TES-26, expressed as a fusion
protein with MBP, was tested in a Lipidex bead assay for binding to
radioactive PE. These beads bind free lipid but not lipid sequestered
by proteins, and this assay has demonstrated PE binding by other TES-26
homologues(24, 25) . Fig. 6shows that TES-26
but not MBP binds directly to labeled PE. This binding can be largely
inhibited by a 10-fold molar excess of cold PE but not by
phosphatidylcholine.
Figure 6:
Phosphatidylethanolamine binding by
recombinant TES-26. Lipidex-1000 beads were used to separate free
[
C]PE from lipid bound to TES-26-MBP fusion
protein (
) or control MBP (
). Positive binding of
[
C]PE to TES-26-MBP is inhibited by 10-fold
excess of free PE (
) but not by free phosphatidylcholine (▪).
For each combination of protein, lipid, and competitor, Lipidex column
separation was performed to yield flow-through (protein-bound) and
methanol-eluted (free lipid bound to column) fractions. The percentage
of protein-bound radioactivity is plotted.
TES-26 Contains Two 36-Residue 6-Cysteine Partial
Repeats
Amino acid residues 23-94 comprise partial repeats
of a cysteine-rich 36-residue consensus sequence, designated the NC6
motif (Fig. 7). This contains the consensus sequence XCXDXAX
CX
CX
RCX
TCX
C previously identified at the C terminus of another major secreted
glycoprotein of T. canis infective larvae, TES-120, and this
motif is also found in several predicted genes from Caenorhabditis
elegans.
Although the 5` and 3` NC6 motifs of TES-26
are homologous, there is a greater similarity between these sequences
and the corresponding motifs in TES-120, indicating that the
duplication to create a 72-amino acid domain was an ancient event (Fig. 7).
Figure 7:
Amino acid sequence alignment of NC6
motifs from TcSL-2 and TES-120 sequences and in the C. elegans genomic sequence zk643.6.
Residues identical in all 5
sequences are shown in solid
boxes.
Abundance of 1.1-kb cDNA
A cDNA library prepared
from T. canis infective larvae (kindly provided by C. Tripp
and R. B. Grieve, Paravax Inc., Fort Collins, CO) was screened with the
850-bp fragment of the 1.1-kb cDNA at high stringency. Hybridization
occurred to 0.8-1.9% of plaques (sample size, 1,387), showing
that this transcript is highly abundant in infective larvae.
DISCUSSION
We describe here a cDNA clone that represents an abundantly
expressed and stage-specific mRNA and a corresponding
excretory/secretory protein in infective larvae of the parasitic
nematode T. canis. Sequence similarity analyses show that
TcSL-2 (TES-26) belongs to a recently defined gene family of PEBPs.
Moreover, TES-26 retains a hydrophobic motif thought in mammalian
homologues to mediate lipid binding. Consistent with these indications
of its function, we have also shown direct PE binding by TES-26, and we
have preliminary evidence for an extracellular (surface) association of
this molecule. It is interesting to note that a homologous protein is
also associated with the surface (cuticle) of O. volvulus adult worms(30) , which, like larval T. canis,
are tissue-dwelling parasites.
Despite the similarities within the
PEBP gene families, TES-26 has numerous distinct features. The most
extensive is that TES-26 alone possesses paired NC6 motifs, suggesting
that it is a hybrid gene composed of domains of diverse ancestry. The
presence of similar paired NC6 sequences on two proteins secreted by
infective larvae suggests that this motif may be associated with some
surface-related function, possibly involving modulation of host
immunity. However, the presence of related sequences in the free-living
nematode C. elegans indicates a more fundamental role in the
biology of the organism.
TES-26 differs in other important respects.
It possesses a 20-residue hydrophobic domain that separates the NC6
motifs from the PEBP homology region and that could permit membrane
association. In other family members, the corresponding sequences
contain numerous charged residues and are unlikely to fufill this
function. All non-nematode homologues lack signal peptides and are
cellular proteins, although the murine PEBP has been shown to be
secreted, being highly abundant in epididymal fluid(25) .
Because nematode cuticles are extracellular structures (46) ,
the acquisition of signal peptide sequences may serve both to direct
the PEBP to a surface location and to allow its secretion into the
environment of the parasite.
A role for this protein in the uptake
and transport of host lipids is possible because parasitic helminths
generally have limited ability to synthesize long chain fatty acids and
cholesterols de novo. For example, the trematodes F.
hepatica and S. mansoni express surface fatty
acid-binding proteins(40, 41) , which sequester fatty
acids from host serum and incorporate them into the tegument. However,
we found no sequence similarity between TES-26 and proteins known to be
involved in lipid transport. A second possibility, arising from the
preferential location of PE on the cytoplasmic leaflet of plasma
membranes, is that a PEBP could asymmetrically associate with
membrane-bound proteins(25) . Finally, PEBPs may be involved in
the segregation of lipids into biologically distinct
compartments(35) . This last possibility is intriguing in the
light of strong experimental evidence that the surface of T. canis larvae is made up of nondiffusing, discontinuous lipid
domains(17, 47) .
We are now studying each of these
possibilities to establish the precise function of the nematode PEBPs
and to understand why T. canis should produce this protein in
such quantity. The extraordinarily high expression of TES-120, the
surface coat mucin, and the major antigen targetted by the host
response can be related to the high turnover of the coat as a mechanism
of immune evasion by this parasite. Unlike TES-120, for which the mRNA
is hyperabundant, the level of expression of TES-26 appears low
relative to its specific message. Moreover, the lack of antibodies in
human infection sera was unexpected, especially because the gene was
originally selected for reactivity with antisera from TES-immunized
mice. It is conceivable that TES-26 is invisible to the human immune
system in the context of an active Toxocara infection and that
a lack of response to this antigen could even be a prerequisite for
establishment. Future studies will therefore aim to assess TES-26 both
as a physiological requirement for parasite survival and as a potential
target to induce immunological protection against infection. These
questions are made especially important by the remarkable ability of T. canis to live for many years in a wide range of mammalian
species.