©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Abundant, trans-spliced mRNA from Toxocara canis Infective Larvae Encodes a 26-kDa Protein with Homology to Phosphatidylethanolamine-binding Proteins (*)

(Received for publication, February 2, 1995; and in revised form, May 15, 1995)

David Gems (§) Cheryl J. Ferguson (¶) Brian D. Robertson(¶)(**) Ren Nieves (§§) Antony P. Page (¶¶) Mark L. Blaxter (A) Rick M. Maizels(A)(B)

From the Wellcome Research Centre for Parasitic Infections, Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5` end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.


INTRODUCTION

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) (^1)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) . (^2)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.^2 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(2)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 10^6 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)(18)-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(4)-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 [^14C]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.^2


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 [^14C]PE from lipid bound to TES-26-MBP fusion protein (bullet) or control MBP (). Positive binding of [^14C]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(2)CX(6)CXRCX(2)TCX(2)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.^2 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.^2 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.


FOOTNOTES

*
This investigation received financial support from a Leverhulme Trust grant, from the Medical Research Council, and from the Wellcome Trust through the Wellcome Research Centre for Parasitic Infections. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U29761[GenBank].

§
Present address: Molecular Biology Program, 311 Tucker Hall, University of Missouri, Columbia, MO 65211.

Supported by Medical Research Council postgraduate studentships.

**
Present address: Dept. of Medical Microbiology, St. Mary's Hospital Medical School, London W2 IPG, UK.

§§
Supported by Minority International Research Training Grant T37-TW00046 from the Fogarty International Center at the National Institutes of Health. Present address: Dept. of Biochemistry, Medical Sciences Campus, University of Puerto Rico, San Juan, PR 00936.

¶¶
Recipient of a Wellcome Prize Studentship. Present address: Wellcome Unit for Molecular Parasitology, University of Glasgow, 56 Dumbarton Road, Glasgow G11 6NU, UK.

A
Present Address: Inst. of Cell, Animal and Population Biology, Ashworth Laboratories, King's Buildings, University of Edinburgh, West Mains Rd., Edinburgh EH9 3JT, UK.

B
To whom correspondence should be addressed. Tel.: 44-131-650-5511; Fax: 44-131-650-5450; R.Maizels{at}ed.ac.uk.

^1
The abbreviations used are: TES, T. canis excretory/secretory product; SL, spliced leader; PCR, polymerase chain reaction; MBP, maltose-binding protein; PEBP, phosphatidylethanolamine-binding protein; PE, phosphatidylethanolamine; kb, kilobase(s); bp, base pair(s).

^2
Gems, D. H., and Maizels, R. M., submitted for publication.


REFERENCES

  1. Sprent, J. F. A. (1958) Parasitology 48, 184-209 [Medline] [Order article via Infotrieve]
  2. Glickman, L. T., and Schantz, P. M. (1981) Epidemiol. Rev. 3, 230-250 [Medline] [Order article via Infotrieve]
  3. Gillespie, S. H. (1988) Parasitol. Today 4, 180-182 [Medline] [Order article via Infotrieve]
  4. Schantz, P. M. (1989) Am. J. Trop. Med. Hyg. 41, (suppl.) 21-34 [Medline] [Order article via Infotrieve]
  5. Beaver, P. C. (1962) Bull. Soc. Pathol. Exot. 55, 555-576
  6. de Savigny, D. H. (1975) J. Parasitol. 61, 781-782 [Medline] [Order article via Infotrieve]
  7. Maizels, R. M., Gems, D. H., and Page, A. P. (1993) in Toxocara and Toxocariasis: Epidemiological, Clinical and Molecular Perspectives (Lewis, J. W., and Maizels, R. M., eds) pp. 141-150, Institute of Biology, London
  8. Maizels, R. M., de Savigny, D., and Ogilvie, B. M. (1984) Parasite Immunol. (Oxf.) 6, 23-37 [Medline] [Order article via Infotrieve]
  9. Glickman, L. T., Schantz, P. M., and Grieve, R. B. (1986) in Immunodiagnosis of Parasitic Diseases: Part 1, Helminthiasis (Walls, K., and Schantz, P. M., eds) pp. 201-232, Academic Press, Orlando, FL
  10. Badley, J. E., Grieve, R. B., Bowman, D. D., Glickman, L. T., and Rockey, J. H. (1987) J. Parasitol. 73, 593-600 [Medline] [Order article via Infotrieve]
  11. Sugane, K., and Oshima, T. (1983) Immunology 50, 113-120 [Medline] [Order article via Infotrieve]
  12. Meghji, M., and Maizels, R. M. (1986) Mol. Biochem. Parasitol. 18, 155-170 [Medline] [Order article via Infotrieve]
  13. Page, A. P., and Maizels, R. M. (1992) Parasitology 105, 297-308 [Medline] [Order article via Infotrieve]
  14. Maizels, R. M., and Page, A. P. (1990) Acta Trop. 47, 355-364 [Medline] [Order article via Infotrieve]
  15. Page, A. P., Rudin, W., Fluri, E., Blaxter, M. L., and Maizels, R. M. (1992) Exp. Parasitol. 75, 72-86 [Medline] [Order article via Infotrieve]
  16. Blaxter, M. L., Page, A. P., Rudin, W., and Maizels, R. M. (1992) Parasitol. Today 8, 243-247
  17. Kennedy, M. W., and Proudfoot, L. (1993) in Toxocara canis: Clinical, Epidemiological and Molecular Perspectives (Lewis, J., and Maizels, R. M., eds) pp. 151-161, Institute of Biology, London _
  18. Page, A. P., Hamilton, A. J., and Maizels, R. M. (1992) Exp. Parasitol. 75, 56-71 [CrossRef][Medline] [Order article via Infotrieve]
  19. Page, A. P., Richards, D. T., Lewis, J. W., Omar, H. M., and Maizels, R. M. (1991) Parasitology 103, 451-464 [Medline] [Order article via Infotrieve]
  20. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  21. Belyavsky, A., Vinogradova, T., and Rajewsky, K. (1989) Nucleic Acids Res. 17, 2919-2932 [Abstract]
  22. Domec, C., Garbay, B., Fournier, M., and Bonnet, J. (1990) Anal. Biochem. 188, 422-426 [Medline] [Order article via Infotrieve]
  23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 13.01-13.102, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Bernier, I., Tresca, J. P., and Jolls, P. (1986) Biochim. Biophys. Acta 871, 19-23 [Medline] [Order article via Infotrieve]
  25. Jones, R., and Hall, L. (1991) Biochim. Biophys. Acta 1080, 78-82 [CrossRef][Medline] [Order article via Infotrieve]
  26. Lipman, D. J., and Pearson, W. R. (1985) Science 227, 1435-1441 [Medline] [Order article via Infotrieve]
  27. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  28. Nilsen, T. W. (1993) Annu. Rev. Microbiol. 47, 413-440 [CrossRef][Medline] [Order article via Infotrieve]
  29. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  30. Lobos, E., Altmann, M., Mengod, G., Weiss, N., Rudin, W., and Karam, M. (1990) Mol. Biochem. Parasitol. 39, 135-146 [CrossRef][Medline] [Order article via Infotrieve]
  31. Lobos, E., Weiss, N., Karam, M., Taylor, H. R., Ottesen, E. A., and Nutman, T. B. (1991) Science 251, 1603-1605 [Medline] [Order article via Infotrieve]
  32. Bernier, I., and Jolls, P. (1984) Biochim. Biophys. Acta 790, 174-181 [Medline] [Order article via Infotrieve]
  33. Schoentgen, F., Saccoccio, F., Jolls, J., Bernier, I., and Jolls, P. (1987) Eur. J. Biochem. 166, 333-338 [Abstract]
  34. Grandy, D. K., Hanneman, E., Bunzow, J., Shih, M., Machida, C. A., Bidlack, J. B., and Civelli, O. (1990) Mol. Endocrinol. 4, 1370-1376 [Abstract]
  35. Perry, A. C. F., Hall, L., Bell, A. E., and Jones, R. (1994) Biochem. J. 301, 235-242 [Medline] [Order article via Infotrieve]
  36. Hori, N., Chae, K.-S., Murakawa, K., Matoba, R., Fukushima, A., Okubo, K., and Matsubara, K. (1994) Gene (Amst.) 140, 293-294 [CrossRef][Medline] [Order article via Infotrieve]
  37. Pikielny, C. W., Hasan, G., Rouyer, F., and Rosbash, M. (1994) Neuron 12, 35-49 [Medline] [Order article via Infotrieve]
  38. Robinson, L. C., and Tatchell, K. (1991) Mol. & Gen. Genet. 230, 241-250
  39. Akeroyd, R., Moonen, P., Westerman, J., Puyk, W. C., and Wirtz, K. W. A. (1981) Eur. J. Biochem. 114, 385-391 [Medline] [Order article via Infotrieve]
  40. Moser, D., Tendler, M., Griffiths, G., and Klinkert, M.-Q. (1991) J. Biol. Chem. 266, 8447-8454 [Abstract/Free Full Text]
  41. Rodriguez-Perez, J., Rodriguez-Medina, J. R., Garcia-Blanco, M. A., and Hillyer, G. V. (1992) Exp. Parasitol. 74, 400-407 [Medline] [Order article via Infotrieve]
  42. Baxa, C. A., Sha, R. S., Buelt, M. K., Smith, A. J., Matarese, V., Chinander, L. L., Boundy, K. L., and Bernlohr, D. A. (1989) Biochemistry 28, 8683-8690 [Medline] [Order article via Infotrieve]
  43. Mori, T., Tsukamoto, T., Mori, H., Tashiro, Y., and Fujiki, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4338-4342 [Abstract]
  44. Linnestad, C., Loenneborg, A., Kalla, R., and Olsen, O. A. (1991) Plant Physiology 97, 841-843
  45. Sadler, J. E., Shelton-Inloes, B. B., Sorace, J. M., Harlan, J. M., Titani, K., and Davie, E. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6394-6398 [Abstract]
  46. Maizels, R. M., Blaxter, M. L., and Selkirk, M. E. (1993) Exp. Parasitol. 77, 380-384 [CrossRef][Medline] [Order article via Infotrieve]
  47. Kennedy, M. W., Foley, M., Kuo, Y.-M., Kusel, J. R., and Garland, P. B. (1987) Mol. Biochem. Parasitol. 22, 233-240 [Medline] [Order article via Infotrieve]

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