(Received for publication, April 5, 1995; and in revised form, May 22, 1995)
From the
DNA encoding a single unit of the DvA-1 polyprotein of the
parasitic nematode Dictyocaulus viviparus was isolated and the
polypeptide (``rDvA-1L'') expressed in Escherichia coli, to give a protein showing high binding affinity for fatty acids
and retinoids. Fluorescent fatty acid probes show substantial changes
in emission spectrum in the presence of rDvA-1L, which can be reversed
by fatty acids (oleic, palmitic, stearic, arachidonic) and retinoids,
but not by tryptophan, squalene, or cholesterol. Moreover, changes in
intrinsic fluorescence of retinol or retinoic acid confirm a retinoid
binding activity. Fluorescence titration experiments indicate
stoichiometric binding to a single protein site per monomer unit with
affinities (K) in the range 3
10
M for
11-((5dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, and,
by competition, 5
10
M for oleic
acid. The extreme blue shift of bound fluorescent fatty acid suggests
an unusually low polarity for the protein binding site. The emission
spectrum of the single tryptophan of rDvA-1L indicates that it is
deeply buried in a non-polar environment, and its spectrum is
unaffected by ligand binding. Far UV circular dichroism of rDvA-1L
reveals a high
-helix content (53%). Differential scanning
calorimetry studies indicate that rDvA-1L is highly stable (T
98 °C), refolding efficiently
following thermal denaturation. DvA-1 therefore represents an example
of a new class of lipid binding protein, and is the first product of a
polyprotein with this activity to be described.
Hydrophobic compounds such as fatty acids and retinoids are
usually transported and protected in intracellular or extracellular
aqueous environments within carrier proteins(1) . The small
(less than 20 kDa) water-soluble fatty acid and retinoid-binding
proteins are usually -strand-rich and include those with either 8
or 10 strands which form so-called
-barrel or
-clam
structures (reviewed in (2) ). Serum albumin is an exception to
the predominance of
-sheet in aqueous phase FABPs (
)in
comprising predominately helical structures, although it is
considerably larger and has binding sites for ligands other than fatty
acids(3) .
One of the most abundant proteins produced by nematodes is an approximately 15-kDa protein similar to the ABA-1 allergen of the large roundworm of humans, Ascaris lumbricoides(4, 5) . These extracellular proteins are produced from large precursors which are subsequently cleaved proteolytically into multiple copies of the 15-kDa polypeptide units(6, 7, 8, 9) . This manner of synthesis and processing led to the term ``nematode polyprotein allergens'' (NPA) for these proteins(10) . The structure of NPA genes is unusual in comprising a head to tail array encoding multiple units of the 15-kDa polypeptides with consensus proteinase cleavage sites at the junctions(6, 7, 8, 9, 10) . No information on the structure and function of NPAs has been published, and data base searches for similarity with proteins of known function have proved fruitless.
Recently we made a fortuitious
observation indicating that the ABA-1 allergen protein binds certain
hydrophobic ligands, which prompted us to examine the behavior of other
NPAs. With very rare exceptions, however, it is not feasible to purify
the NPA from parasitic nematodes because of the small size of the
organisms or the difficulty in obtaining them in sufficient quantities,
particularly from human sources. Here we circumvented this problem by
isolation of NPA-encoding nucleic acid from parasites, and expression
of the polypeptide in bacteria to provide material for biochemical and
structural analysis. In this case we have concentrated on the NPA array
of Dictyocaulus viviparus, the highly pathogenic parasite of
the lungs of cattle. Using recombinant polypeptide representing a
single unit of the polyprotein array, we have been able to demonstrate
that the protein has high affinity lipid binding activity and
structural characteristics distinguishing it from the -barrel
family of small fatty acid/retinoid-binding proteins of vertebrates.
It has been established from cDNA sequencing that the DvA-1
polyprotein comprises different forms of the approximately 15-kDa DvA-1
polypeptide units(11) , but the expression system used here was
designed to provide only the polypeptide encoded by the most 3` unit of
the 12-member array, designated DvA-1L. The recombinant polypeptide
(molecular weight 16,410) was designated rDvA-1L, and judged to be
homogeneous on the basis of a single band on SDS-polyacrylamide gel
electrophoresis or a single symmetrical peak in gel filtration. The
single gel filtration peak eluted at M 29,000
± 1,000, and SDS-polyacrylamide gel electrophoresis yielded a
single band of M
15,000, indicating that the
native protein may form a dimer in solution.
The fatty acid binding activity of purified rDvA-1L was examined using fluorescent lipid analogues whose emission spectra alter upon entry into binding proteins. The fluorescence emission of DAUDA is polarity-sensitive, and its intensity is increased and shifted to a shorter emission wavelength when bound to liver FABP(18) . Control experiments with dansylamide revealed minimal binding to DvA-1L, indicating that the fluorophore group itself is probably not contributing significantly to the binding of the dansylated fatty acid. In buffer alone, the peak emission of DAUDA occurred at 543 nm, but moved to 490 nm upon addition of rDvA-1L (Fig. 1), together with a marked increase in emission intensity. This blue shift was greater than that reported for serum albumin (495 nm; (19) ) and rat liver FABP (500 nm; (18) ).
Figure 1:
Binding of
DAUDA to recombinant DvA-1 and competition with a fatty acid.
Fluorescence emission spectra (uncorrected) ( = 345 nm) of 3.3 µM DAUDA alone or with 3
µM of the rDvA-1L monomer. Also shown is the reversal of
changes in DAUDA emission by competition with arachidonic acid (1.6 or
3.2 µM) added to the rDvA-1L
DAUDA
complex.
Blue shifting of the emission wavelength of the dansyl fluorophore is usually taken as a measure of the polarity of the binding site in binding proteins(20) , which can be calibrated semiquantitatively by reference to the emission spectrum of fluorescent probe in a series of organic solvents. The fluorescence spectrum of DAUDA in ethanol, dimethylformamide and cyclohexane showed emission peaks at 506, 505, and 475 nm, respectively, in order of decreasing polarity (data not shown). The observed blue shift to 490 nm of DAUDA in rDvA-1L thus indicates a binding site which is highly apolar or one in which unusual ligand orientation constraints or interactions between DAUDA and protein occur.
Similar blue shifts in fluorescence were
found with a fluorescent fatty acid probe in which the dansyl
fluorophore is attached to the carbon
(dansyl-DL-
-aminocaprylic acid), rather than to the
hydrocarbon (
) terminal, as in DAUDA (data not shown). This
similarity in behavior of the two probes could be taken to indicate
that the ligand is held entirely within the binding site of rDvA-1L and
isolated from polar solvent. X-ray crystallographic studies of
intestinal, muscle, and myelin FABPs show the removal of ligand from
solvent(21, 22, 23) . This feature appears,
therefore, to be widespread among FABPs and would have the advantage of
protecting oxidation-sensitive ligands such as retinoids and
unsaturated fatty acids during transport within an organism or cell.
The binding of natural, non-fluorescent fatty acids and other
hydrophobic ligands was determined from their competitive effects on
the fluorescence of the rDvA-1LDAUDA complex. For example,
progressive additions of arachidonic acid brought about a reversal of
the fluorescence effect, presumably by displacement of the DAUDA from
the binding site (Fig. 1). Similar experiments revealed strong
competitive inhibition by oleic, stearic, and palmitic acids, retinol
and retinoic acid, but none with tryptophan, squalene, or cholesterol.
Fluorimetric titration of rDvA-1L with DAUDA (Fig. 2A) gave a progressive increase in relative
fluorescence intensity consistent (within experimental uncertainty
limits) with binding of DAUDA to a single binding site (n = 0.949) on each rDvA-1L monomer, with apparent
dissociation constants (K) of
approximately 3.0
10
M. This K
is of a similar order to those observed
for lipid-binding proteins FABPs(24) , and a similar value was
obtained for oleic acid when this was progressively added to a
rDvA-1L/DAUDA mixture (K
= 5
10
M; Fig. 2B).
Figure 2:
Titration curves for the binding of DAUDA
and oleic acid to rDvA-1L. A, change in relative fluorescence
intensity at 490 nm (corrected for dilution; = 345 nm) of 1.6 µM rDvA-1L monomer on
addition of increasing concentrations of DAUDA. The solid line is the theoretical binding curve for complex formation with a
dissociation constant, K
= 3.0
(± 1)
10
M, and apparent
stoichiometry n = 0.95 per monomer unit. B,
the decrease in relative fluorescence due to displacement of DAUDA from
rDvA-1L by oleic acid. Increasing concentrations of oleic acid were
added to a mixture containing 1.15 µM DAUDA and 1.1
µM DvA-1. The solid line is a theoretical curve
for simple competitive binding of oleic acid in the DAUDA binding site
of DvA-1, with apparent K
(oleic)
5
(±2)
10
M.
The binding characteristics of rDvA-1L were also investigated using
the fluoresceinated fatty acid OAF. This probe has a low level of
fluorescence in aqueous solution and binding to proteins can be
detected by an increase in fluorescence without a change in
. Addition of rDvA-1L to this probe (Fig. 3)
gave a substantial increase in fluorescence which occurred with none of
the other reference proteins except
-lactoglobulin (
-LG) and
serum albumin (BSA), both of which have well established fatty acid
binding activities(3, 25) . As with the dansylated
probes, the effect of rDvA-1L on OAF fluorescence was reversed upon
addition of oleic acid or retinoic acid (data not shown).
Interestingly, DAUDA and OAF differentiated the fatty acid binding
activities of rDvA-1L and BSA from that of
-LG, in that the former
two bound both DAUDA and OAF, whereas
-LG bound only OAF.
Figure 3:
Binding of OAF to rDvA-1L. Fluorescence
emission spectra ( = 480 nm) of approximately
1 µM OAF alone or with 1.8 µM rDvA-1L
monomer, or 50 µg ml
BSA,
-lactoglobulin,
or each of the other control proteins (ovalbumin, transferrin,
ribonuclease A, hemocyanin, and hemoglobin, all at a concentration of
50 µg ml
).
The
binding of retinoids to rDvA-1L, indicated by the above competition
experiments, was confirmed directly by the effect on their intrinsic
fluorescence. Fig. 4shows the emission spectra of retinol and
retinoic acid in which the addition of rDvA-1L resulted in a
substantial increase in fluorescence. BSA or -LG, but none of the
other standard proteins used above, had similar effects on the
fluorescence of the retinoids (not shown). The change in the
fluorescence of both retinoids when bound to rDvA-1L was reversed by
the addition of oleic acid, indicating that the binding site for
retinol was the same as, or sterically interactive with, that for fatty
acids (data not shown).
Figure 4:
Binding of retinol and retinoic acid to
rDvA-1L. Fluorescence emission spectra ( =
350 nm) of 1 µM retinol (lower graph) or retinoic
acid (upper graph) in the absence or presence of 1.0
µM rDvA-1L monomer.
The far UV CD spectrum of rDvA-1L is
illustrated in Fig. 5, and shows a strong helix signal.
Analysis of the data over the range 240 to 190 nm by the CONTIN
procedure (15) yielded the following estimates of secondary
structure: 53 ± 2%
-helix, 43 ± 2%
-sheet, and
the remainder 4 ± 3%. In our experience, the application of the
CONTIN procedure to proteins with a significant content of
-helix
can overestimate the
-sheet content considerably(26) .
This is presumably a consequence of the smaller signals which arise
from
-sheet compared with
-helix and the difficulties caused
by noise observed below 195 nm. In such circumstances, however, the
estimate of
-helix is fairly reliable. Addition of increasing
concentrations of GdnHCl led to a progressive loss in secondary
structure as detected by CD (Fig. 5, inset), with the
midpoint of the unfolding occurring between 2 and 3 M GdnHCl.
Figure 5: Circular dichroism (CD) spectrum recorded at 20 °C on a 33.1 µM solution of rDvA-1L monomer in 8.3 mM sodium phosphate and 83.3 mM sodium chloride, path length 0.02 cm. Inset, change in ellipticity at 225 nm of a 7.3 µM sample of rDvA-1L monomer with increasing concentration of guanidine hydrochloride (GuHCl), path length 0.05 cm, overlaid with the change in fluorescence emission of Trp-15 under the same conditions (see Fig. 7). The sigmoidal curves are merely for guidance.
Figure 7:
DSC data
for rDvA-1L (95 µM monomer) in 50 mM
NaP, 0.5 M NaCl, pH 7.2. Upper
graph, repeated scans illustrating the high temperature transition
both in the initial scan (uppermost curve) and in five
re-scans (progressively decreasing magnitude) after re-heating to 110
°C and cooling to 20 °C in the DSC. Lower graph, the
first scan with the dashed lines showing the theoretical
curves for deconvolution into two independent unfolding
transitions.
The predominance of helical structures within rDvA-1L sets it apart
from the small FABPs of the -barrel family. Acyl-coenzyme
A-binding protein is known from crystal studies also to be
predominately helical, but it does not bind free fatty
acids(27) , the nucleotide group of the coenzyme being required
to shield the aliphatic tail from the solvent environment.
Some
fatty acid-binding proteins, such as BSA, -LG, and intestinal FABP
have a tryptophan residue either within the binding cavity and involved
in interaction with ligand, or in close proximity to this site (2, 3, 25) . DvA-1L and its homologs in other
species of nematode have a single conserved tryptophan (Trp-15), the
environment of which was examined here by fluorescence analysis. The
intrinsic fluorescence spectrum of the protein excited in the region of
290 nm showed a sharp emission peak at 307 nm (probably due to
tyrosine, of which there are five in DvA-1L) and a shoulder at 318 nm,
presumably due to Trp-15 (Fig. 6).
Figure 6:
Intrinsic protein fluorescence spectrum of
a 7.3 µM solution of rDvA-1L monomer in increasing
concentrations of guanidine hydrochloride (GuHCl), as
indicated. = 290
nm.
Emission by Trp-15 at such
a short wavelength indicates that it is isolated from solvent
water(28) , and deeply buried within the protein's
structure. In order to examine the alternative possibility that Trp-15
is instead surface proximal but in an environment in which solvent is
excluded, or in which the side chain is under rotational
constraint(28, 29) , the accessibility of Trp-15 to
the water environment was further tested using quenching of
fluorescence by succinimide at excitation and emission wavelengths of
295 and 330 nm, respectively, in order to examine the tryptophan
selectively(29) . The results showed that addition of
succinimide led to minimal quenching, confirming the highly buried
position of the tryptophan. The Stern-Volmer coefficient (K) is much lower (0.12 M
) than that for tryptophan residues in
other proteins, such as phosphoglycerate mutase of Schizosacchromyces pombe (2.2 M
) (13) and pig heart citrate synthase (1.68 M
). (
)Increasing concentrations
of GdnHCl, however, led to a red shift in the emission maximum
corresponding to the increased exposure of the tryptophan with
progressive denaturation of the protein (Fig. 6). In 6 M GdnHCl, the K
for quenching by succinimide
was 3.1 M
, which is of the same order of
magnitude as that of N-acetyltryptophanamide in 6 M GdnHCl (5.2 M
)(30) . The major
changes in the fluorescence spectrum occurred over the same range
(2-3 M GdnHCl) as the most radical changes in the far UV
CD spectrum (Fig. 5, inset), suggesting that the losses
in secondary and tertiary structure ran largely in parallel.
One of
the possible reasons for the isolation of Trp-15 from solvent is its
involvement in the fatty acid binding site of rDvA-1L. Intrinsic
fluorescence spectra with excitation at 290 nm for BSA and -LG,
however, were found to peak at 345 and 336 nm, respectively, indicating
that their Trp side chains are only partially excluded from water (data
not shown). When oleic acid was added to each of these proteins, the
fluorescence intensity of the Trp in
-LG increased (as previously
reported; (31) ), and decreased in BSA (data not shown). This
is consistent with a change in the environment of the tryptophans due
either to direct interaction with the ligand, a change in the
conformation of the protein, or displacement of water from the binding
site(32) . In contrast, there was no such change in the
spectrum upon addition of oleic acid to rDvA-1L (data not shown), so
Trp-15 may not be involved in the binding site.
DSC experiments on
purified rDvA-1L in neutral buffers consistently showed a high
temperature, endothermic thermal transition with midpoint at T = 98 °C (Fig. 7). The
shape of the curve indicated that more than one transition may have
occurred. By deconvolution(17) , the overall transition could
be modelled in terms of two independent unfolding processes with T
of 91 and 100 °C, respectively, and
H
for each transition in the region of
50-60 kcal mol
. This might indicate the
existence of two discrete domains within the structure, as reflected by
the possible internal duplication which has been noted in the amino
acid sequence of the 15-kDa units of NPAs(9) . Repeated DSC
scans showed that the major transition was largely reversible, even
after heating to 110 °C (Fig. 7). In contrast, control
experiments with BSA and
-LG showed transition temperatures around
60-70 °C, with no evidence of reversibility.
The
mechanism of ligand binding to the -barrel family of FABPs and
retinol-binding proteins is understood from crystal studies, and the
NPAs such as DvA-1 present a potentially valuable system with which to
investigate the binding of hydrophobic ligands to helical proteins. The
stability of rDvA-1L bodes well for such studies and the availability
of functionally active recombinant material also means that
site-directed mutagenesis can be used to identify the crucial amino
acid side chains involved in ligand binding and specificity, as has
been done with members of the
-barrel family of FABPs(2) .
DvA-1L is unusual, if not unique, among products of polyproteins described to date in having lipid binding properties. The advantage to the parasite of this manner of synthesis is obscure but it would permit the efficient production of large quantities of binding proteins. This in turn emphasizes the importance to the organisms of acquisition of fatty acids and retinoids from the host, assuming that the binding propensities shown here are an accurate reflection of the natural condition. Another important consideration is that many anthelmintic drugs are hydrophobic and may therefore interact with DvA-1 and its homologs, and we are currently examining this using the fluorescence-based methods described here. Finally, in all cases examined, the NPAs appear to be released by parasitic nematodes cultured in vitro(6, 7, 33) . Assuming that this also occurs in vivo, the binding of arachidonic acid may indicate that NPAs bind both it and its metabolites, and thereby modulate the local immunological and inflammatory environment.