From the a School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, Great Britain, the b Department of Biological Sciences, Salford University, Salford M5 4WT, Great Britain, the c Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, and the d Department of Chemistry, Joseph Black Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, the e Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G4 0NR, Scotland, and the f Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, Great Britain
Received for publication, June 24, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Parasitic nematodes of humans and
plants secrete a structurally novel type of fatty acid- and
retinol-binding protein, FAR, into the tissues they occupy. These
proteins may interfere with intercellular lipid signaling to manipulate
the defense reactions of the host or acquire essential lipids for the
parasites. The genome of the nematode Caenorhabditis
elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8). These
fall into three discrete groups as indicated by phylogenetic sequence
comparisons and intron positions, the proteins from parasitic nematodes
falling into group A. Recombinant Ce-FAR-1 to -7 were produced in
Escherichia coli and tested for lipid binding in
fluorescence-based assays. Ce-FAR-1 to -6 bound DAUDA
(11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid),
cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids
relatively poorly. Each protein produced a characteristic shift in peak
fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift
greater than has been observed previously for any lipid-binding
protein. Selected Ce-FAR proteins were analyzed by circular dichroism
(CD) and differential scanning calorimetry, were found to be
helix-rich, and exhibited high thermal stability (transition midpoint,
82.7 °C). CD and secondary structure predictions, however, both
indicated that Ce-FAR-7 possesses substantially less helix than the
other FAR proteins. The genes encoding the Ce-FAR proteins were found
to be transcribed differentially through the life cycle of C. elegans, such that Ce-far-4 was transcribed at
highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.
Nematodes are probably the most abundant and ecologically diverse
group of multicellular organism on Earth, and parasitic forms directly
cause more human disease and economic damage to domestic animals and
crop plants than any other group of metazoan organisms, with the
possible exception of insects. Lipid-binding proteins released by
nematode parasites have attracted increasing interest because of their
potential role in nutrient acquisition, manipulation of the tissues
they occupy, and countering host defense reactions (1-3). Nematodes
produce two different types of small retinol- and fatty acid-binding
proteins (~14-20 kDa), neither of which have recognizable
counterparts in other animal groups, including mammals. Both are
helix-rich, in contrast to the The nematode FAR proteins were originally identified in parasitic
species; Ov-FAR-1 (originally named Ov20) is secreted by Onchocerca volvulus, the causative agent of river blindness
(8). Gp-FAR-1 is associated with the surface of the potato cyst
nematode Globodera pallida and is thought to interfere with
plant lipid-based defense signaling reactions (9). Ov-FAR-1 binds both
fatty acids and retinol (10), and it was the latter activity that drew
particular attention, for three reasons. First, retinol is known
to play an important role in gene activation, cell signaling, and
tissue differentiation and repair (11). The localized depletion of this
vitamin by Ov-FAR-1 secreted by the parasite could therefore be a
contributing factor to the severe eye and skin damage associated with
the infection (3, 12, 13). Second, there is evidence that parasitic
nematodes may require retinol for their proper development and
reproduction (14-18), and secreted FAR proteins such as Ov-FAR-1 could
be essential for the acquisition of this resource by the parasite.
Third, retinol deficiency can alter the character of the host immune
response (19-23), and it is possible that such changes could be
beneficial to the parasite (24, 25).
Genes encoding FAR proteins have now been described from many species
of parasitic nematode (8, 9, 26, 27), and inspection of expressed
sequence tag and other surveys (see nema.cap.ed.ac.uk/Nest.html and
Ref. 28) indicates that that some or all parasitic nematodes may
produce more than one type of FAR, although it is not yet clear whether
these represent alleles at a single locus or multiple genes. To
understand the function of FAR proteins in parasitism, it is important
to establish how many genes encoding FAR proteins exist in a given
species, the function of the encoded proteins, and which are important
in host-parasite interactions. At present such investigations would be
difficult if not impossible in parasites, so we have begun an
examination of the FAR proteins of the free-living nematode
Caenorhabditis elegans, to understand the extent and biological functions of the FAR protein family. This has additional significance in that, of the 58% of the genes of this organism that
appear to encode novel protein types (29), the FAR proteins constitute
one of only two protein types to which biochemical activities have been
ascribed (1).
Screening the genome sequence of the free-living soil nematode C. elegans has identified eight genes encoding FAR-like proteins. The
amino acid sequences of these proteins are diverse, and a key question
is whether the different proteins perform different functions in the
biology of the organism. In addition, anticipating that parasitic
nematodes may also be found to have multiple far genes, what
might be the role of the encoded proteins in parasitism? To this end,
we have cloned cDNA encoding all but one of the discernible FAR
protein family members from C. elegans, examined the lipid binding and biophysical properties of the recombinant proteins, and
followed the patterns of far gene transcription in the life cycle of the organism. Despite their considerable amino acid sequence diversity, the C. elegans FAR proteins were found to have
similar lipid binding activities, although some possess distinctive
properties. In terms of their amino acid sequences, we find that the
C. elegans FAR proteins fall into three distinct groups and
that the proteins so far described from parasitic nematodes cluster
with one of these groups. This group may therefore have been important
in the evolution of parasitism and justifies further examination using
C. elegans as a surrogate for establishing the roles of FAR
proteins in the biology of nematodes.
Identification of C. elegans far Genes--
C. elegans
far genes were identified by blastp and tblastn (30) searches of
the C. elegans sequence data base available online at
(www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml) using the
amino acid sequence of Ov-FAR-1 as the probe. High scoring matches were
inspected to determine the relevance of the match, and a total of eight
far genes were identified. These eight genes were designated
Ce-far-1 through Ce-far-8 and correspond to the following loci that were predicted by GENEFINDER (29):
Ce-far-1 is F02A9.2, Ce-far-2 is F02A9.3,
Ce-far-3 is F15B9.1, Ce-far-4 is F15B9.2,
Ce-far-5 is F15B9.3, Ce-far-6 is W02A2.2,
Ce-far-7 is K01A2.2, and Ce-far-8 is K02F3.3.
RT-PCR and RACE experiments corrected the GENEFINDER ORF predictions
for F15B9.3 and K02F3.3. Additional blastp and tblastn searches of the
C. elegans data base using the eight C. elegans
FAR proteins as probes did not reveal the existence of any additional
far genes in this organism.
Growth and Maintenance of C. elegans--
The C. elegans strains used in this study were supplied by the
Caenorhabditis Genetics Centre and were the wild-type Bristol N2 strain
and the mutant him-8(el489). The nematodes were
maintained at 20 °C and were cultured as previously described
(31).
Cloning, Expression, and Purification of Recombinant
Proteins--
Total RNA was prepared from a mixed-stage population of
wild-type C. elegans using TRIzol (Invitrogen) in
accordance with the manufacturer's instructions. cDNA was
synthesized from total RNA by standard procedures (32). DNA encoding
the C. elegans FAR proteins was amplified for cloning
purposes by PCR using PCR primers that were designed to omit the
putative signal peptide from those proteins that were predicted to have
one. The sequences of the PCR primers that were used are as follows:
Ce-far-1 forward primer, 5'-GGT ATT GAG GGT CGC GCT CCA GTC
CCG GAG GTC C-3'; Ce-far-1 reverse primer, 5'-AGA GGA GAG
TTA GAG CCT TAG TTG AGG TAT TGT CCG ACC A-3'; Ce-far-2
forward primer, 5'-CAT ATG CCA ATC CCA GAG GTT CCA C-3';
Ce-far-2 reverse primer, 5'-GGA TCC TTA GTT GAC GTA TTG TCC
GAC-3'; Ce-far-3 forward primer, 5'-CAT ATG GCT CCA GCT GAT
GAT TCT TCT C-3'; Ce-far-3 reverse primer, 5'-GGA TCC ATT
GTT CTT CTC AAG AAG CTT GG-3'; Ce-far-4 forward primer, 5'-GGT ATT GAG GGT CGC TTT CCA TTC GGA GAA CCA CA-3';
Ce-far-4 reverse primer, 5'-AGA GGA GAG TTA GAG CCT TAT TTT
CCC ATA CCC ATT CCC-3'; Ce-far-5 forward primer, 5'-GGT ATT
GAG GGT CGC GCT GAT GGA ATA TTT GAA GCT G-3'; Ce-far-5
reverse primer, 5'-AGA GGA GAG TTA GAG CCT CAC AAC AAT TTA TCA ATC TCA
GT-3'; Ce-far-6 forward primer, 5'-GGT ATT GAG GGT CGC GCT
CCA ATT TCA CGC TTA CC-3'; Ce-far-6 reverse primer, 5'-AGA
GGA GAG TTA GAG CCT TAG TAG TTC ACG ATC TGT TGG ATG-3';
Ce-far-7 forward primer, 5'-GGT ATT GAG GGT CGC ATG AGC GTT
GCT TCA CTT CC-3'; Ce-far-7 reverse primer, 5'-AGA GGA GAG
TTA GAG CCT CAA TCC GGA TTA TTC TTT CTC AGC-3'. The PCR products that
were obtained for Ce-far-2 and Ce-far-3 were
cloned into TOPO TA (Invitrogen) in accordance with the manufacturer's instructions, and they were then sequenced. The Ce-far-2 and
Ce-far-3 sequences were subcloned from TOPO TA into pET15b
(Novagen) using the NdeI and BamHI restriction
sites that had been incorporated in the PCR primers. The two
recombinant pET15b plasmids were maintained in Escherichia
coli DH5 Analysis of C. elegans far Gene Transcription Patterns--
The
temporal expression pattern of each of the far genes was
investigated by semi-quantitative PCR (sqRT-PCR). First-strand cDNA
for the sqRT-PCR was generated from total RNA that had been isolated
from embryos and from synchronous L1, L2, L3, L4, and young adult
cultures of wild-type C. elegans, as previously described (33, 34). The stage-specific cDNA samples were kindly provided by
Dr. Antony Page (Wellcome Centre for Molecular Parasitology, University
of Glasgow). For each developmental stage investigated, the abundance
of a control gene, ama-1, was also estimated by sqRT-PCR.
The primers that were used for the sqRT-PCR reactions all spanned at
least one intron so that any contaminating genomic DNA would give PCR
products that could be easily identified. The number of PCR cycles that
gave linear amplification signals for each gene was determined
empirically and varied between 30 and 35. The products of the sqRT-PCR
reactions were visualized by electrophoresis on 1% agarose gels
containing 0.5 µg/ml ethidium bromide, and their relative abundance
was estimated by densitometric analysis of the ethidium bromide-stained
bands. The gene transcription levels were calculated as the ratio of
the abundance of the far gene sqRT-PCR products to that of
ama-1 at each developmental stage. The sequences of the
primers that were used in the sqRT-PCR reactions are as follows:
Ce-far-1 forward primer, 5'-GCT CCA GTC CCG GAG GTC C-3';
Ce-far-1 reverse primer, 5'-TTA GTT GAG GTA TTG TCC GAC
CA-3'; Ce-far-2 forward primer, 5'-CAT ATG CCA ATC CCA GAG
GTT CCA C-3'; Ce-far-2 reverse primer, 5'-GGA TCC TTA GTT
GAC GTA TTG TCC GAC-3'; Ce-far-3 forward primer, 5'-CAT ATG
GCT CCA GCT GAT GAT TCT TCT C-3'; Ce-far-3 reverse primer, 5'-GGA TCC ATT GTT CTT CTC AAG AAG CTT GG-3'; Ce-far-4
forward primer, 5'-CTC GAG TTT CCA TTC GGA GAA CCA CA-3';
Ce-far-4 reverse primer, 5'-GGA TCC TTA TTT TCC CAT ACC CAT
TCC C-3'; Ce-far-5 forward primer, 5'-GGT ATT GAG GGT CGC
GCT GAT GGA ATA TTT GAA GCT G-3'; Ce-far-5 reverse primer,
5'-AGA GGA GAG TTA GAG CCT CAC AAC AAT TTA TCA ATC TCA GT-3';
Ce-far-6 forward primer, 5'-CAT ATG GCT CCA ATT TCA CGC TTA
CC-3'; Ce-far-6 reverse primer, 5'-GGA TCC GTA GTT CAC GAT
CTG TTG GAT GA-3'; Ce-far-7 forward primer, 5'-GGT ATT GAG
GGT CGC ATG AGC GTT GCT TCA CTT CC-3'; Ce-far-7 reverse
primer, 5'-AGA GGA GAG TTA GAG CCT CAA TCC GGA TTA TTC TTT CTC AGC-3';
ama-1 forward primer, 5'-GTT CAG TTT GGA ATT CTC GG-3';
ama-1 reverse primer, 5'-GAT TTG TCG ATG AGA AGC C-3'. Experiments were also undertaken to determine whether any of the C. elegans far genes are up-regulated in males. The
procedure was as described above, but first-strand cDNA for the
sqRT-PCR was generated from total RNA that had been isolated from a
mixed-stage culture of C. elegans
him-8(el489).
Spectrofluorometry and Fluorescence-based Ligand Binding
Assays--
Fluorescence emission spectra were recorded at 20 °C
with a SPEX FluorMax spectrofluorometer (Spex Industries, Edison,
NJ) using 2-ml samples in silica cuvettes. Raman scattering by solvent water was corrected for where necessary using appropriate blank solutions. The fluorescent fatty acid analogs,
11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid
(DAUDA) and dansyl-DL- Gas Chromatography and Mass Spectrometry--
The protein
solutions were acidified with 5 M HCl and then extracted
with an equal volume of ethyl acetate. The samples were centrifuged to
disperse the resultant emulsion, and the organic layer was then
transferred to a glass vial and evaporated to dryness under a stream of
nitrogen. N,O-Bis(trimethylsilyl) acetamide (50 µl) was added to the sample, which was then heated at 80 °C for 20 min. After cooling, 2 µl of the sample was injected into a
Thermoseparations Automass Multi GC-MS instrument, which was fitted
with a ZB-1 column (15-m length, 0.25-mm internal diameter, and
0.25-µm film). The GC oven was programmed as follows:
100 °C for 1 min, then 10 °C increase per min to 300 °C. The
head pressure used was 20 kPa. The mass spectrometer was operated in the electron impact mode at 70 eV and was scanned from 50-500 atomic
mass units.
Circular Dichroism--
The CD spectra of Ce-FAR-2 and Ce-FAR-3
were recorded at 20 °C in the far UV (260-195 nm) using a Jasco
J-600 spectropolarimeter and compared with earlier data on the Ov-FAR-1
protein from Onchocerca volvulus (10). For each protein,
spectra were recorded in 0.02-cm path length cells at three different
concentrations over the range 10-45 µM. Analysis of the
CD spectra was undertaken using the CONTIN (36) and SELCON (37)
procedures, which produced similar results, but the values quoted are
from the latter, because in our experience the CONTIN method has a
tendency to overestimate the proportion of Differential Scanning Calorimetry--
Experiments were
performed by standard procedures (38) using a Microcal VP-DSC
instrument at a scan rate of 60 °C h Sequence Comparisons and Secondary Structure
Predictions--
Sequence analysis was performed using programs
available through the ExPASy molecular biology server
(www.expasy.ch/tools). The SignalP program (40) was used to predict the
presence and location of any signal peptide cleavage sites, and the
PSORT program (41) was used to predict protein localization and sorting
signals. The molecular weight and isoelectric point of the proteins
were estimated using the ProtParam program, and all of the sequences were scanned for the occurrence of motifs stored in the PROSITE data
base (42). The sequences of the eight C. elegans FAR
proteins were aligned with each other using the ClustalW program (43), and the alignment was then examined to locate positions where amino
acids are conserved or positions where the hydrophobic or hydrophilic
nature of the amino acids is conserved. Secondary structure predictions
of the individual or the multiple aligned sequences were performed
using the PHD and the Jpred programs (44). The ClustalW program was
also used to generate an alignment of the eight C. elegans
FAR sequences and the Ov-FAR-1 and Gp-FAR-1 sequences of O. volvulus and G. pallida. Phylogenetic analysis was
conducted on the alignment using the maximum likelihood protein sequence parsimony method (45) and the unweighted pair group method
with arithmetic mean (46). The trees were rooted against the FAR
protein sequence of Ascaris suum (derived from an expressed sequence tag: accession number BF169343) or Ce-FAR-8. All methods produced trees of the same topology, and a typical result with bootstrap values is presented.
Sequence Comparisons and Structural Predictions--
The
C. elegans sequence data base was screened for the
occurrence of Ov-FAR-1-like proteins by blastp and tblastn searches, yielding eight protein sequences that were designated Ce-FAR-1 to -8. RT-PCR and RACE experiments (data not shown) corrected the GENEFINDER
ORF predictions for Ce-far-5 and Ce-far-8, which were originally predicted to be parts of larger (hybrid) proteins (F15B9.3 and K02F3.3, respectively). No PCR products were obtained for
the full-length F15B9.3 and K02F3.3 gene predictions, and neither
Ce-far-5 nor Ce-far-8 was found to be
trans-spliced to the nematode-spliced leaders SL1 or SL2.
The amino acid sequences of the eight C. elegans FAR
proteins are aligned in Fig.
1A. The sequences
are highly diverse, although with clear evidence of amino acid
positions that are conserved absolutely or by biochemical character.
Ce-FAR-1 to -6 all appear to have cleavable N-terminal leader/signal
peptides predicted with acceptable probabilities by the SignalP and
PSORT programs. SignalP predicted a leader for Ce-FAR-8 but not
Ce-FAR-7, and PSORT predicted leaders for neither. C. elegans FAR proteins 1 through 6, and possibly also 8, are
therefore likely to be secreted from the synthesizing cell, as are the
FAR proteins of filarial parasites (8, 27) and the plant parasitic
nematode, G. pallida (9). In addition to lacking a putative
signal peptide at the N terminus, Ce-FAR-7 is also significantly
truncated at the C terminus relative to the other members of the
C. elegans FAR protein family. Our RACE experiments,
however, confirmed the GENEFINDER prediction for this gene (data not
shown). A further distinctive feature of Ce-FAR-7 and -8 is the
possession of four and three cysteines, respectively, in the predicted
mature proteins (excluding leader/signal peptides). The other C. elegans FAR proteins possess no cysteines, except for Ce-FAR-3,
which has one in the putative leader/signal peptide. The cysteine
positions in Ce-FAR-7 and Ce-FAR-8 are dissimilar, and the third
cysteine in Ce-FAR-8 may be lost if the C-terminal extension of this
protein is trimmed post-translationally. Given the weighting usually
attributed to the occurrence and position of cysteines in protein
families, it is arguable that Ce-FAR-7 and -8 should not be included
within the FAR family. We nevertheless continued to consider them,
because they may represent specialized or ancestral forms of FAR
protein with distinctive functions. An unusual feature of Ce-FAR-4 and Ce-FAR-8 is the possession of C-terminal extensions, that of Ce-FAR-4 being most unusual in being glycine- and methionine-rich. Our data base
searches with these extension peptides in isolation provided no
indication of their function. Computer-based analysis predicted that
the C. elegans FAR proteins are rich in
Part of the rationale for this study was to evaluate the suitability of
C. elegans as a surrogate to investigate the biological functions of FAR proteins in parasitic nematodes. It is therefore pertinent to establish which of the FAR proteins of the organism are
homologous and paralogous to FAR proteins from parasites, such as
Ov-FAR-1 and Gp-FAR-1. A phylogenetic tree showing the relationship
between the C. elegans FAR proteins and the parasite FAR
proteins from O. volvulus and G. pallida is
given in Fig. 1B. This shows that the sequences fall into
clusters, such that Ce-FAR-1, -2, and -6 belong to a discrete group
(group A) along with Ov-FAR-1 and Gp-FAR-1 proteins of parasites. Which
of these C. elegans FAR proteins shares the closest common
ancestor with the parasite-derived proteins cannot be determined with
confidence because of the degree of sequence divergence that has
occurred over time. However, it is notable that the similarities are
apparent despite the fact that the three species concerned are in
separate major clades of Nematoda (26).
Ce-FAR-3, -4, and -5 fall into another distinct grouping (group B), and
both groups A and B are distant from Ce-FAR-7 and Ce-FAR-8 (group C),
the latter protein being the most distant. Inspection of the intron
splice site positions in the eight C. elegans far genes
shows that Ce-FAR-1 through -6 have intron positions in common,
although no gene possesses a full set (Fig. 1C). Thus, introns 3 and 6 are unique to group A, and introns 1, 2, and 4 are
unique to group C. Intron 5 appears in both groups A and B, but the
intron positions of groups A plus B and group C are mutually exclusive.
This further emphasizes the remoteness of group C proteins from groups
A and B. The identity of the Ce-FAR gene/protein that is the true
homolog by descent of the parasite FARs may be more clearly
identifiable when the positions of their introns are established. Given
this analysis, we chose to express all of Ce-FAR-1 through -6 in
bacteria for biochemical and biophysical analysis, plus Ce-FAR-7 as an
outlier, but ignored Ce-FAR-8 as being too divergent a member of the family.
Ligand Binding--
The seven proteins were tested for lipid
binding using fluorophore-tagged or intrinsically fluorescent natural
compounds. With the exception of Ce-FAR-7, all were found to bind DAUDA
(a saturated fatty acid conjugated to the environment-sensitive dansyl fluorophore) strongly, eliciting similarly substantial shifts in the
wavelengths of maximum emission (Fig.
2A). Moreover, DAUDA was
displaceable from all the proteins by addition of oleic or arachidonic
acids, although the efficiency of competitive displacement varied
considerably from protein to protein (Table
I). Ce-FAR-1, -2, -3, -4, and -6 induced
similar shifts in DAUDA fluorescence emission (from 543 nm for DAUDA in
buffer alone to between 482 and 490 nm upon addition of protein),
indicating that the degrees of apolarity of their binding sites are
similar (47) despite considerable disparities in their amino acid
sequences. Ce-FAR-5 was notable in the degree of blue shift it caused
in DAUDA, which (from 543 to 466 nm) is the largest shift in DAUDA
emission yet recorded for a binding protein, and is indicative of a
binding site environment that is unusually apolar. To place this in
context, the peaks of fluorescence emission of DAUDA in rat liver fatty acid-binding protein, human tear lipocalin, horse
endometrial P19/uterocalin, and bovine serum albumin are
500 nm (48), 490 nm (49),2
483 nm (50), and 495 nm (51), respectively, and that for DAUDA in an
apolar organic solvent (cyclohexane) is 475 nm (51). Similar results
were found with DACA (data not shown), a dansylated fatty acid in which
the fluorophore is attached to the
In contrast to the other FAR proteins, Ce-FAR-7 exhibited minimal
binding of DAUDA, although subtraction of DAUDA emission spectra
recorded in the presence and absence of protein revealed a minor peak
at a similar wavelength as for the other proteins (~482 nm; Fig.
2A). Ce-FAR-7 might therefore have a binding site environment similar to that of the other proteins, but excludes DAUDA
because of steric interference by the bulky dansyl fluorophore. Ce-FAR-7 induced fluorescence emission enhancements in
cis-parinaric acid and retinol but less so than did the
other proteins (Fig. 2, B and C). So, either
Ce-FAR-7 is indeed functionally discrete from the others, or a
substantial fraction of this particular recombinant protein may fail to
fold correctly, possibly as a consequence of inappropriate
cross-linking of its cysteines.
Protein:ligand dissociation constants were estimated for two of the
C. elegans FAR proteins from each of groups A and B
(Ce-FAR-1 and -2, and Ce-FAR-3 and -5) for binding of DAUDA,
cis-parinaric acid, and retinol. Fig.
4 shows typical saturation binding curves for each of the three fluorescent ligands for one of these (Ce-FAR-5). The dissociation constants ranged from 8 × 10
Samples of each of the Ce-FAR proteins, prepared as for all of the
above experiments, were treated to extract hydrophobic ligands and
subjected to GC-MS, as detailed under "Experimental Procedures."
None of the samples revealed any co-purifying compounds. All of these
protein preparations had been produced by lysozyme lysis of the
bacteria, whereas we have previously found that resident fatty acid
ligands co-purify with a bacterial recombinant FAR protein
(Ov-FAR-1/Ov20) (10). We therefore took a selection of clones
expressing Ce-FAR proteins (one from each of groups A and B, Ce-FAR-6
and Ce-FAR-3, and Ce-FAR-5, also from group B, but which induces the
greatest recorded blue shift in DAUDA) and released the recombinant
protein using the sonication method. The proteins were
affinity-purified as described but were not passed down a detergent
removal column. This was also carried out on a sample of Ov-FAR-1
prepared in an identical manner. This time, all four samples were found
to contain a similar pattern of hydrophobic compounds, the major one
being palmitic acid, followed in abundance by oleic and palmitoleic
acids (two isomers). There was one unusual fatty acid present that
appeared to be a monounsaturated heptadecanoic acid. Presumably,
therefore, disruption of the bacteria with sonication exposes the
recombinant proteins to fatty acid from the bacteria, which does not
happen with the less aggressive lysozyme method. The disparity between
the treatments was unexpected but means that none of the above ligand
binding experiments on the Ce-FAR proteins, including the
Kd estimations, should have been compromised by
resident ligand derived from the expression host. But, under
appropriate protein recovery conditions, the proteins co-purify
with natural hydrophobic ligands of the expected type.
Circular Dichroism--
Analysis of one Ce-FAR protein from each
of groups A and B (Ce-FAR-2 and Ce-FAR-3) by circular dichroism
revealed that the proteins have a high content of
CD analysis of Ce-FAR-7, however, indicated that its secondary
structure content is unlike that of the others (Fig. 5). Application of
both SELCON and CONTIN analysis to its CD spectrum indicated that
Ce-FAR-7 had considerably less Microcalorimetry--
Ce-FAR-2 was subjected to differential
scanning calorimetry (Fig. 6), which
indicated co-operative thermal unfolding of the protein with a midpoint
transition temperature, Tm = 82.7 °C. A ratio
of ~2:1 between the van't Hoff enthalpy and the calorimetric enthalpy of unfolding
( Stage- and Gender-specific Transcription Patterns--
The
temporal and gender specificity of transcription of each of
Ce-far-1 to -7 genes was investigated by
sqRT-PCR, using RNA isolated from embryos, L1, L2, L3, and L4 larvae,
young adult hermaphrodites, and mixed-stage him-8 mutants
that give rise to male-enriched cultures (63). Transcript levels for
each of Ce-far-1 to -7 are presented in Fig.
7, expressed relative to that of a control transcript, ama-1, which encodes the large subunit
of RNA polymerase II (64). Transcript levels for ama-1
exhibit minimal fluctuation during the life cycle of C. elegans (65), and the suitability of this gene as a reference
transcript for sqRT-PCR has previously been established (33, 34). The
results of this study indicate that the far genes have
discrete transcription patterns, with some being particularly evident
in certain developmental stages or gender. For instance,
Ce-far-3 and -7 transcripts are notably abundant
in males, Ce-far-4 transcripts are abundant in L4
hermaphrodites, and there is a progressive increase in
Ce-far-2 transcript levels with development in
hermaphrodites.
We find that the FAR proteins of C. elegans represent a
family of eight proteins, six of which (Ce-FAR-1 to -6) have
overlapping lipid binding properties, and two are outliers (Ce-FAR-7
and -8) in terms of their amino acid sequences, genomic intron
positions, possession of cysteines, lipid binding function, and
secondary structure content. Other unifying characteristics within the
family from C. elegans and the two FAR proteins that have
been examined from parasitic nematodes (one of humans, one of plants)
are their richness in
The lipid binding properties of the C. elegans FAR proteins,
and the environment of their binding sites, show considerable similarities despite their amino acid sequence diversity. Assuming that
our analysis included biologically relevant ligands, then the question
arises as to what advantage the nematode derives from producing so many
different isoforms. By analogy with mammals, which produce multiple
isoforms of the FABP/P2/CRBP/CRABP family of cytoplasmic lipid-binding
proteins (6, 66), it is conceivable that the FAR proteins will possess
different tissue or cell type expression patterns or be under
differential developmental or gender-specific control. Our analysis of
the transcription of each of the far genes throughout the
life cycle of C. elegans is consistent with this idea, in
that they are clearly under differential developmental control, and two
appear to be strongly up-regulated in males. But it is also clear that
a single life cycle stage transcribes more than one far gene
at a time. It may therefore be that the Ce-FAR proteins differ in their
ligand binding functions in ways undetectable by the assays we used. A
more exciting possibility is that the proteins interact with different
specific cell surface receptors/targets in the delivery or collection
of their ligands and that this is reflected in their differential
regulation and disparate amino acid sequences. An essential next step
will therefore be to establish which cells and tissues produce each of
the C. elegans FAR proteins through the life cycle of the
organism and the phenotypic effect of deletion or disablement of their
encoding genes. It will also be essential to establish whether
differential expression of the C. elegans far genes is
merely apparent through differential mRNA stabilities and
transcription rates, so it will be important to determine the relative
levels of each FAR protein in the different developmental stages and sexes.
Ce-FAR-1 through -6, like both Ov-FAR-1 and Gp-FAR-1 of parasites, bind
retinol and fatty acids at similar affinities. But what role do
retinoids play in nematode biology? C. elegans can be
propagated in monoxenic culture in a medium in which neither the
E. coli food source, nor the growth medium itself, provides a source of retinoids (67). Worms grown in such a medium do, however,
exhibit abnormalities, including physical deformities, metabolic
perturbations, and reduced fertility (68). It is possible that the diet
of C. elegans in their natural habitat of the soil includes
retinoids or their precursors, but that other compounds can substitute
for their functions in the absence of retinoid precursors. In this
context, it may be pertinent that the C. elegans genome
possesses a gene encoding an enzyme similar in sequence to
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-rich small lipid transporter
proteins of vertebrates such as the lipocalins (which are extracellular
and secreted) (4) and members of the FABP/P2/CRBP/CRABP1 family
(which are cytoplasmic lipid transporters, with exceptions only from
nematodes) (5, 6). One of these two families of helix-rich
lipid-binding proteins of nematodes is synthesized as a large
polyprotein that is post-translationally cleaved into multiple copies
of the functional protein of ~14 kDa (2, 7). The other family
comprises the FAR proteins (fatty acid and
retinol binding), which are the subjects of this report.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cells (Novagen) and transformed into E. coli BL21(DE3) cells (Novagen) for expression of the recombinant fusion proteins. The PCR products that were obtained for the other C. elegans FAR genes were cloned into pET30 Xa/LIC (Novagen)
in accordance with the manufacturer's instructions, and they were then
sequenced. The recombinant pET30 Xa/LIC plasmids were maintained in
E. coli NovaBlue cells (Novagen) and transformed into
E. coli BLR(DE3) cells (Novagen) for expression of the
recombinant fusion proteins. The expression and purification of the
proteins was performed in accordance with the kit manufacturer's
instructions for high yield, the major proportion of each protein being
water-soluble, and only batches exhibiting a single band under SDS-PAGE
with Coomassie Blue staining were used. No detergents were added during the extraction procedures. The purified proteins were exhaustively dialyzed at 4 °C against phosphate-buffered saline (PBS; 171 mM NaCl, 3.35 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4; pH 7.2) and subsequently passed down a
column of Extracti-Gel D resin (Pierce) to remove any contaminating
detergent. Preliminary experiments showed that the biochemical
properties of the recombinant fusion proteins were indistinguishable
from those proteins from which the N-terminal fusion partner had been
cleaved, either with thrombin for the pET15b clones or with Factor Xa
for the pET30 Xa/LIC clones. In the interests of reducing manipulation,
therefore, all the assays reported in this report were performed with
the intact fusion proteins.
-aminocaprylic acid (DACA), were
obtained from Molecular Probes (Eugene, OR) and Sigma (Poole, Dorset), respectively. All-trans-retinol and oleic acid were obtained
from Sigma, and cis-parinaric acid was obtained from
Molecular Probes. The excitation wavelengths used for DAUDA, DACA,
retinol, and cis-parinaric acid were 345, 345, 350, and 319 nm, respectively. The dansylated fatty acids were stored as
stock solutions of ~10 mM in ethanol in the dark at
20 °C and freshly diluted in PBS to ~1 µM by
serial dilution in PBS for use in the fluorescence experiments. Retinol
solutions were freshly prepared in ethanol at ~0.1 mM and
were added directly to solutions of the proteins in the fluorescence
cuvettes immediately before use to minimize the degradation of retinol
in aqueous solution before binding occurred. Oleic acid for competition
studies was prepared as a stock solution in ethanol at ~10
mM and diluted 1:10, 1:100, and 1:1000 in PBS for use in
the assays. The dissociation constants for DAUDA,
cis-parinaric acid, and retinol were estimated by
fluorescence titration experiments as previously described (10). For
DAUDA and cis-parinaric acid, the protein solutions were
added in small aliquots (typically 5 µl) to 2 ml of PBS containing
the fluorescent ligands. For retinol, the fluorescent ligand was added
incrementally (5 µl) to 2 ml of PBS containing the protein to be
assayed. The concentrations of the proteins used in the titrations were
estimated spectrophotometrically using a calculated theoretical
extinction coefficient (using ProtParam software available at
ca.expasy.org/tools/protparam.html) or by quantitative amino acid
hydrolysis, and the concentrations of the fluorescent ligands were
estimated using extinction coefficients of 52,480 at 325 nm
for retinol, 76,000 at 276 nm for cis-parinaric acid, and
4,800 at 335 nm for DAUDA, using solutions diluted in ethanol or
methanol as appropriate. The fluorescence titration data were corrected
for dilution where necessary and fitted by standard non-linear
regression techniques (using Microcal Origin software) to a single
non-competitive binding model to give estimates of the dissociation
constant (Kd) and maximal fluorescence intensity
(Fmax). The Kd values for
retinol were estimated with correction for the fluorescence of free
retinol added to a cuvette containing only PBS, as previously described
(35).
-sheet in helix-rich
proteins. Progressive denaturation with GdnHCl was carried out and
followed by CD as previously described (10).
1 over a
20-110 °C range using sample concentrations of 0.7 mg ml
1. Normalized excess heat capacity data for thermal
transitions were analyzed in terms of the standard non-two-state model,
using Microcal ORIGIN software. This gives independent estimates of the
calorimetric and van't Hoff unfolding enthalpies,
Hcal and
HVH, from
which the cooperativity of the transition may be evaluated (39).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix (Fig.
1A), in common with the FAR proteins of animal and plant parasitic nematodes (9, 10, 27). Another feature that is shared by the
parasite FAR proteins and all of the C. elegans FAR
proteins, apart from Ce-FAR-8, is the possession of a consensus casein
kinase II phosphorylation site at a conserved position (Fig.
1A) (9, 10, 27).
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Fig. 1.
Ce-FAR protein relationships.
A, multiple sequence alignment and secondary structural
predictions for the C. elegans FAR proteins. The putative
hydrophobic leader/signal peptides that were predicted by the SignalP
program are shown in lowercase letters. Consensus
N-linked glycosylation sites are underscored, and the
consensus casein kinase II phosphorylation site is in boldface
type. In the consensus line, uppercase letters refer to those amino acids that are conserved
at that position in all of the sequences, and lowercase
letters refer to amino acids that are conserved at that position
in more than half of the sequences. The symbol # denotes conservation
of the amino acids N, D, Q, and E at that position in the alignment.
The Jpred line shows the secondary structure prediction from
submission of the multiple alignment to the Jpred secondary structure
prediction program. H represents a prediction for -helix,
and the gaps indicate regions for which no structural
prediction emerged. No
-structure was predicted by Jpred or any
other secondary structure prediction programs. The bottom
line indicates conserved hydrophobic (O) and
hydrophilic (+) positions, grouped according to the Dayhoff
substitution matrix (69). B, phylogenetic relationships
between the FAR protein sequences of C. elegans and those
known from O. volvulus and G. pallida. The
phylogenetic tree was constructed as detailed under "Experimental
Procedures." Bootstrap values are shown at each node. C,
position of intron splice sites in the Ce-far genes. Intron
positions mentioned in the text are as numbered from the 5'-end of the
genes (to the left), as derived from the C. elegans genome, and corrected in this study.
carbon, rather than the omega
carbon as in DAUDA, with Ce-FAR-5 again causing the most extreme blue
shift in emission (to 466 nm). The ligand is therefore likely to be
taken into the binding site of the proteins in its entirety and removed
from contact with solvent water. Such complete enclosure of ligand,
despite the possession of a charged carboxylate group, is typical of
most small fatty acid transporter proteins (52-55), with only a few
exceptions (56). The C. elegans FAR proteins also bound
cis-parinaric acid and retinol (Fig. 2, B and
C), which are intrinsically fluorescent and exhibit
environment-sensitive fluorescence emission. It was also found that the
addition of oleic acid could displace retinol from the binding site
(see Fig. 3 for Ce-FAR-2 and Ce-FAR-3 as representing the extremes, and Table I), indicating that the retinol
and fatty acid binding sites are congruent, overlapping, or
interactive. Marked differences between the proteins were observed in
the efficiency of competitive displacement of DAUDA or
cis-parinaric acid by oleic acid (Table I).
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Fig. 2.
Diverse binding site environments
of the recombinant Ce-FAR proteins. A, recombinant
Ce-FAR proteins (final concentrations between 3 and 5 µM)
were added to ~1 µM DAUDA in 2 ml of PBS in the
fluorescence cuvette. The spectra obtained for Ce-FAR-1 to -6 were
adjusted to the same peak value of fluorescence to illustrate with
clarity the different blue shifts in peak fluorescence emission. All
the spectra obtained upon addition of Ce-FAR proteins have been
corrected by subtraction of the emission spectrum of DAUDA in buffer
alone. B, similar experiment carried out under identical
conditions but with ~3.6 µM cis-parinaric
acid. The spectra were not adjusted as in panel A. C, ethanolic solutions of retinol were added to the Ce-FAR
proteins as used above or to buffer alone, to a final concentration of
~1 µM retinol.
Differential efficiency of ligand displacement by oleic acid
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Fig. 3.
Differential efficiency of competition
between retinol and oleic acid for FAR protein binding
sites. Identical quantities of oleic acid (final concentration,
6.5 µM) were added to preformed complexes of 1 µM retinol and 0.9 µM Ce-FAR-2
(A) or 1 µM Ce-FAR-3 (B).
7 to
1 × 10
8 M for all the combinations of
ligand and protein, so were very similar within experimental error. The
results were consistent with 1:1 protein:ligand binding stoichiometry,
although the n values obtained were less than unity
(0.5-0.9) presumably due to a proportion for the recombinant proteins
being misfolded (inappropriate proline isomerization?) by production in
a prokaryote system. Dissociation constants of this order of magnitude
are observed for other small soluble lipid transporter proteins from a
range of sources and biological functions (48, 50, 57, 58).
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Fig. 4.
Titration curves for the binding of DAUDA,
cis-parinaric acid, and retinol to recombinant
Ce-FAR-5. Typical saturation binding curves for each of DAUDA,
cis-parinaric acid, and retinol with Ce-FAR-5 protein.
Similar binding curves were obtained for Ce-FAR-1, -2, -3, -4, and -6. The titrations and the dissociation constant calculations were carried
out as described under "Experimental Procedures."
-helix (54 and
61%, respectively) and only a very small content of
structure
(Fig. 5). No
concentration-dependent changes in the secondary structure
of the proteins were detected over the range of concentrations
evaluated (10-45 µM). Ce-FAR-2 and Ce-FAR-3 exhibited
high probabilities of coiled coil structure by the Coils algorithm
(59). However, although the
222/
208 ratio
from the Ce-FAR-2 and -3 spectra (both 0.97) was greater than that
recorded for isolated helices (0.86), it was less than that recorded
for coiled coils (1.03) (60-62). The results imply that the helical
elements in Ce-FAR-2 and Ce-FAR-3 associate with each other, but the
evidence at present is not sufficient to prove the presence of
coiled-coil structures. Comparison of the spectra of Ce-FAR-2 and
Ce-FAR-3 with the previously published spectrum of Ov-FAR-1 (10) shows
negligible differences, indicating that the three proteins have very
similar secondary structures, despite considerable divergence in their
primary structures. Addition of increasing quantities of GdnHCl led to
progressive loss of secondary structure as detected by CD (Fig. 5,
inset), with the midpoints occurring at ~3 and 3.25 M GdnHCl for Ce-FAR-2 and -3, respectively. By this
measure, the two Ce-FAR proteins are only slightly less robust to
GdnHCl denaturation than Ov-FAR-1, the midpoint for which occurs at
~3.5 M GdnHCl (10).
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Fig. 5.
Helix richness of FAR proteins. Circular
dichroism analysis of one Ce-FAR protein from each of groups A
(Ce-FAR-2) and B (Ce-FAR-3) and C (Ce-FAR-7) in comparison with data
previously obtained for the FAR protein secreted by the O. volvulus parasite (Ov-FAR-1; Ov20) (10). With the exception of
Ce-FAR-7, the spectra are essentially identical, with the differences
likely to be due to small disparities in the estimation of protein
concentrations. See the text for a fuller description of the CD
spectral analysis. The inset shows the progressive loss of
secondary structure with increasing concentrations of GdnHCl, as
measured by changes in molar ellipticity ( ) values measured at 225 nm for Ce-FAR-2, Ce-FAR-3, and Ov-FAR-1; Ce-FAR-7 began to unfold
detectably at only 1 M GdnHCl (see text) and is not
included in the inset. The sigmoid-fitted line is for
guidance only.
-helix content and more
structure
(SELCON 30.4%
-helix, 11.1% antiparallel
sheet; CONTIN 32%
-helix, 26%
sheet) than Ov-FAR-1, Ce-FAR-2, or Ce-FAR-3. The
222/
208 for Ce-FAR-7 is ~0.83, which
suggests that the helices in this protein are unlikely to interact in a
coiled-coil arrangement. Secondary structure prediction algorithms
(e.g. GOR IV and HNN, accessed through www.expasy.ch/tools)
also reflect this difference between Ce-FAR-7 and the group A and B
proteins in predicting about 37.0% helix for Ce-FAR-7, whereas the
values for the other FAR protein sequences lay between 62 and 72%
helix. Moreover, although not shown in Fig. 5, Ce-FAR-7 was
considerably more sensitive to unfolding by GdnHCl in that loss of
secondary structure was observed to begin at 1 M
denaturant, and unfolding was 60% complete by 2 M GdnHCl.
These findings reinforce the idea that the group C proteins are
structurally and functionally distinct from the others, albeit
descended form a common ancestor.
HVH/
Hcal)
indicated that native Ce-FAR-2 might exist and unfold cooperatively as
a dimer (38). Our preliminary hydrodynamic results have shown that
Ov-FAR-1 (group A, as is Ce-FAR-2) is a tight homodimer, but that a
group B FAR protein (Ce-FAR-5) exists as a
monomer.3 After completion of
the scan to 110 °C and subsequent cooling to 10 °C, rescanning
yielded a similar symmetrical peak with the same
Tm but of reduced amplitude (~0.66 of first
scan), indicative of a high but incomplete degree of refolding
following the thermal transition (complete denaturation). The extreme
stability of Ce-FAR-2 to thermal unfolding resembles that previously
reported for Ov-FAR-1, which was also found to be relatively resistant
to GdnHCl denaturation (10).
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Fig. 6.
High thermal stability of Ce-FAR-2.
Differential scanning calorimetry analysis of recombinant Ce-FAR-2
protein was carried out at a scan rate of 60 °C h 1
over a range of 20-110 °C using a sample concentration of 0.7 mg
ml
1. Normalized excess heat capacity data were calculated
by standard procedures, using Microcal Origin software. The
smooth line shows a theoretical fit to these data with
transition midpoint (Tm = 82.7 °C),
Hcal = 3.57 × 104 cal
mol
1, and
HVH = 7.57 × 104 cal/mol
1.
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Fig. 7.
Temporal and gender-specific differences in
Ce-far transcript levels. The abundance of
transcript derived from each of the Ce-far genes was
estimated as described under "Experimental Procedures" and are
expressed relative to transcript levels for the ama-1 gene
obtained in parallel PCR reactions.
-helix and apparent absence of
-extended
structure. We therefore predict that Ce-FAR-1, -2, or -6 have
the closest common ancestry with, and are functional homologs of, the
FARs that have been detected as major secreted products of parasitic nematodes. Consequently, these three C. elegans FAR proteins
should receive particular attention to elucidate their biological
function, thus providing an understanding of the role of FAR proteins
in the success of nematode parasitism.
-carotene
15,15'-dioxygenase (29). Moreover, putative retinoid receptors and
dehydrogenase enzymes have also been identified from the genome
sequence (29), implying a role for retinoids in the control of gene
expression, although the operative ligands have not been identified.
FAR proteins may be particularly important to animal parasitic
nematodes, supplying the parasite with retinoids and causing localized
depletion of these molecules in the surrounding host tissue. Given the
central role of retinoids in cellular and tissue differentiation and
repair (11), and immune processes (19-23), the subversion of retinol
signaling in mammalian hosts by secreted FAR proteins clearly demands attention.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Christoph Janssen of Glasgow University for expert assistance in the final checks of M.W.K.'s phylogenetic analysis. Many thanks must also go to Freya Fowkes, who helped with the early analysis of the proteins.
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FOOTNOTES |
---|
* This work was supported by grants from the Universities of Salford and Nottingham (to J. E. B.) and the Wellcome Trust (to M. W. K.). The circular dichroism and biological calorimetry facilities were supported by the University of Glasgow and the Biotechnology and Biological Sciences Research Council (UK), the fluorometry facilities were supported by the University of Glasgow, and the GC-MS instrument was provided by a Wellcome Trust grant (to D. G. W. and M. W. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
g Both authors contributed equally to this work.
h To whom correspondence should be addressed. Tel.: 44-141-330-5819; Fax: 44-141-330-5971; E-mail: malcolm.kennedy@bio.gla.ac.uk.
i To whom correspondence should be addressed. Tel.: 44-115-951-3207; Fax: 44-150-951-3251; E-mail: jan.bradley@nottingham.ac.uk.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M206278200
2 M. W. Kennedy, unpublished data.
3 A. Solovyova, M. W. Kennedy, and O. Byron, unpublished.
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ABBREVIATIONS |
---|
The abbreviations used are:
FABP/P2/CRBP/CRABP, members of the cytoplasmic lipid transporter family
of proteins;
CD, circular dichroism;
dansyl, 5-dimethylaminonaphthalene-1-sulphonyl;
DAUDA, 11-((5-dimethylaminonaphthalene-1-sulphonyl)amino)undecanoic acid;
DSC, differential scanning calorimetry;
DACA, dansyl-DL--aminocaprylic acid;
FAR, nematode-specific
family of fatty acid and retinol-binding proteins;
far, gene
encoding a FAR protein;
GC-MS, gas chromatography and mass
spectrometry;
GdnHCl, guanidinium hydrochloride;
L1, L2, L3, and
L4, first, second, third, and fourth larval stages in the nematode
lifecycle;
ORF, open reading frame;
PBS, phosphate-buffered saline;
RACE, rapid amplification of cDNA ends;
retinol, all-trans retinol;
RT, reverse transcriptase;
sqRT, semi-quantitative RT.
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