(Received for publication, November 8, 1996, and in revised form, January 17, 1997)
From the Early development of the parasitic nematode,
Ascaris suum, occurs inside a highly resistant eggshell,
and the developing larva is bathed in perivitelline fluid.
Two-dimensional gel analysis of perivitelline fluid from infective
larvae reveals seven major proteins; a cDNA encoding one of these,
As-p18, has been cloned, sequenced, and protein expressed in
Escherichia coli. The predicted amino acid sequence of
As-p18 exhibits similarities to the intracellular lipid-binding protein
(iLBP) family including retinoid- and fatty acid-binding proteins
(FABP). As-p18 is unusual in that it possesses a hydrophobic leader
that is not present in the mature protein, the developmental regulation
of its expression, and in terms of its predicted structure.
Recombinant As-p18 is a functional FABP with a high affinity for both a
fluorescent fatty acid analog
(11(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino) undecanoic
acid) and oleic acid, but not retinol. Circular dichroism of rAs-p18
reveals a high As-p18 is not found in unembryonated eggs, begins to be synthesized at
about day 3 of development, reaches a maximal concentration with the
formation of the first-stage larva and remains abundant in the
perivitelline fluid of the second-stage larva. Since As-p18 is not
present in the post-infective third-stage larva or adult worm tissues,
it appears to be exclusive to the egg. Surprisingly, however, Northern
blot analysis yields mRNA for As-p18 not only in the early larval
stages, but also the unembryonated egg, third-stage larvae, and ovaries
of adult worms, even though the protein is not detectable from any of
those sources. As-p18 may play a role in sequestering potentially toxic
fatty acids and their peroxidation products, or it may be involved in
the maintenance of the impermeable lipid layer of the eggshell.
Parasitic nematodes cause medical and economic damage on a global
scale, in part the result of the longevity and environmental robustness
of their dispersal stages, their eggs. Arguably the most successful and
widely distributed nematode parasite of humans is Ascaris
lumbricoides, which infects over 1 billion people. A
morphologically indistinguishable species, Ascaris suum,
occurs in pigs and has become the best understood nematode in
biochemical terms. The eggs contaminate soil, and the embryos develop
to infective, second-stage larvae (L2s)1
within the eggs. The L2 undergoes developmental arrest until ingestion
by the host, and may survive for up to 7 years, but little is known of
the biochemical basis for such long term survival. The problems faced
by the L2 include maintaining both the egg's impermeability to water
and dissolved ions (they can survive in dilute formalin and sulfuric
acid) and its permeability to oxygen, as well as the potential
accumulation of toxic products of lipid peroxidation. The ascarid L2
develops in the perivitelline fluid enclosed within the highly
resistant, chitinous eggshell (1). The innermost lipid layer of the
eggshell accounts for the extreme impermeability and resistance and is
composed of three unusual, structurally related glycosides, the
ascarosides (1-3). Perturbation of this ascaroside layer appears to be
involved in the initiation of the hatching process upon infection of a
new host (4-6). The organism, therefore, has particular need of a
mechanism to transport and store hydrophobic compounds between the
larva and the eggshell.
The present study was initiated to identify major proteins in the
perivitelline fluid of A. suum infective larvae,
specifically those potentially involved with the maintenance of the
highly resistant lipid layer. Toward this end, we have identified a
novel fatty acid-binding protein (FABP), designated As-p18, in the
perivitelline fluid surrounding the infective L2. Based on sequence and
structure analysis, and its fatty acid binding function, As-p18 is an
unusual member of lipid-binding protein (LBP) family in having a
hydrophobic leader sequence, being under strong developmental
regulation, and in terms of its predicted structure.
Adult A. suum females were obtained
from Routh Packing (Sandusky, OH). Unembryonated eggs (day 0) were
collected from uteri and incubated at 25 °C for 10 and 30 days to
obtain first-stage larvae (L1s) and second-stage larvae (L2s),
respectively, as described previously (7). Third-stage larvae (L3s)
were recovered from rabbit lungs 7 days post-infection with 3 × 105 L2s as described previously (8, 9).
L2s were
artificially hatched, as modified from Urban et al. (7).
Briefly, L2s were treated with 5.25% sodium hypochlorite for 10 min at
room temperature. Eggshells were disrupted with glass beads by gentle
stirring at 37 °C in Earle's balanced salt solution with protease
inhibitor mixture A (20 µg/ml pepstatin A, 20 µg/ml chymostatin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 mM EDTA, 2 mM
EGTA). The suspension was centrifuged at 200 × g for
10 min, and the resulting supernatant was centrifuged at 155,000 × g for 1 h at 4 °C. The hatching fluid was
concentrated and exchanged by ultrafiltration (Centricon 10, Amicon)
into 20 mM MOPS, pH 7.2, containing protease inhibitor mixture A.
Larvae were disrupted by treatment in a French pressure cell at 20,000 p.s.i. in 20 mM MOPS, pH 7.2, containing protease inhibitor mixture B (mixture A plus 20 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, 40 µg/ml antipain, and 20 µg/ml caproic acid). All adult tissues were homogenized in 20 mM MOPS, pH 7.2, with protease inhibitor mixture B. Larval and adult homogenates were brought to 1% with Triton X-100 on ice for 30 min and centrifuged at
10,000 × g for 30 min at 4 °C before concentration
of the supernatant by ultrafiltration, as outlined above. Protein
concentrations were estimated by the Bradford method (Bio-Rad). All
samples were aliquoted, lyophilized, and stored at Isoelectric focusing
electrophoresis was performed on precast Immobiline dry strips (linear
gradient, pH 3-10, 11 cm) with a Multiphor II electrophoresis system
(Pharmacia Biotech Inc.) following the manufacturer's instructions
with minor modifications according to Westermeier (10) and Ji et
al. (11). Briefly, the Immobiline dry strips were rehydrated in 8 M urea, 0.2% dithiothreitol (DTT), 0.5% Triton X-100, and
0.5% Nonidet P-40 overnight. Lyophilized homogenates (300 µg) were
dissolved in 100 µl sample buffer (9 M urea, 1% DTT, 2%
Ampholine, pH 3.5-9.5, 2% Nonidet P-40, and 0.005% bromphenol blue),
loaded on the acidic end of the strip and run for 21,125 volt-hours
(V-h) in three continuous phases: phase I, 75 V-h (150 V, 30 min),
phase II, 1500 V-h (300 V, 5 h); and phase III, 19,550 V-h (1150 V, 17 h). The strips were stored at Proteins separated by two-dimensional
gel electrophoresis were transferred to a Problott membrane (Applied
Biosystems) in 10 mM CAPS buffer, pH 11, with 10% methanol
according to the manufacturer's instructions. Proteins of interest
were excised from the membrane and the NH2-terminal amino
acid sequences were analyzed with a model 477A Protein Sequencer
(Applied Biosystems).
The full-length cDNA of
As-p18 was cloned from cDNA prepared with a Marathon kit
(Clontech). Briefly, cDNAs were synthesized from total RNA isolated
from day 5 A. suum eggs, and the double-stranded cDNAs
then were adapted with adapter primer 1 (AP-1) and primer 2 (AP-2).
With these adapted double-stranded cDNAs as a template, a
degenerate sense primer (primer A, 5 Total RNAs from different developmental
stages and adult tissues were isolated by acid guanidinium
thiocyanate-phenol-chloroform extraction according to Chomczynski
et al. (14). Total RNA (20 µg) was separated on a 1%
agarose formaldehyde denatured gel and transferred to a Nytran Plus
positively charged nylon membrane (Schleicher & Schuell), and the
membrane was baked in a vacuum oven at 80 °C. The full-length As-p18
cDNA was used as a probe after labeling with
[32P]dCTP and purification on a NICK column (Pharmacia).
The membrane was hybridized and autoradiographed at A cDNA
encoding the full-length mature As-p18 protein was expressed in the
pQE-30 vector (QIAGEN), which is designed to produce a recombinant
protein bearing a 6xHis tag at the NH2 terminus. A
BamHI site was added to the 5 E.
coli with the pQE-30/As-p18 were cultured in LB medium (10 g of
tryptone, 5 g of yeast extract, and 5 g of NaCl/liter) until
the absorbance at 600 nm reached 0.6 units. Expression of recombinant
As-p18 (rAs-p18) was induced by 1 mM
isopropyl-thio- Purified rAs-p18 was
separated on 12% SDS-polyacrylamide gels and used to immunize rabbits
as described previously (15). Antibodies against rAs-p18 were
affinity-purified using protein bound to CNBr-activated Sepharose 4B
(Sigma) according to Kent (16). For immunoblotting, samples were
separated on 12% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes. Membranes were blocked with 1% bovine serum
albumin (BSA), incubated with primary antibody and alkaline
phosphatase-conjugated goat anti-rabbit IgG secondary antibody, and
developed with nitro blue tetrazolium and 5 Fluorescence measurements were made at 20 °C with a
Spex FluorMax spectrofluorimeter (Spex Industries), using 2 ml samples in a silica cuvette. Raman scattering by the solvent 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- Circular dichroism (CD) spectra were
recorded at 20 °C in a JASCO J-600 spectropolarimeter using quartz
cells of path length 0.02 or 0.05 cm. The protein concentration was 0.3 mg ml Protein sequence comparisons were
carried out with the BLAST (19) program to search the protein sequence
data base (blastp). MaxHom (20) through SwissProt and Wisconsin
Sequence Analysis Package (GCG) were used for protein sequence
alignment. Secondary structure predictions were made using the PHD
(21-23) program at SwissProt and GOR (24). Molecular mass and
theoretical pI values for As-p18 were calculated with the ProtParam
program through the SwissProt and GCG packages.
The As-p18 structure was modeled on the structure of mouse adipocyte
lipid-binding protein (ALBP, Protein Data Bank code 1ADL) determined at
1.6-Å resolution by Lalonde et al. (25). A sequence alignment based on that produced by the PHD program on the
Predict-Protein server (23) was used to direct the mutation of residues
in the mouse protein to those of As-p18 using the program O (26). Three insertions were added using the lego-loop option, selecting by inspection the loop with the smallest root-mean-square deviation of
base residue positions that had no significant Van der Waals overlap of
atoms. Following energy minimization of the entire molecule, these
loops were subjected to 2000 iterations (1 ps) of two-step Verlet
molecular dynamics at 300 K; 2000 iterations at 200 K and 2000 at 100 K. This was followed by restrained energy minimization to convergence
of the entire molecule and finally unrestrained energy minimization to
convergence. Molecular simulation calculations were performed using the
X-PLOR program and the CHARMM force field (27).
Perivitelline fluid was recovered from infective
A. suum eggs after mechanical disruption and analyzed by
two-dimensional gel electrophoresis and NH2-terminal
sequencing of selected proteins (Fig. 1). Seven abundant
proteins with apparent molecular weights of about 50, 43a, 43b, 18, 16, 13, and 12.5 kDa were discernible by this procedure, in addition to a
number of minor components. The NH2-terminal amino acid
sequence of the protein, which migrated at about 18 kDa and a pI of
7.8, exhibited similarity to a group of small, water-soluble iLBPs
including fatty acid- and retinoid-binding proteins identified from a
variety of different organisms (Figs. 2 and
3). This protein was designated As-p18 and selected for further study.
The cDNA encoding a fragment of As-p18 was isolated by PCR using
degenerate oligonucleotides designed from the amino-terminal amino acid
sequence generated from material eluted from two-dimensional gels.
Full-length sequence for the As-p18 cDNA was then obtained by 3 Comparison of the As-p18 sequence with protein sequences available in
the data bases indicated that it had greatest similarity to iLBPs from
variety of other sources ranging from flatworms to mammals and it was
most similar to proteins predicted from the Caenorhabditis
elegans genome project (see Fig. 3 for alignment), which are
presumed to be LBPs based on their alignment with LBPs from
non-nematode parasitic helminths and mammals. At the amino acid level,
As-p18 exhibited 43% identity to C. elegans LBP-1 (GenBankTM accession no. U40420[GenBank], protein identification code 1065518),
42% to C. elegans LBP-2 (GenBankTM accession no. U40420[GenBank],
protein code 1065519), 33% to mouse ALBP, 32% to rabbit myelin P2
protein, 30% to Fasciola hepatica FABP, 29% to mouse brain
FABP, 28% to Schistosoma mansoni FABP and
Echinococcus granulosus FABP, and 27% to bovine heart FABP
(25, 30-35). The nematode sequences were the only ones arising from
the data base searches which had hydrophobic leader peptides. The
nematode sequences had many more residues in common than with the other
iLBPs, although residues conserved across the entire array were
apparent. The nematode proteins also had two insertions of three and
four amino acids in similar positions, which are absent in the other
iLBPs. The cleavage site for the leader peptide is between Ala-20 and Lys-21 of As-p18, as defined by the NH2-terminal amino acid
analysis of protein isolated from A. suum eggs, and this is
also the most probable cleavage site identified by the Signalp program,
based on neural networks trained on eukaryotic sequences (36). The Signalp program also predicts cleavage sites in the C. elegans proteins precisely aligning with that for As-p18.
Early
A. suum larval stages and adult tissues were examined for
As-p18 by immunoblotting with affinity-purified antiserum against
rAs-p18. Immunoblotting analysis revealed that As-p18 was absent in day
0 unembryonated eggs, began to be synthesized at about day 3 of
development, was abundant at day 10 (when the L1 is formed), and
remained abundant in eggs containing the infective L2. However, As-p18
could not be detected either in the parasitic L3 or adult A. suum muscle, ovaries, testis, or intestine (Fig. 4A).
Northern blotting using the radiolabeled full-length cDNA as a
probe identified a single mRNA species of about 0.7 kb in total RNA
extracted from Day 3 eggs, L1, and L2 (Fig. 4B).
Surprisingly, significant bands of this size were also observed in
Northern blots of RNA extracted from unembryonated eggs, ovaries, and
especially L3s, even though the As-p18 protein could not be
demonstrated by immunoblotting of these stages. To confirm the identity
of the mRNA observed in ovaries and L3s on Northern blots,
As-p18-specific primers were designed and used for PCR of cDNA
pools prepared from mRNA isolated from both ovaries and L3s. The
sequences obtained from both sources were identical to that of
full-length As-p18 (data not shown).
To localize As-p18 during early larval development, infective L2 were
hatched artificially, and both hatched larvae and hatching fluid were
analyzed by SDS-PAGE and immunoblotting with affinity-purified antiserum against rAs-p18. As expected, As-p18 was present primarily in
the hatching fluid, but a weak signal was also observed from the
hatched L2s rinsed free from hatching fluid (data not shown). Immunolocalization and in situ hybridization experiments are
currently in progress to identify the cells responsible for As-p18
synthesis.
The sequence of As-p18 aligns with iLBPs
whose structures are known from x-ray crystallographic studies (37).
These iLBPs are
Modeling of the As-p18 sequence to mouse ALBP (25), the iLBP most
similar to As-p18, demonstrated that the protein can be successfully
modeled as a
The final model of As-p18 has 96.1% of residues in the two
most-favored regions of the Ramachandran plot and an acceptable overall
geometry, both determined with Procheck (41). There is a single major
cavity in the protein interior as determined by the program Voidoo
(42). The volume available to a probe of radius 1.4 Å in this cavity
is 335 Å3, also determined using Voidoo. It is an artifact
of the modeling process used that a cavity within a protein will
contract during the energy minimization stage. The alternative is to
model the protein with a ligand filling the cavity, thus partially
predefining the results of the experiment. This was considered less
acceptable than a small artificial contraction of the ligand-binding
cavity. Nevertheless, the cavity remains of a suitable size to bind
fatty acid ligands; oleic acid is approximately 300 Å3
while retinol occupies 330 Å3, as calculated from the
volume which these ligands occupy within the known crystal structures
of their binding proteins. The cavity is shown in Fig. 6B.
In mouse ALBP, Arg-126 is available to bind a fatty acid carboxylate
group, while in As-p18 the corresponding Arg (Arg-158) is
charge-neutral in combination with Asp-36, invariably a serine in other
similar proteins, and so less available for carboxylate binding. The
remainder of the ligand-binding cavity is lined with either hydrophobic
residues or with hydrogen-bonded hydrophilic side chains. The only
exceptions to this are Arg-46, which has its side chain directed toward
the cavity, and Lys-90. Both these anomalies can be explained from the
differences between the consensus sequence and that of As-p18. Arg-46
probably replaces Arg-158 in binding the fatty acid carboxylate group,
while Lys-90 forms a charge pair with Asp-100, making this region of
the ligand binding pocket also charge-neutral.
Additional evidence for the accuracy of this model, especially the
insertions, is found in the positioning of the nonpolar hydrophilic
side chains in the ligand-binding cavity. The positioning of Tyr-160,
Asn-67, Asn-80, His-88, and the backbone of Glu-101 creates a four link
hydrogen-bond chain forming one "wall"' of the ligand-binding
cavity.
As-p18 was
expressed in E. coli without its putative leader peptide but
with a His6 affinity tag at its amino terminus and six
additional amino acids derived from the vector (see "Experimental Procedures"). rAs-p18 was soluble under nonreducing conditions and
was purified to apparent homogeneity by chromatography on Ni-NTA-agarose. Under SDS-PAGE on 12% gels, its apparent molecular mass was 22 kDa, while the calculated molecular mass is 18,309 Da (data
not shown).
The fatty acid binding capacity of rAs-p18 was examined using
fluorescent lipid analogs, as described previously (43, 44). The
emission spectrum of DAUDA is altered upon entry into fatty acid-binding proteins (45) and binding to rAs-p18 was accompanied by a
substantial increase of fluorescence intensity and a shift in the
fluorescence emission peak from 543 nm to 498 nm (Fig. 7). To place this blue shift in context, rat liver FABP,
BSA, and the ABA-1 allergen of A. suum produce shifts in
DAUDA emission to 500, 495, and 475 nm, respectively (43, 45). The
degree of the shift in the dansyl fluorophore emission is considered to
be a measure of the polarity of the binding site (46), and that brought
about by rAs-p18 is similar to other iLBPs. Titration of rAs-p18 with
DAUDA yielded an apparent dissociation constant (Kd)
of (1.2 ± 0.5) × 10
We also investigated binding of rAs-p18 to a fluorescent fatty acid,
DACA, in which the dansyl fluorophore is attached at the Several of iLBPs bind retinol in addition to fatty acids, so As-p18 was
also tested for retinol binding by fluorescence. Since retinol is
highly unstable in water, the fatty acid binding assay was modified so
that retinol was added directly to the rAs-p18 solution in the
fluorescence cuvette. This assay yielded no evidence of binding by
rAs-p18, although control proteins BSA, bovine lactoglobulin and rABA-1
all showed strong binding, as revealed by a dramatic increase in the
intensity of fluorescence of retinol (data not shown).
Many proteins that bind fatty acids have a tryptophan residue in or
near the binding pocket of the protein, the fluorescence emission of
which is altered upon ligand binding, presumably through direct
involvement in ligand binding, alteration in the environment of the
tryptophan, or local changes in protein conformation. This has been
observed for We have demonstrated that the perivitelline fluid surrounding the
developing larva of the parasitic nematode, A. suum, has a
relatively simple polypeptide composition and have identified from it a
novel, abundant fatty acid-binding protein, As-p18. The predicted amino
acid sequence of As-p18 exhibits significant similarity to iLBPs
isolated from a variety of different organisms, ranging from flatworms
to mammals.
All LBPs appear to have similar conformations with 8 or 10 anti-parallel Sequence differences aside, however, As-p18 is clearly a functional
fatty acid-binding protein. It has a high affinity for oleic acid, and
there is strong evidence that it is structurally very similar to iLBPs,
based on the preponderance of Another unusual feature of As-p18 is the tight developmental control of
its expression, which is not common among LBPs, although a few cases
have been reported (51, 52). As-p18 is not present in unembryonated
eggs, but begins to be synthesized at about day 3 and is abundant in
the perivitelline fluid by the time the L1 is formed on day 10. The
reasons for the apparent lack of As-p18 protein in these stages is
unclear, but the data suggest that the maternal As-p18 mRNA in the
unembryonated egg (53) may not be translated. For the L3, however, an
alternative explanation may be that As-p18 is synthesized and
immediately secreted, so it does not accumulate within the larva.
As-p18 appears to be present in the perivitelline fluid prior to
development of any of the secretory structures possessed by later
stages of A. suum, and no As-p18 immunoreactivity has been
detected in the duct leading to the secretory pore in infective
L2.3 As-p18 must therefore arise in the
perivitelline fluid by an unexpected route, and studies are under way
to characterize the cells responsible for As-p18 synthesis and
secretion into the perivitelline fluid before the development of a
functional secretory system. In view of the similarity between As-p18
and the putative LBPs from C. elegans, it would seem
valuable to investigate the developmental control, function and site of
their synthesis in this organism. It is conceivable that one or both
homologues are important to the survival of the embryo within the
eggshell.
Although the function of As-p18 is unclear, a number of interesting
possibilities are apparent. The early development of A. suum
from unembryonated egg to infective L2 takes place entirely within a
highly resistant eggshell, and the quiescent L2 can remain viable for
several years while it awaits ingestion by an appropriate host (54).
Carbohydrate is utilized during the first 5 days of development and
then resynthesized from stored triglycerides by the glyoxylate cycle
(55). Stored triglycerides are the only energy source for quiescent
L2s, and the appearance of As-p18 in the perivitelline fluid parallels
the increasing utilization of triglycerides as development proceeds
(55, 56). As-p18 may therefore play a role by sequestering potentially
toxic fatty acids and their peroxidation products accumulating around a
larva enclosed within an impermeable eggshell. The resistance of the eggshell is thought to be due to a highly impermeable lipid layer comprised of unusual glycosides (ascarosides) secreted by the fertilized egg prior to the expression of As-p18 (3). We are currently
testing the binding capacity of As-p18 for ascarosides and potential
products of lipid peroxidation. As-p18 also may play a role in the
altered permeability of the lipid layer that accompanies the onset of
hatching. This altered permeability permits the diffusion of trehalose
out of the perivitelline fluid, and this change in osmotic pressure is
presumed to activate the quiescent larva whose subsequent movement may
further mechanically disrupt the lipid layer (1, 5).
FABPs have been isolated from a number of flatworms, including S. mansoni, E. granulosus, and F. hepatica and
have received attention as being potentially important in helminth
parasitism. Indeed, the flatworm FABPs appear to be important antigens
released during the course of infection with these helminths and have
been identified as promising vaccine candidates (31, 33-34, 57). In
addition, an ethanolamine-binding protein recently has been identified
as a major antigen released during the larval migration of the related
dog ascarid, Toxocara canis (58). Finally, the ABA-1
allergen produced by A. suum also displays strong fatty acid
binding activity, even though it is quite different in structure from
the LBPs (43, 59).
As-p18 is, therefore, a member of the The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U51906[GenBank]. The predicted structure model for As-p18 (code 1AS1) has been
submitted to Protein Data Bank, Brookhaven National Laboratory, Upton,
NY. We are indebted to Prof. N. C. Price and Dr.
S. Kelly at Stirling University, Stirling, United Kingdom for
assistance with the circular dichroism, Dr. Alan Cooper at the
University of Glasgow for help with the analysis of ligand binding
data, Drs. Y.-J. Huang and E. Duran at The University of Toledo for
helpful discussions, and Dr. Y. Xia and Fiona McMonagle at the
University of Glasgow for supplying the recombinant ABA-1 protein. The
circular dichroism facility at Stirling University is supported by the
Biotechnology and Biological Sciences Research Council (United
Kingdom). We also thank personnel in Routh Packing (Sandusky, OH) for
allowing us to collect adult A. suum.
Department of Biology, The University of
Toledo, Toledo, Ohio 43606-3390, the § Division of Infection
and Immunity,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-sheet content (62%), which is consistent with
secondary structure for the protein predicted from sequence algorithms,
and the structure of iLBPs. Unusual features are apparent in a
structural model of As-p18 generated from existing crystal structures
of iLBPs.
Parasites
80 °C until
use.
80 °C for less than 3 months before use. Before running the second dimension, each strip was
equilibrated in 10 ml of 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/ml DTT, and 0.0015%
bromphenol blue for 20 min with gentle agitation. The second dimension
was run on 12% SDS-polyacrylamide gels according to Laemmli (12). The
gels were either stained with Coomassie Blue to visualize proteins or
transferred to Problott membranes (Applied Biosystems) for
NH2-terminal amino acid sequencing.
-GA(C/T) AA(A/G) TT(C/T) (C/T)T(N)
GG(N) AC(N) TT(C/T) AA-3
) designed from NH2-terminal amino
acid sequence of As-p18 (DKFLGTFK) and the AP-1 primer were used for
3
-RACE. The 3
-RACE PCR products were analyzed on a 1% agarose gel
and a weak band at about 600 base pairs was purified from the gel by
GENO-BIND (Clontech). The purified DNA was used as template for nested
PCR with primer A and AP-2 primer. The nested PCR products were
analyzed on a 1% agarose gel. The DNA at about 600 base pairs was
purified from the gel by GENO-BIND (Clontech), cloned into pNoTA/T7
vector (5
-3
, Inc.), and both strands sequenced by the
dideoxynucleotide chain termination method of Sanger et al.
(13) using Sequenase 2.0 (U.S. Biochemical Corp.). According to the
sequence of the nested PCR product, two complementary gene-specific
primers (primer B, 5
-TCC TTG TGA TGC GTG TCT TTC-3
; primer C, 5
-GAA
AGA CAC GCA TCA CAA GGA-3
) were designed. Primer B and primer C were
used with AP-1 for 5
-RACE and 3
-RACE, respectively, with the adapted
day 5 cDNA. These 5
-RACE and 3
-RACE products were fused to
prepare the full-length cDNA, which was both strands sequenced as
described above.
80 °C.
end immediately before the
nucleotide (nt 72) coding for the first amino acid of the mature
protein, and a PstI site was added to the 3
-untranslated
region at nt 603-608 by PCR with primer D (5
-G GTT
AAG ACT CTA CCC GAC-3
) and primer E (5
-GC AAT
TAA CAG ATG ATC AG-3
), respectively.
The PCR fragment at about 550 base pairs was cloned into pCR II vector
(Invitrogen). The 532-base pair BamHI/PstI fragment digested from pCR II/As-p18 plasmid DNA was cloned into pQE-30
vector via BamHI/PstI sites. This pQE-30/As-p18
plasmid was transformed into M15 (pREP4) strain of Escherichia
coli (QIAGEN) and both strands sequenced, as described above, to
verify the insert sequence.
-D-galactopyranoside for 4 h at
37 °C. rAs-p18 was purified by affinity chromatography on
Ni-NTA-agarose (QIAGEN) under nonreducing conditions. Briefly, bacteria
were sonicated on ice in 50 mM
NaH2PO4, 300 mM NaCl, pH 7.8 and
the lysate was centrifuged at 12,000 × g for 20 min at
4 °C. Triton X-100 (0.5% final concentration) was added and the
solution was gently stirred with Ni-NTA-agarose for 60 min on ice. The
resin was then loaded into a column, washed with 50 mM
NaH2PO4, 300 mM NaCl, 10%
glycerol, pH 6.0, and eluted with a gradient of 0-300 mM
imidazole. The purified rAs-p18 was concentrated in 10 mM
Tris-HCl, pH 7.5, by ultrafiltration (Centricon 10, Amicon) and stored
at
80 °C in small aliquots. This procedure provided approximately
16 mg of rAs-p18/liter of culture. Recombinant ABA-1 (rABA-1) allergen
from A. lumbricoides was produced by similar methods, as
will be described in detail
elsewhere.2
-bromo-4-chloro-3-indolyl
phosphate.
-aminocaprylic acid (DACA) were obtained from
Molecular Probes and Sigma, respectively. All-trans-retinol
and oleic acid were also obtained from Sigma. The dansylated fatty
acids were stored as stock solutions (~1 mg ml
1 in
ethanol) in the dark at
20 °C. They were freshly diluted to 1 µM with phosphate-buffered saline (PBS; 171 mM NaCl, 3.35 mM KCl, 10 mM
Na2PO4, 1.8 mM
KH2PO4) pH 7.2, before use in the fluorescence
experiments. Competitors of fluorescent fatty acid binding were
prepared as stock solutions (~10 mM in ethanol) and diluted in PBS before use. Retinol binding to proteins was tested by
adding retinol (6 µl of a freshly prepared 180 µM
solution in ethanol) directly to a cuvette containing protein in PBS.
Reference proteins BSA,
-lactoglobulin (bovine), ribonuclease A,
ovalbumin (chicken), and transferrin (bovine) were obtained from Sigma
and prepared as 10 mg ml
1 stock solutions in PBS. For
estimation of the dissociation constant, successive 5- or 10-µl
samples of rAs-p18 (at a monomer concentration (assuming monomeric
dispersion) of 26.9 µM) were added to 2 ml of 0.7 µM DAUDA, and the fluorescence measured at 476 nm
(
exc = 345 nm). The concentration of the ethanol stock
solution of DAUDA was checked by absorbance of a 1:10 dilution in
methanol at 335 nm, using an extinction coefficient
335
of 4400 M
1 cm
1. The
concentration of rAs-p18 was estimated by absorbance at 280 nm, using
an extinction coefficient of
280 = 27310 M
1 cm
1, based on the amino acid
composition of the recombinant protein (17). Fluorescence data were
corrected for dilution, and fitted by standard nonlinear regression
techniques (using Microcal ORIGIN software) to a single noncompetitive
binding model to give estimates of the dissociation constant
(Kd) and maximal fluorescence intensity
(Fmax). Similar nonlinear regression methods
were used to analyze results of competition experiments in which oleic
acid was progressively added to DAUDA/rAs-p18 mixtures. A stock
solution of oleic acid in ethanol was freshly diluted to 10.6 µM in PBS, and increasing concentrations of oleic acid
were added to a mixture containing 0.71 µM DAUDA and 0.94 µM rAs-p18. For all of the fluorescence-based experiments, residual detergent was removed from rAs-p18 solutions by
passage through an Extracti-Gel D column (Pierce).
1, as estimated by absorbance at 280 nm, in PBS.
Molar ellipticity values were calculated using a value of 118 Da for
the mean residue weight calculated from the amino acid sequence of
rAs-p18 protein. Analysis of the secondary structure of the protein was
performed using the CONTIN procedure (18) over the range from 240 to
195 nm.
The As-p18 Protein and Cloning of Its Encoding
cDNA
Fig. 1.
Two-dimensional gel electrophoresis of
perivitelline fluid prepared from infective A. suum larvae.
A. suum L2s were artificially hatched as described under
"Experimental Procedures." Perivitelline fluid (300 µg of
protein) was analyzed by two-dimensional gel electrophoresis and
stained with Coomassie Blue, as described under "Experimental
Procedures." The arrow indicates As-p18.
[View Larger Version of this Image (36K GIF file)]
Fig. 2.
Nucleotide and predicted amino acid sequence
of As-p18. cDNA encoding As-p18 was cloned and sequenced as
described under "Experimental Procedures." The 17-amino acid
NH2-terminal sequence determined by direct sequencing of
the As-p18 spot obtained from a two-dimensional gel is
underlined, as is a consensus polyadenylation signal in the
3-untranslated region.
[View Larger Version of this Image (50K GIF file)]
Fig. 3.
Predicted amino acid sequence of As-p18
aligned with iLBP sequences from other sources. Dots
indicate identity with As-p18. The boxed area is the
NH2-terminal sequence determined by direct sequencing of
As-p18. The amino acids of As-p18 are numbered.
[View Larger Version of this Image (53K GIF file)]
-
and 5
-RACE from day 5 egg cDNAs prepared as described under
"Experimental Procedures." The sequence contained 628 nucleotides with a portion of the 22-nucleotide spliced leader sequence (SL1) characteristic of many nematode mRNAs (5
-CCA AGT TTG AG-3
, Fig. 2) (28). Downstream of the SL1 sequence, no 5
-untranslated region was
apparent, as the initiation ATG codon immediately followed the spliced
leader, and an open reading frame of 492 nucleotides ended with the
termination codon TAA. The 3
-untranslated region of 125 nucleotides
contained a sequence identical to the consensus polyadenylation signal,
AATAAA, and a short poly(A) tail after the polyadenylation signal. The
open reading frame predicts a protein with a molecular mass of 19,077 Da including a hydrophobic leader peptide of 20 amino acids which was
not observed at the amino terminus of the mature, egg-derived protein
(Fig. 3). This leader peptide exhibited features characteristic of
secretory signals identified in other organisms (29). The predicted
molecular mass of the mature As-p18 was 16,911 Da, and its theoretical
pI was 7.1 by the ProtParam program and 7.8 by the GCG packages, respectively.
Fig. 4.
Developmental regulation of As-p18.
A, immunoblot of A. suum larval stages and adult
tissues with affinity-purified antiserum against rAs-p18. Homogenates
(35 µg of protein) of A. suum unembryonated eggs
(D0), day 3 (D3), L1, L2, L3, adult muscle (M), ovaries (O), testis (T), and
intestine (I) were separated on a 12% SDS-polyacrylamide
gel and transferred to a nitrocellulose membrane. The membrane was
incubated with affinity-purified antiserum against rAs-p18 and
developed as described under "Experimental Procedures."
B, Northern blot of A. suum RNA isolated from
different larval stages and adult tissues using a cDNA clone for
As-p18 as probe. Total RNA (20 µg) from larval stages and adult
tissues as in panel A was separated on a formaldehyde
denatured 1% agarose gel and transferred to a nylon membrane. The
membrane was hybridized with a full-length 32P-labeled
As-p18 cDNA probe and exposed for 3 days at 80 °C.
[View Larger Version of this Image (35K GIF file)]
-sheet-rich proteins, which have 10 antiparallel
-sheets forming so-called
-barrel or
-clam structures, with
one or two short helices closing one end of the barrel. The ligand is
held within a cavity in the center of the barrel, where it is removed from solvent and thereby protected from oxidation. The PHD program predicts As-p18 to be
-rich (19.0% helix, 35.0%
extended/
-structures, and 46.0% remainder) based on multiple
alignments assembled from the As-p18 sequence and similar mammalian
proteins by the MaxHom routine, and GOR predicts 14.2% helix, 59.1%
extended/
-structure. The predictions of predominance of
-sheet
structures are borne out by circular dichroism of rAs-p18. The far-UV
circular dichroism spectrum of rAs-p18 (Fig. 5) showed a
strong
-signal, and analysis of the data obtained over the range 240 to 195 nm by the CONTIN procedure provided the following estimates of
secondary structure: 62%
-sheet and less than 5%
-helical
content. This estimate is close to the known content of
-structure
from crystal structures of iLBPs (37-40).
Fig. 5.
Circular dichroism spectrum of rAs-p18.
The spectrum was recorded at 20 °C in PBS, path length 0.02 cm.
[View Larger Version of this Image (14K GIF file)]
-barrel with two short helices, although modifications
have to be made to accommodate the amino acid insertions (Fig.
6A). These have been made in loop regions
with the first insertion adding an extra turn of helix to the second
-helical region. The second insertion is close to a loop region. The
third loop insertion is between
-strands 7 and 8 and has the effect of increasing the enclosure of the lipid-binding site, although a
separate, currently unsuspected, function could still be attributed to
this region.
Fig. 6.
Structural modeling of As-p18. A,
model of the structure of As-p18. Ribbon representation of the
predicted structure, based on the crystal structure of mouse ALBP. The
positions of the three insertions which occur in As-p18, but not in
mouse ALBP or other similar proteins, are shown. B, the
predicted structure of the ligand binding site of As-p18 and the space
available for ligand occupancy. The labeled residues projecting into
the cavity are responsible for the formation of the predominately
hydrophobic cavity and maintenance of the charge distribution on the
cavity surface. Tyr-160, Asn-67, Asn-80, His-88, and the backbone of Glu-101 create a four-link hydrogen-bond chain forming one face of the
ligand-binding cavity.
[View Larger Version of this Image (32K GIF file)]
7 M (Fig.
8). The binding of a natural, nonfluorescent ligand
(oleic acid) was then determined from its competitive effects on the fluorescence of the rAs-p18·DAUDA complex. The progressive addition of oleic acid to the rAs-p18·DAUDA complex caused a stoichiometric reversal of the fluorescence effect, presumably by the displacement of
DAUDA from As-p18. Analysis of the oleic acid titration curve yielded
an apparent Kd (oleic) of
2 × 10
8 M (Fig. 8), which is within the range of
Kd found for other iLBPs (47).
Fig. 7.
Fatty acid binding activity of rAs-p18.
Fluorescence emission spectra (uncorrected, exc = 345 nm) of 1 µM DAUDA in PBS or with 2.2 × 10
7 M rAs-p18. Also shown is the reversal of
changes in DAUDA emission by competition with 5.85 × 10
7 M oleic acid added to the rAs-p18·DAUDA
complex. The difference spectrum is the subtraction of the spectrum of
DAUDA alone from DAUDA + rAs-p18, and shows the fluorescent emission of
DAUDA while in the binding site of the protein.
[View Larger Version of this Image (23K GIF file)]
Fig. 8.
Titration curves for the binding of DAUDA and
oleic acid to rAs-p18. A, increase in relative fluorescence
intensity on progressive addition of recombinant As-p18 to DAUDA. The
solid line is the theoretical binding curve for rAs-p18 to
DAUDA with an apparent dissociation constant of about 1.2 × 107 M. B, stoichiometric decrease
in relative fluorescence intensity due to the displacement of DAUDA
from rAs-p18 by oleic acid. The solid line is the
theoretical curve for simple competitive binding of oleic acid in the
DAUDA binding site of As-p18 with an apparent KI(oleic) of approximately 2 × 10
8 M.
[View Larger Version of this Image (15K GIF file)]
carbon,
rather than the hydrocarbon terminal, as is DAUDA. The DACA probe was
found not to alter its fluorescence characteristics when mixed with
rAs-p18, although it bound well to BSA and rABA-1 in control
experiments (data not shown). This lack of DACA binding may indicate
that the mechanism of binding of fatty acids is different from BSA and
ABA-1, or merely that the dansyl group interrupts the hydrogen bonding
network which anchors the carboxylate group in iLBPs (48).
-lactoglobulin and for serum albumin (43, 49), and, in
the case of the former, this effect has been used to measure the
affinity of ligand binding (49). As-p18 has three tryptophan residues,
and excitation of these at 287 nm produced an emission spectrum that
peaked at 339 nm (data not shown). Although this is a compound spectrum
from the emission from three tryptophan residues, the spectrum was
simple, and there was a clear blue shift away from that of
solvent-exposed tryptophan in denatured protein (355 nm, data not
shown). Presumably this means that the tryptophans are removed from
solvent water even in the apo-protein. For comparison, the
peak emissions of the single tryptophans in
-lactoglobulin, BSA, and
ABA-1 are 335, 344, and 308 nm, respectively (data not shown and Ref.
43). There was no indication of a change in the tryptophan fluorescence
of As-p18 upon addition of oleic acid ligand (data not shown). This
suggests that the tryptophans in As-p18 are not close to the ligand,
and none are associated with ligand binding.
-strands and two short
-helices, even though some members of the group exhibit as little as 20% sequence identity (37-38, 48, 50). However, conserved positions have been identified. As-p18 exhibits a number of significant differences in these conserved regions and, with the exception of presumptive LBP-encoding sequences recently identified through the C. elegans genome project,
is the only member of this group with a putative secretory signal. Of
the 39 conserved polar and nonpolar positions in most iLBPs, 8 are not
conserved in As-p18. For example, two invariant residues that
correspond to Ser-55 and Phe-57 of the human ALBP are at a proposed
portal region for ligand entry (37) and are not conserved in As-p18
(Glu-79 and Leu-81).
-sheet as indicated by the CD analysis
and computer-based predictions of its secondary structure. However,
while the modeling study does show that the sequence conforms well to a
10-stranded iLBP, there are indications of modifications to the binding
site which could relate to ligand specificity. In addition, the unusual
extra loops exposed on the surface of the protein might be involved in
unsuspected protein-protein interactions.
-barrel lipid-binding
proteins, which is unusual in the possession of a leader peptide in its
precursor, and its lack of retinol binding. It is also the first member
of this family from nematodes to be characterized as an FABP, and makes
it probable that the proteins identified by similarity from C. elegans indeed represent functional FABPs. It clearly possesses
some unique features, which may reflect adaptation to a specific
function within the egg.
*
This work was supported by National Institutes of Health
Grant AI 18427 (to R. K.), and by Grant 044156 from the Wellcome Trust
(to M. W. K. and A. Cooper (University of Glasgow)).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.
To whom correspondence should be addressed: Dept. of Biology,
The University of Toledo, 2801 W. Bancroft St., Toledo, OH 43606-3390. Tel.: 419-530-2065; Fax: 419-530-7737; E-mail: rkomuni{at}uoft02.utoledo.edu.
1
The abbreviations used are: L2, second-stage
(infective) larva; ALBP, adipocyte lipid-binding protein; AP-1, adapter
primer 1; AP-2, adapter primer 2; BSA, bovine serum albumin; CAPS,
3-(cyclohexylamino)-1-propanesulfonic acid; DACA,
dansyl-DL--aminocaprylic acid; dansyl,
dimethylaminonaphthalene-1-sulfonyl; DAUDA,
11-(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino)undecanoic acid; DTT, dithiothreitol; FABP, fatty acid-binding protein; iLBP, intracellular lipid-binding protein; L1, first-stage larva; L3, third-stage larva; LBP, lipid-binding protein; MOPS,
3-(N-morpholino)propanesulfonic acid; NTA, nitrilotriacetic
acid; PBS, phosphate buffered saline; PCR, polymerase chain reaction;
rABA-1, recombinant ABA-1; rAs-p18, recombinant As-p18; RACE, rapid
amplification of cDNA ends; SL1, nematode spliced leader RNA; V-h,
volt-hour(s).
3
B. Mei, P. R. Komuniecki, and R. Komuniecki,
unpublished observation.
2
Y. Xia, N. Heaney, A. Cooper, and M. W. Kennedy,
manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.