Eur. J. Biochem.250, 2692275 (1997) FEBS1997

Characterisation and properties of an intracellular lipid-binding proteinfrom the tapeworm Moniezia expansa

John BARRETT1, Nahid SAGHIR1, Anna TIMANOVA 2, Katie CLARKE3 and Peter M. BROPHY3

1 Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, UK2 Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Sofia, Bulgaria3 Department of Pharmaceutical Sciences, De Montford University, Leicester, UK

(Received 25 July/3 October1997) 2 EJB 971064/3

The tapewormMoniezia expansacontains an extremely abundant cytoplasmic lipid-binding protein(LBP). It is a small protein consisting of 66 amino acids with a molecular mass of 794361.5 Da. Theamino acid sequence has been established by Edman degradation and confirmed by PCR analysis. TheMoniezia LBP shows no sequence similarity with any previously described binding protein, but doesshow similarity with antigen B fromEchinococcus glanulosusandEchinococcus multilocularisand withTaenia crassicepsantigen. The predicted structure forMonieziaLBP shows four helices and a putativetyrosine kinase site on the loop between helix1 and 2. Each of the four helices has a well definedhydrophobic face. Studies with fluorescent probes suggest a single hydrophobic binding site. Resultsindicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand binding, andcalculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic envi-ronment.

Keywords:lipid-binding protein;Moniezia expansa; hydrophobic binding site ; cestode; fatty acid.

The intracellular lipid-binding proteins (LBP) are members not their CoA derivatives), sterols and retinoids. A number ofanthelmintics, including bithionol, hexachlorophene, niclosam-of a group of widely distributed, low-molecular-weight (82

15 kDa) proteins which bind, amongst other things, fatty acids, ide, nitroscanate and oxyclozanide, were strongly bound withKd

values in the micromolar range. The interaction of parasite bind-their CoA esters, sterols, lysophospholipids, and a range of non-polar organic ions. They are extremely abundant proteins, con- ing proteins with anthelmintics could, therefore, be important in

determining the site of drug action and in the development ofstituting up to 5% of the total cytosolic protein. A variety offunctions, including the uptake, transfer and storage of fatty drug resistance.

In this paper we report the complete amino acid sequenceacids, targetting of fatty acids to specific organelles and path-ways, sequestration of toxic compounds and regulation of gene and predicted structure ofMonieziaLBP and studies on the na-

ture of the hydrophobic binding site.action have been proposed for LBP, but their exact function re-mains unclear (Veerkamp et al.,1991, 1992; Sacchettini andGordan,1993). Substrates bound to binding proteins may havea kinetic advantage and be preferentially metabolised, this mayMATERIALS AND METHODSinvolve targetted transport and substrate channelling or modified

Preparation of Moniezia LBP. Adult M. expansawere ob-enzyme activity (Srivastava and Bernhard,1985; Vancura andtained from the intestines of freshly slaughtered sheep at a localHaldar,1992).abbatoir, and LBP was isolated by a combination of ammoniumRecently we have purified an LBP from the cytosol of thesulphate fractionation, gel filtration, and ion-exchange chroma-tapewormMoniezia expansa(Janssen and Barrett,1995). Thetography, as described by Janssen and Barrett (1995). The homo-protein was present in large amounts in the cytosol and appearsgeneity of the preparation was monitored by SDS/PAGE andto represent a distinct class of binding protein. On SDS/PAGEprotein concentration determined by the Coomassie-Blue-G-gels, the protein was observed to have a molecular mass of ap-2502binding assay, using BSA as the protein standard (Sedmakproximately 8 kDa, but in solution it formed oligomers of aboutand Grossberg,1997).250 kDa. The partial amino acid sequence showed no similarity

Protein sequence.Protein sequencing was carried out by Drto any previously described binding protein (Janssen and Barrett,K. Bailey, Queens Medical Centre, Nottingham, using an Ap-1995). TheMoniezia protein binds long-chain fatty acids (butplied Biosystems Model 473A automatic analyser. N-terminal

Correspondence toJ. Barrett, Institute of Biological Sciences, Uni- sequencing of the native protein yielded residues1231. Theversity of Wales, Aberystwyth, Ceredigion SY23 3DA, UK protein was cleaved, and the peptides were resolved by HPLC

Fax: 144 1970 622350. using a Phenominex Primesphere C18 column (250 mm32 mm).E-mail : jzb@aber.ac.uk

Acid hydrolysis (70% trifluoroacetic acid at 55°C) cleaved theURL: http://www.aber.ac.uk/~dbswww/protein between residues 28 and 29 (Asp-Pro), and one of theAbbreviations.ANS, 8-anilinonaphthalene1-sulphonic acid;16-AP,resulting fragments corresponded to amino acids 29263. Tryp-16-(9-anthroyloxy)palmitate; DAUDA,11-[(5-dimethylaminonaphtha-tophan cleavage withO-iodosobenzoic acid yielded two frag-lene-1-sulphonyl)amino]-undecanoic acid; LBP, lipid-binding protein;

OAF, 5-octadecanoylaminofluorescein. ments: the N-terminal sequence of the larger corresponded to

270 Barrett et al. (Eur. J. Biochem. 250)

Fig.2. Amino acid sequence and predicted secondary structure ofMonieziaLBP. The shaded boxes are predicted helical regions, the openbox is the predicted tyrosine kinase site.

Fig. 1. Nucleotide and deduced protein sequence ofMoniezia LBP.The protein sequence was predicted from the reading frame incorporat-ing the LBP N-terminal primer sequence following amplification ofM.expansafirst-strand cDNA by the PCR conditions outlined in Materialsand Methods. Upstream and downstream PCR primer regions are un-derlined. The asterisk indicates the termination condon.

residues1233; the second peptide corresponded to 42266. En-zymatic cleavage with endopeptidase Lys C gave four identifi-able fragments corresponding to residues13219, 20233, 37242 and 56261. Cleavage of the protein with CNBr yielded nofragments, confirming the absence of methionine from the pro-tein.

Molecular-mass determinations by electrospray mass spec-Fig.3. Transformed data from the positive-ion electrospray-ioniza-trometry were carried out by Dr K. Ortori, Pharmaceutical Sci-tion mass spectrum of purified Moniezia LBP.ences, Nottingham, using a Fisons Micromass Platform.

Isolation of cDNA. RNA was isolated fromM. expansa(0.1 g tissue) by guanidinium thiocyanate andN-sarcosine ex- for similarities with other known proteins in the GenBank,traction followed by oligo(dT)-cellulose chromatography ac-EMBL and SwissProt databases (Altschul et al.,1990).cording to the manufacturer’s instructions (Pharmacia). Synthe- Spectrofluorimetry. Fluorescent measurements were per-sis of first-strand cDNA was catalysed from mRNA using Malo-formed at 20°C in a Shimadzu RF-5301PC spectrofluorimeterney murine leukemia virus reverse transcriptase (Sambrook etusing 2-ml samples in a quartz cuvette. Raman scattering by theal., 1989). The synthesis of first-strand cDNA was completedsolvent was corrected for, where necessary, by using appropriateusing an oligo(dT)-based primer [5′-d(AACTGGAAGAATTC- solutions. All spectra are uncorrected unless otherwise stated.GCGGCCGCAGGAAT18)-3′], and this sequence was also used 11-[(5-Dimethylaminonaphthalene-1-sulphonyl) amino]un-as the downstream PCR primer (Brophy et al.,1994). A 20-base decanoic acid (DAUDA), 5-(octadecanoyl) aminofluoresceindegenerate PCR primer (5′-GAA

GCAAGGAA

GACNAACTCCNAT-3′) (OAF), and 16-(9-anthroyloxy) palmitate (16-AP), were ob-

directed against the N-terminal sequence ofMoniezia LBP tained from Molecular Probes Inc., 8-anilino-1-naphthalene sul-(Janssen and Barrett,1995) was used as the upstream primer.phonic acid (ANS) was obtained from Sigma Ltd. The fluores-PCR was carried out on 0.5µg of a RNA · cDNA heteroduplex cent ligands were stored at10 mM in ethanol in the dark atsample (heat denatured at 90°C for 5 min). A 25-µl reaction 220°C. They were freshly diluted to 0.1 mM with 50 mM po-was prepared containing the above sample, standard PCR buffer,tassium phosphate, pH 7.4 prior to use (Kennedy et al., 1995a).1.5 mM magnesium chloride, 0.8 mM dNTPs, 20 pmol each10 mM oleate in ethanol was stored at220°C in the dark, andprimer and1.5 U Taq polymerase (Hoffman-La Roche). Ampli-diluted to 50µM with ethanol prior to competition experiments.fication of LBP cDNA took place under the following condi- The quenching of trytophan and DAUDA fluorescence bytions: denaturation at 94°C for 1 min, annealing at 40°C for succinimide and acrylamide, respectively, was carried out ac-1min and extension at 72°C for 1 min, for 45 cycles, followed cording to the methods described by Eftink and Ghiron (1976,by 72°C for 4 min to complete extension. A 330-bp product1984), with appropriate corrections for dilution and inner-filterwas visualised on1.5% agarose gel electrophoresis by ethidiumeffects. Aliquots (5220 µl) of 5 M acrylamide or 2 M succinam-bromide staining. The PCR product was excised from gel byide were added to 2 ml protein solution (5µg/ml in 50 mM po-Sephaglas (Pharmacia) and, after treatment with Klenow frag-tassium phosphate, pH 7.4). After mixing and equilibration forment and T4 polynucleotide kinase, blunt-end ligated into the5 min, the change in emission fluorescence was recorded (forSmaI site of pUC18. Sequencing of both strands was completedtryptophan, Ex max 5 295 nm, Em max5 320 nm; for DAUDA,on an automated AB1 DNA sequencer. Ex max 5 350 nm,Em max5 480 nm). The results were plotted ac-

Molecular modelling. Secondary-structure predictions werecording to the Stern-Volmer equation (Lakowicz and Weber,made using the SOPMA programme (Geourjon and Deleage,1973).1994, 1995) and nnPredict (University of California). TheMo- F0/F 5 1 1 Ksv [Q]niezia sequence was compared with protein sequences in theProsite data base to identify possible functional domains based This describes the collision mechanism of quenching due to the

diffusion of a quencher through the protein matrix.F0 and Fon amino acid sequence (Bairoch,1991). Tertiary structure pre-dictions were made using ProMod at the Swiss-Model Protein are the fluorescent intensities in the presence and absence of

quenching agent, respectively, [Q] is the concentration ofModelling Server (Peitsch,1996; Peitsch and Jongeneel,1993).Multiple-alignment techniques (TBLASTN) were used to look quenching agent in molarity andKsv is the Stern-Volmer quench-

271Barrett et al. (Eur. J. Biochem. 250)

Fig. 4. Amino-acid-sequence alignment ofM. expansaLBP with antigen B from E. granulosus(Shepherd et al., 1991; Frosch et al., 1994)and E. multilocularis (Frosch et al., 1994) and the major diagnostic antigen fromT. crassiceps(Zarlengo et al., 1994).Host organisms :M.expansa, sheep;E. granulosus, 1,4,5 human,2 camel,3 cattle; T. crassiceps, cattle.

Fig. 5. Helical wheels for the four predicted helices ofMoniezia LBP. Hydrophobic residues are indicated by filled circles, hydrophilic residuesby open circles.

ing constant. In denaturation studies with guanidine hydrochlo- cept for positions 64 and 66, which were Arg and Leu by Edmandegradation, and Gln and Glu, respectively, from cDNA (Figs1ride the protein was allowed to equilibrate for15 min before

readings were taken. and 2).The molecular mass of the protein determined by electro-Fluorescent titrations were carried out as described pre-

viously (Janssen and Barrett,1995). The fluorescent ligand was spray was 7943.661.58 Da (Fig. 3), which is very close to thepredicted molecular mass of 7943.19 Da calculated from theadded in small aliquots (225 µl) to 2 ml protein in 50 mM po-

tassium phosphate, pH 7.4. Because of time-dependent effects, amino acid sequence.Comparison of theMoniezia LBP sequence with those offluorescent measurements with OAF and16-AP were made

5 min after addition. The fluorescent measurements were cor- other proteins using multiple-alignment techniques showed sig-nificant sequence similarity with three other proteins, antigen Brected for dilution and solvent effects and for the fluorescent

contribution of the unbound ligand. Fluorescent data (bound fromE. granulosusandE. multilocularisand the immunodiag-nostic antigen fromT. multiceps(Fig. 4).ligand versus unbound ligand) were fitted by standard non-linear

regression techniques to a single-non-competitive-site modelStructural predictions. The MonieziaLBP is anA-class proteinwith non-specific binding to give estimates of the dissociationwith four predicted helical regions, and a putative tyrosine ki-constant (Kd) and number of binding sites (n) on eachMoniezianase site on the loop between helix1 and helix 2 (Fig. 2). Each

LBP monomer. of the predicted helices has relatively well defined hydrophobicfaces and the simplest hypothesis is that they all orientate

RESULTS towards the core of the protein (Fig. 5). Trp41, which appearsSequence analysis.The amino acid sequences derived from Ed- to be involved in binding is located in the hydrophilic region of

helix 3.man degradation and predicted from cDNA were identical, ex-

272 Barrett et al. (Eur. J. Biochem. 250)

ANS binds toMonieziaLBP with an approximately 90-fold flu-orescent enhancement and a blue shift in emission wavelengthof 42 nm (from Em max5 514 nm to Em max5 472; Ex max 5380 nm). Analysis of the binding data (Fig. 7B) shows a singlebinding site (n 5 0.7460.15) with a Kd of 0.5460.1 µM. 16-AP shows a sevenfold fluorescent enhancement on binding, butwith no change in fluorescent emission maximum (Em max 5447 nm, Ex max 5 360 nm). Analysis of the binding data wascomplicated by a high degree of non-specific binding (Fig. 7C),but showed a single binding site for16-AP (n 5 0.860.1) witha Kd of 260.2µM. OAF shows a tenfold increase in fluores-cence on binding toMonieziaLBP, but no significant change inemission maximum (Em max5 520 nm; Ex max 5 480 nm). FreeOAF forms micellar structures in water (Haughland,1992),which are detected as a slow rise in background fluorescence.

Fig. 6. Predicted tertiary structure of Moniezia LBP using ProMod The fluorescent enhancement accompanying protein binding isat the Swiss-Model Protein Server.

due to the reversal of self-quenching when OAF molecules aretransferred from aqueous micelles to the protein environment.The time-dependent nature of these changes make OAF unsuit-An attempt was made to model the tertiary structure usingable for kinetic studies. All four probes (DAUDA, ANS,16-AP,ProMod at the Swiss-Model Protein Server. The protein wasOAF) are displaced from the binding site by increasing concen-modelled in four sections approximately corresponding to thetrations of oleate (Fig. 8), indicating that they bind to the fatty-four helical regions. Good matches were found for the first twoacid-binding site of the protein.helical regions but there were only relatively poor matches for

Previous studies (Janssen and Barrett,1995) and the pre-regions three and four. The four sections were joined using acyl-dicted protein structure (Fig. 5) suggest that the single trypto-CoA2binding protein and lipoprotein A as templates (Fig. 6).phan residue in the molecule (Trp41) is involved in ligand bind-ing. The accessibility of this tryptophan to water was investi-Nature of the hydrophobic binding site. The hydrophobicgated by determining the Stern-Volmer quenching constantbinding site of purifiedMonieziaLBP was investigated using a(Fig. 9A). Succinimide produced only modest quenching, con-series of polarity-sensitive fluorescent probes. DAUDA has beenfirming that the tryptophan is buried within the molecule (Ksv 5shown previously (Janssen and Barrett,1995) to bind to the pro-0.7960.04 M21). Increasing concentrations of guanidine hydro-tein with an increase in fluorescence and a blue shift in emis-chloride lead to a red shift in the emission maximum of thesion-wavelength maximum of 70 nm (fromEm max5 550 nm totryptophan residue corresponding to an increase in exposureEm max5 480 nm;Ex max 5 350 nm). A double-reciprocal plot ofwith increasing denaturation of the protein. In 6 M guanidinefluorescence against protein concentration shows an approxi-hydrochloride the red shift is approximately10 nm (320 nm tomate 60-fold fluorescent enhancement. Examination of the bind-330 nm) and theKSV increases to1.9860.02 M21. The accessi-ing data for DAUDA (Fig. 7A) suggests a single binding sitebility of bound DAUDA to water was investigated using acryl-(n 5 0.760.3, Kd 5 3.160.3µM) and not two as suggestedamide quenching. Acrylamide produced only modest quenchingpreviously. Dansylamide did not bind toMonieziaLBP, showing

that the dansyl group alone was not contributing to binding. of the bound ligand (Ksv 5 0.3860.02 M21), suggesting that

Fig. 7. Analysis of fluorescent-probe binding to purified Moniezia LBP. Fluorescent ligands were added in small aliquots (225 µl) to fixedamounts of protein (approximately 0.5µM) in 50 mM potassium phosphate, pH 7.4. The fluorescent measurements were corrected for dilution andsolvent effects and for the contribution of the unbound ligand. It was assumed that the maximal fluorescence achieved in the presence of excessLBP represents100% binding and that the amount of probe bound is proportional to the relative intensity. The lines were fitted by non-linearregression techniques to a single-non-competitive-site model with non-specific binding to give estimates ofKd andn. (A) DAUDA, Ex max 350 nm,Em max 480,Ex andEm slit 5 nm; (B) ANS,Ex max 380 nm,Em max 472, Ex and Em slit 5 nm; (c)16-AP, Ex max 360 nm,Em max 447 nm,Ex andEm slit3 nm.

273Barrett et al. (Eur. J. Biochem. 250)

Fig. 8. Binding of fluorescent probes to purifiedMoniezia LBP, showing fluorescent enhancement and peak shift, and decrease in relativefluorescence due to displacement of the probe by oleic acid.(A) DAUDA, Ex max 350 nm,Ex andEm slit 5 nm; (B) ANS,Ex max 380 nm,Ex andEm slit 5 nm; (C)16-AP,Ex max 360 nm,Ex and Em slit 3 nm; (D) OAF,Ex max 480 nm,Ex andEm slit 3 nm.

DAUDA was deeply burried in the molecule and not accessible larity toS. mansoniLBP has been isolated from a cDNA expres-sion library of E. granulosus(Esteves et al.,1993). A secretedto solvent (Fig. 9B).LBP has been reported recently fromAscaris suumeggs. Itspredicted amino acid sequence exhibits similarities to mamma-DISCUSSIONlian retinoid binding and fatty-acid-binding proteins (Mei et al.,

Parasitic helminths have a limited lipid metabolism (noβ- 1997). A number of polyprotein antigens from nematodes (Asca-oxidation of fatty acids and limited synthetic capabilities). Theris lumbricoides, Dictyocaulus viviparus, Brugia malayi) havepresence of high levels of LBP in these organisms suggests im-been shown to bind fatty acids. These polyprotein antigentsportant roles for these proteins and their study could lead to newshow no sequence similarity with mammalian LBP, althoughpossibilities in anthelmintic design and delivery systems. LBPthey both bind a similar range of substrates (Kennedy et al.,have been isolated from a number of parasitic helminths. The1995a2c). The nematode polyprotein antigens form dimers inLBP from digeneans (Fasciola hepatica, Fasciola gigantica, solution and appear to have a four-bundle helix structure, ratherSchistosoma mansoni, Schistosoma japonicum) show similarities than the β-barrel configuration characteristic of mammalianwith mammalian liver fatty-acid-binding protein in their aminofatty-acid-binding proteins. The LBP fromMonieziahas, similaracid sequences and binding properties (Moser et al.,1991 ; Ro- to the nematode proteins, a four-helix rather than aβ-barreldriguez-Perez et al.,1992; Becker et al.,1994; Smooker et al., structure, but it shows no sequence similarity with mammalian,

nematode or digenean LBP. TheMoniezia protein is signifi-1997). A stage-specifically expressed gene with sequence simi-

274 Barrett et al. (Eur. J. Biochem. 250)

The Moniezia LBP binds a wide range of hydrophobic li-gands. Detailed studies with fluorescent probes suggest a singlenon-polar binding site with aKd in the micromolar range. Theblue shift in the emission spectrum of the dansyl fluorophore ofDAUDA can be taken as a measure of the polarity of the bindingsite (Macgregor and Weber,1986). The fluorescence spectra ofDAUDA in organic solvents of decreasing polarity, i.e. ethanol,dimethylformamide and cyclohexane, show blue shifts of 37, 38and 70 nm, respectively (Kennedy et al.,1995a). On binding toMonieziaLBP, DAUDA shows a blue shift of 70 nm (Fig. 8A),which is similar to the blue shift of 68 nm reported forAscarisABA-1 antigen (also a helix-bunch LBP) by Kennedy et al.(1995a). The binding pocket of these helical proteins is con-siderably more hydrophobic than the fatty-acid-binding site ofserum albumin (blue shift 48 nm) or rat liver fatty-acid-bindingprotein (blue shift 43 nm).

The MonieziaLBP shows sequence similarity with antigenB from E. granulosusandE. multilocularisand with the immu-nodiagnostic antigen fromT. crassiceps(Shepherd et al.,1991 ;Frosch et al.,1994; Zarlengo et al.,1994). These are importantdiagnostic antigens and potential vaccine candidates. The simi-larity would suggest that these antigens are themselves LBP. An-tigen B is known to be able to disrupt neutrophil chemotaxis(Shepherd et al.,1991). It is, therefore, possible that in helminthsLBP have an additional function in disrupting the hosts immuneresponse. However, inMoniezia there is no evidence that theLBP is secretedin vivo or that it is glycosylated.

This work was supported in part by Wellcome Trust Grant 047546(to P. M. B.) and Royal Society Grant 63003 (to J. B.).

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