©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The DvA-1 Polyprotein of the Parasitic Nematode Dictyocaulus viviparus
A SMALL HELIX-RICH LIPID-BINDING PROTEIN (*)

(Received for publication, April 5, 1995; and in revised form, May 22, 1995)

Malcolm W. Kennedy (§) Collette Britton (¶) Nicholas C. Price (2) Sharon M. Kelly (2) Alan Cooper (1)

From the  (1)Wellcome Laboratories for Experimental Parasitology and Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and the (2)Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 10M for 11-((5dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, and, by competition, 5 10M 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 alpha-helix content (53%). Differential scanning calorimetry studies indicate that rDvA-1L is highly stable (T approx 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.


INTRODUCTION

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 beta-strand-rich and include those with either 8 or 10 strands which form so-called beta-barrel or beta-clam structures (reviewed in (2) ). Serum albumin is an exception to the predominance of beta-sheet in aqueous phase FABPs (^1)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 beta-barrel family of small fatty acid/retinoid-binding proteins of vertebrates.


EXPERIMENTAL PROCEDURES

Production of Recombinant DvA-1L Polypeptide

DNA encoding dva-1 repeat unit l was amplified from plasmid pCB10 (GenBank accession number U02568; (11) ) using primers complementary to the 5` and 3` ends of the repeat unit (primer L1, ACGACGggatccTTACGAAATAGACGTG; primer L2, ATGTTggatccCCTAATGACGTCGTGCTTTCG; lower case denotes BamHI sites). Amplification was carried out for 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The DNA fragments were purified from agarose gel using a siliconized glass wool spin column and Magic clean-up resin (Promega, Southampton, United Kingdom). These were then digested with BamHI and ligated into BamHI digested and dephosphorylated pET 15b expression vector (Novagen, Witney, UK). Transformants containing insert DNA in the correct orientation, as determined by DNA sequencing, were grown to an A of 0.6 then induced with 1 mM isopropyl beta-D-thiogalactopyranoside (Sigma, Poole, Dorset) at 37 °C for 2 h. Cells were collected by centrifugation at 4,000 g, resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl, pH 7.9) containing 0.1% Nonidet P-40 (Sigma) and sonicated on ice for five 1-min bursts. Following centrifugation at 15,000 g, the supernatant was applied to His-Bind metal chelation resin (Novagen) packed in a 2-ml column. The washing procedure and elution of the fusion protein were carried out according to the manufacturer's instructions. The N-terminal histidine tag was cleaved by treatment with thrombin at a concentration of 1 unit/mg protein and the cleaved tag was then removed by filtration through a Centricon 10 device (Amicon, Stonehouse, Scotland, UK). This leaves a polypeptide representing the entire DvA-1L with a small residual stretch of amino acids remaining after thrombin cleavage or encoded by the BamHI restriction sites engineered into the plasmids used: GSHMLEDP at the N terminus and IP at the COOH terminus. Residual detergent was removed by passage down an Extracti-Gel D column (Pierce). The concentrations of solutions of rDvA-1L protein were determined spectrophotometrically using an value of 12,090 M cm, calculated from the tyrosine and tryptophan content of the protein (12) . Fast protein liquid chromatographic analysis was carried out with a Superose 12 column (Pharmacia, Sweden) calibrated over the appropriate molecular size range(11) .

Fluorescent Probes and Competitors

The fluorescent fatty acid analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and 5-(octadecanoyl)aminofluorescein) (OAF) were obtained from Molecular Probes, Eugene, OR. Dansyl-DL-alpha-aminocaprylic acid and dansylamide were obtained from Sigma, as were all-trans-retinol, all-trans-retinoic acid, arachidonic acid, oleic acid, stearic acid, palmitic acid, squalene, cholesterol, and L-tryptophan. The fluorophore-conjugated fatty acids and the retinoids were stored as 10 mM stock solutions in ethanol, in the dark at -20 °C, and freshly diluted in 171 mM NaCl, 3.3 mM KCl, 17 mM Na(2)HPO(4), 1.84 mM KH(2)PO(4), pH 7.2 (PBS), to approximately 1 µM for use in the fluorescence experiments. Competitors of fluorescent fatty acid binding were prepared as stock solutions in ethanol at approximately 10 mM (except for tryptophan, which was prepared in PBS) and diluted in PBS for use. Reference and control proteins were obtained from Sigma and prepared as stock solutions at 10 mg ml in PBS.

Spectrofluorimetry

Fluorescence measurements were made at 20 °C in a Perkin-Elmer LS50 or SPEX FluorMax spectrofluorimeter (Spex Industries, Edison, NJ), using 2-ml samples in a quartz cuvette. Raman and background scattering by the solvent was corrected for where necessary using appropriate blank solutions. Quenching of protein fluorescence by succinimide was performed and analyzed as described previously(13) , using succinimide recrystallized from ethanol before use. Control experiments using dansylamide in a similar system indicated minimal binding of the fluorophore group alone to NPA proteins.

Binding Affinity

2-ml samples of rDvA-1L at a monomer concentration of 1.6 µM were titrated by addition of 10-µl aliquots of DAUDA at a concentration of 33 µM, and the fluorescence measured at 490 nm, with = 345 nm, in comparison to similar mixtures in the absence of protein. The concentration of the ethanol stock solution of DAUDA was confirmed by absorbance of a 1:10 dilution in methanol at 335 nm, using an extinction coefficient of 4400 M cm(14) . Fluorescence data (F/F(max)), corrected for dilution, were analyzed by standard nonlinear regression techniques (using Microcal ORIGIN software) in a simple noncompetitive binding model (F(max)/F = 1 + K/(C(L) - nC(p)F/F(max)); C(L) = total ligand (DAUDA) concentration; C(p) = total protein (rDvA-1L) concentration) to give estimates of the extrapolated maximal fluorescence intensity (F(max)), dissociation constant (K), and number of binding sites (n) per rDvA-1L monomer unit. The apparent dissociation constant (K) for oleic acid was estimated from the decrease in fluorescence at 490 nm by the progressive addition of increasing concentrations of oleic acid to a mixture of 1.1 µM rDvA-1L and 1.15 µM DAUDA, where K = K(1 + [DAUDA]/K) and K is the competitive inhibition constant for oleic acid.

Circular Dichroism

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. Protein concentrations were typically in the range 0.1 to 0.5 mg ml. Molar ellipticity values were calculated using a value of 115 for the mean residue weight calculated from the amino acid sequence of the protein. Analysis of the secondary structure of the protein was performed using the CONTIN procedure (15) over the range 240 to 190 nm. Guanidinium hydrochloride (GdnHCl) (ultrapure grade) was purchased from Life Technologies, Inc., Paisley, Scotland, and the concentrations of solutions checked by refractive index measurements(16) .

Differential Scanning Calorimetry (DSC)

Experiments were performed by standard procedures (17) using a Microcal MC2-D instrument at a scan rate of 60 °C h over a 20-110 °C range using sample concentrations of 0.5-1.5 mg ml. Normalized excess heat capacity data were analyzed by standard procedures, using Microcal ORIGIN software, to give the midpoint transition temperatures (T) and calorimetric (DeltaH) transition enthalpies.


RESULTS AND DISCUSSION

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(r) 29,000 ± 1,000, and SDS-polyacrylamide gel electrophoresis yielded a single band of M(r) 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-1LbulletDAUDA 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 alpha carbon (dansyl-DL-alpha-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-1LbulletDAUDA 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 10M. 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 10M; 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) 10M, 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) approx 5 (±2) 10M.



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 (max). 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 beta-lactoglobulin (beta-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 beta-LG, in that the former two bound both DAUDA and OAF, whereas beta-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, beta-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 beta-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 alpha 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% alpha-helix, 43 ± 2% beta-sheet, and the remainder 4 ± 3%. In our experience, the application of the CONTIN procedure to proteins with a significant content of alpha-helix can overestimate the beta-sheet content considerably(26) . This is presumably a consequence of the smaller signals which arise from beta-sheet compared with alpha-helix and the difficulties caused by noise observed below 195 nm. In such circumstances, however, the estimate of alpha-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 beta-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, beta-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). (^2)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 beta-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 beta-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 Tof 91 and 100 °C, respectively, and DeltaH 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 beta-LG showed transition temperatures around 60-70 °C, with no evidence of reversibility.

The mechanism of ligand binding to the beta-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 beta-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.


FOOTNOTES

*
This work was supported in part by Wellcome Trust Grant 031559/Z/90/A (to M. W. K.). The biological calorimetry services at Glasgow University and the circular dichroism facility at Stirling University are funded by the Biotechnology and Biological Sciences Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Glasgow G61 1QH, Scotland, United Kingdom. Tel.: 44-141-330-5819; Fax: 44-141-942-7215; gbzc02{at}udcf.gla.ac.uk.

Supported by a grant from Hoechst Veterinär (to M. W. K.). Present address: Dept. of Pathology, Laboratory of Molecular Pathology, University of California-San Francisco, 4150 Clement St., 113B, San Francisco, CA 94121.

(^1)
The abbreviations used are: FABP, fatty acid-binding protein; beta-LG, beta-lactoglobulin; BSA, bovine serum albumin; CD, circular dichroism; dansyl, dimethylaminonaphthalene-1-sulfonyl; DAUDA, 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid; DSC, differential scanning calorimetry; GdnHCl, guanidine hydrochloride; K, Stern-Volmer coefficient; NPA, nematode polyprotein allergen; OAF, 5-(octadecanoyl)aminofluorescein); PBS, phosphate-buffered saline.

(^2)
N. C. Price, unpublished observations.


ACKNOWLEDGEMENTS

We are indebted to Alan B. McCruden for carrying out the FPLC analysis, Andy Brass for much useful discussion and for involvement in the productive collaboration which led to the suggestion that DvA-1L is a hydrophobic binding protein, and to Freya Kennedy for other assistance.


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