The Ov20 Protein of the Parasitic Nematode Onchocerca volvulus
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29442
ISSN1083-351X
AutoresMalcolm W. Kennedy, Lisa Garside, Lucy E. Goodrick, Lindsay McDermott, Andy Brass, Nicholas C. Price, Sharon M. Kelly, Alan Cooper, Janette E. Bradley,
Tópico(s)Biotin and Related Studies
ResumoOv20 is a major antigen of the parasitic nematodeOnchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-dl-α-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized β-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a α-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of β structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 °C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy. Ov20 is a major antigen of the parasitic nematodeOnchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-dl-α-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized β-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a α-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of β structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 °C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy. Infection with Onchocerca volvulus can result in perhaps the most distressing of diseases caused by parasitic nematodes, river blindness. The infection is a major cause of eye pathology in areas in which the parasite is endemic, and degenerative and disfiguring skin conditions also result (1Rodger F.C. Bull. W. H. O. 1962; 27: 429-448PubMed Google Scholar). One of the first indications of eye damage in onchocerciasis is night blindness (1Rodger F.C. Bull. W. H. O. 1962; 27: 429-448PubMed Google Scholar), a symptom which would be consistent with a deficiency in available retinoids. The parasites are known to accumulate retinol to a concentration an order of magnitude greater than the tissue they occupy (2Sani B.P. Vaid A. Comley J.C.W. Montgomery J.A. Biochem. J. 1985; 232: 577-583Crossref PubMed Scopus (22) Google Scholar, 3Sani B.P. Vaid A. Biochem. J. 1988; 249: 929-932Crossref PubMed Scopus (19) Google Scholar), and retinol-binding proteins (RBPs) 1The abbreviations used are: RBP, retinol-binding protein; DACA, dansyl-dl-α-aminocaprylic acid; dansyl, dimethylaminonaphthalene-1-sulfonyl; DAUDA, 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid; FABP, fatty acid-binding protein; GC, gas chromatography; MS, mass spectrometry; GdnHCl, guanidine hydrochloride; retinol, all-trans-retinol; NPA, nematode polyprotein allergen/antigen; rOv20, recombinant Ov20 protein produced inE. coli; PBS, phosphate-buffered saline; PCR, polymerase chain reaction. 1The abbreviations used are: RBP, retinol-binding protein; DACA, dansyl-dl-α-aminocaprylic acid; dansyl, dimethylaminonaphthalene-1-sulfonyl; DAUDA, 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid; FABP, fatty acid-binding protein; GC, gas chromatography; MS, mass spectrometry; GdnHCl, guanidine hydrochloride; retinol, all-trans-retinol; NPA, nematode polyprotein allergen/antigen; rOv20, recombinant Ov20 protein produced inE. coli; PBS, phosphate-buffered saline; PCR, polymerase chain reaction. from O. volvulus have also been reported to bind ivermectin, the drug which is the cornerstone of the program for the eradication of the infection (2Sani B.P. Vaid A. Comley J.C.W. Montgomery J.A. Biochem. J. 1985; 232: 577-583Crossref PubMed Scopus (22) Google Scholar, 3Sani B.P. Vaid A. Biochem. J. 1988; 249: 929-932Crossref PubMed Scopus (19) Google Scholar). RBPs are, therefore, potentially relevant to the survival of the parasite, the development of blindness in sufferers, and to therapeutic interventions against the infection.Retinol is usually transported and protected from chemical degradation in intracellular and extracellular aqueous environments within carrier proteins. The retinol-binding proteins described to date are all β-strand-rich proteins which form β-barrel structures having a single binding site for retinol in their interiors. These proteins can be further subdivided into the predominately extracellular proteins with eight β-strands (the lipocalins), which include plasma retinol-binding protein and milk lactoglobulin (4Papiz M.Z. Sawyer L. Eliopoulos E.E. North A.C.T. Findlay J.B.C. Sivaprasadarao R. Jones T.A. Newcomer M.E. Kraulis P.J. Nature. 1986; 324: 383-385Crossref PubMed Scopus (847) Google Scholar), and the predominately intracellular 10 β-stranded FABP/P2/CRBP/CRABP family of hydrophobic ligand transporter proteins (5Cowan S.W. Newcomer M.E. Jones T.A. Proteins. 1990; 8: 44-61Crossref PubMed Scopus (298) Google Scholar, 6Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar). Members of each family bind retinoids and/or fatty acids and have been described throughout the animal phyla, ranging from vertebrates to insects and flatworms (7Haunerland N.H. Jacobson B.L. Wesenberg G. Rayment I. Holden H.M. Biochemistry. 1994; 33: 12378-12385Crossref PubMed Scopus (38) Google Scholar, 8Mei B. Kennedy M.W. Beauchamp J. Komuniecki P.R. Komuniecki R. J. Biol. Chem. 1997; 272: 9933-9941Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Moser D. Tendler M. Griffiths G. Klinkert M.-Q. J. Biol. Chem. 1991; 266: 8447-8454Abstract Full Text PDF PubMed Google Scholar). Other proteins with retinol binding activities include interphotoreceptor retinol-binding protein, which is confined to the interphotoreceptor matrix (sub-retinal space) of the retina, and serum albumin, although retinol binding is thought to be a minor function for albumin in vivo, and both of these are substantially larger and more complex in structure than the β-barrel proteins.Here we describe a new class of helix-rich retinol-binding protein, which is exemplified by the Ov20 protein of O. volvulus.Ov20 originally attracted attention as a glycoprotein secreted by the parasite and has been used in immunological analyses of human immune responses to the infection (10Bradley J.E. Elson L. Tree T.I.M. Stewart G. Guderian R. Calvopiña M. Parades W. Araujo E. Nutman T.B. J. Infect. Dis. 1995; 172: 831-837Crossref PubMed Scopus (32) Google Scholar, 11Tree T.I.M. Gillespie A.J. Shepley K.J. Blaxter M.L. Tuan R.S. Bradley J.E. Mol. Biochem. Parasitol. 1995; 69: 185-195Crossref PubMed Scopus (48) Google Scholar). Genes encoding Ov20-like proteins have also been found in several other parasitic nematodes that are highly pathogenic in humans, and in a plant parasite. 2Unpublished data base entries. 2Unpublished data base entries. Ov20 is shown to possess strong retinol binding, but it is distinct from all the other known RBPs in terms of its structure and the characteristics of its binding site.RESULTS AND DISCUSSIONThe cDNA encoding Ov20 of O. volvulus predicts a 20580.6-Da protein with a hydrophobic leader which is presumably removed posttranslationally (11Tree T.I.M. Gillespie A.J. Shepley K.J. Blaxter M.L. Tuan R.S. Bradley J.E. Mol. Biochem. Parasitol. 1995; 69: 185-195Crossref PubMed Scopus (48) Google Scholar). The Signalp program, trained on eukaryotic sequences, predicts that this cleavage should occur between Ala16 and Asn17 to give a 18781.2-Da polypeptide, assuming no further modifications. DNA encoding Ov20 was amplified by PCR from cDNA using primers designed to exclude the hydrophobic leader, and recombinant protein with a 6xHis affinity tag plus 20 vector-encoded amino acids, was produced in bacteria and purified as detailed under "Experimental Procedures." The recombinant polypeptide (rOv20; molecular mass 20944.6) was judged to be homogeneous on the bases of a single band on SDS-polyacrylamide gel electrophoresis. Data base searching revealed no proteins of known function similar to Ov20, but a short N-terminal amino acid sequence of a retinol-binding protein from the dog heart worm Dirofilaria immitis showed similarities to the Ov20 sequence. 3T. V. Rajan, personal communication. The beginning of this sequence aligns exactly at the predicted cleavage site for the removal of the hydrophobic leader in Ov20.Ligand BindingThe retinol binding activity of rOv20 was investigated using changes in retinoid fluorescence that occur upon interaction with binding proteins (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar). The fluoresence emission of retinol was minimal in buffer alone, but was dramatically enhanced when added to a solution of Ov20 (Fig. 1). Similar changes were observed with bovine milk lactoglobulin, human serum retinol-binding protein, and recombinant ABA-1 allergen ofAscaris lumbricoides (data not shown), all of which have well described retinol binding activities (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar). Transferrin, ovalbumin, or RNase had no effect on retinol fluorescence (data not shown). The fluorescence enhancement brought about by Ov20 was reversed upon addition of oleic acid, presumably through displacement of retinol from a protein-binding site into solvent. This therefore indicated that Ov20, in common with many retinol-binding proteins, will also bind fatty acids and that the binding sites for retinol and fatty acids are coincident or interact competitively.The dissociation constant of retinol:Ov20 binding was estimated by fluorescence titration in which increasing quantities of retinol were added to an rOv20 solution in the cuvette. Fig.2 shows the dissociation curve, corrected for free retinol, consistent with a single retinol-binding site per molecule of rOv20 (assuming monomeric dispersion), which provided a K d of 8.5 × 10−8m.Figure 2Titration curve for the binding of retinol to rOv20. Change in relative fluorescence intensity at 470 nm of 1.06 μm rOv20 monomer on addition of increasing concentrations of retinol. λexc = 350 nm. The line for free retinol was obtained with increasing concentrations of retinol in PBS in the absence of protein, and the best fit line is shown. This was used to correct for free retinol to produce the corrected curve. The line drawn for the corrected data is the theoretical binding curve for complex formation with a dissociation constant, K d = 8.5 × 10−8m, and apparent stoichiometryn = 0.8 per monomer unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The fatty acid binding activity of purified Ov20 was examined using solvent polarity-sensitive fluorescent lipid analogues. The fluorescence emission intensity of DAUDA is increased and shifted to shorter wavelengths when bound to fatty acid-binding proteins (FABPs) such as rat liver FABP (27Wilkinson T.C.I. Wilton D.C. Biochem. J. 1986; 238: 419-424Crossref PubMed Scopus (65) Google Scholar). In buffer alone, the peak emission of DAUDA occurred at 541 nm, but moved to 485 nm upon addition of rOv20 (Fig. 3), together with a marked increase in emission intensity. Blue shifts of this magnitude are taken as indicative of entry of the fluorophore into a highly apolar environment and removal from collisional contact with water (28Macgregor R.B. Weber G. Nature. 1986; 319: 70-73Crossref PubMed Scopus (222) Google Scholar). This blue shift was greater than that reported for serum albumin (495 nm) (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar) and rat liver FABP (500 nm) (27Wilkinson T.C.I. Wilton D.C. Biochem. J. 1986; 238: 419-424Crossref PubMed Scopus (65) Google Scholar), and approached that reported for DAUDA binding to the ABA-1 fatty acid-binding protein ofAscaris (475 nm) (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar). Control experiments with dansylamide revealed minimal binding to rOv20, indicating that the fluorophore group itself does not contribute to the binding of the dansylated fatty acid.Figure 3Binding of DAUDA to recombinant rOv20 and competition with fatty acid. Fluorescence emission spectra (λexc = 345 nm) of 0.64 μm DAUDA alone or upon addition of 1.84 μm rOv20. Also shown is the reversal of changes in DAUDA emission by competition with oleic acid (5.7 μm) added to the rOv20·DAUDA complex. The wavelengths of peak emission by DAUDA when in PBS or when bound to the protein are as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Similar blue-shifts in fluorescence emission (from 543 to 483 nm) upon interaction with Ov20 were found with a fluorescent fatty acid probe in which the dansyl fluorophore is attached to the α carbon (dansyl-dl-α-aminocaprylic acid), rather than to the hydrocarbon (ω) terminal, as in DAUDA (data not shown). Assuming that the binding of both the dansylated fatty acids is representative of natural ligands, this similarity in behavior of the two probes indicates that the ligand is held entirely within the binding site of rDvA-1L and isolated from polar solvent. X-ray crystallographic studies of the β-barrel retinol- and fatty acid-binding proteins show similar isolation of ligand from solvent (4Papiz M.Z. Sawyer L. Eliopoulos E.E. North A.C.T. Findlay J.B.C. Sivaprasadarao R. Jones T.A. Newcomer M.E. Kraulis P.J. Nature. 1986; 324: 383-385Crossref PubMed Scopus (847) Google Scholar, 6Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar) (with exception of liver fatty acid-binding protein, which holds one of two ligand molecules close to the surface of the protein) (29Thompson J. Winter N. Terwey D. Bratt J. Banaszak L. J. Biol. Chem. 1997; 272: 7140-7150Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), which would serve to protect oxidation-sensitive ligands, such as retinol and other hydrophobic ligands during transport within an organism or cell.Fluorimetric titration of rOv20 with DAUDA (Fig.4A) gave a progressive increase in relative fluorescence intensity, with an apparent dissociation constant (K d ) of 9.7 × 10−7m. This value approximates that obtained for other lipid-binding proteins (30Thumser A.E.A. Evans C. Worrall A.F. Wilton D.C. Biochem. J. 1994; 297: 103-107Crossref PubMed Scopus (38) Google Scholar), and a similar value was obtained for progressive addition of a natural, nonfluorescent fatty acid (oleic acid) to a rOv20:DAUDA mixture (K I apparent = 3.6 × 10−7m; Fig. 4 B). A titration with the naturally fluorescent fatty acid parinaric acid gave a value of 8.6 × 10−7m, which is of the same order of magnitude to the values for DAUDA and oleic acid. Competition was also used to test for binding with other natural ligands, which showed that arachidonic acid, but not cholesterol, binds to rOv20 (data not shown).Figure 4Titration curves for the binding of DAUDA and oleic acid to rOv20. A, change in relative fluorescence intensity at 483 nm (corrected for dilution; λexc = 345 nm) of 1.26 μm DAUDA on addition of increasing concentrations of rOv20. The solid lineis the theoretical binding curve for complex formation with a dissociation constant, K d = 9.7 × 10−7m, and apparent stoichiometry consistent with one binding site per monomer unit. B, decrease in relative fluorescence due to displacement of DAUDA from rOv20 by oleic acid. Increasing concentrations of oleic acid were added to a mixture containing 1.26 μm DAUDA and 2.45 μm rOv20. The solid line is a theoretical curve for simple competitive binding of oleic acid in the DAUDA-binding site of rOv20, with apparent K I (oleic) 3.6 × 10−7m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the hydrophobic ligands that bind to Ov20 in a biological context, a sample of Ov20 which had been affinity-purified as described under "Experimental Procedures," but with no further treatment, was subjected to extraction with ethyl acetate, and the extracted material was subjected to GC-MS. The mass spectra revealed single ions consistent with the presence of palmitoleic, palmitic, heptenoic, oleic, and stearic acids. No evidence for significant amounts of retinol was found, which would be consistent with the fact thatE. coli does not synthesize retinoids.Structural Analysis and StabilityThe far UV CD spectrum of Ov20 is illustrated in Fig.5 A, and shows a strong α-helix signal. Analysis of the data over the range 190 to 240 nm by the CONTIN procedure yielded the following estimates of secondary structure: 60 ± 1.3% α-helix, 32 ± 1.6% β-sheet, and the remainder 8 ± 2.7%. In our experience, the application of the CONTIN procedure to proteins with a significant content of α-helix can overestimate the β-sheet content considerably (31White M.F. Fothergill-Gilmore L.A. Kelly S.M. Price N.C. Biochem. J. 1993; 295: 743-748Crossref PubMed Scopus (22) Google Scholar), but the estimate of the α-helix is reliable. Addition of increasing concentrations of GdnHCl led to a progressive loss in secondary structure as detected by CD (Fig. 5 B), with the greater part of the unfolding occurring between 3 and 4 m GdnHCl. Ov20 therefore appears to be more robust to this means of denaturation than are many proteins, including the ABA-1-type proteins (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar, 32Kennedy M.W. Britton C. Price N.C. Kelly S.M. Cooper A. J. Biol. Chem. 1995; 270: 19277-19281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), although detailed quantification of the stability of Ov20 and its reversibility remains to be carried out.Figure 5CD analysis of rOv20. A, CD spectrum recorded at 20 °C on a 10.2 μm solution of rOv20 monomer in 10 mm sodium phosphate and 8 mm sodium chloride, path length 0.02 cm. B,inset, change in ellipticity at 225 nm of a 7.2 μm sample of rOv20 monomer with increasing concentration of GdnHCl, path length 0.05 cm. The sigmoidal curve is merely for guidance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This conformational stability was confirmed in the absence of denaturants by differential scanning calorimetry experiments which indicated an endothermic unfolding transition T m in excess of 90 °C, but accompanied by exothermic irreversible aggregation for Ov20 in PBS, which precludes any detailed thermodynamic analysis (data not shown). This would indicate that the stability of the protein to chemical denaturation is mirrored by extreme thermal stability, which is also in excess of that exhibited by the ABA-1-type proteins (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar, 32Kennedy M.W. Britton C. Price N.C. Kelly S.M. Cooper A. J. Biol. Chem. 1995; 270: 19277-19281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).Structural PredictionsAn alignment of the amino acid sequence of Ov20 with similar proteins of other nematodes (animal-parasitic, plant-parasitic, and free-living) is given in Fig.6. No significant matches to other proteins were found from data base searching. Secondary structure prediction algorithms applied to the whole alignment predicted a highly helical conformation (86% helix), 13.5% loop structures, but no β/extended structure (Fig. 6). When plotted on helical wheels (Fig.7), some of the predicted helical stretches can be seen to be strongly amphipathic. This applies both for the hydrophobic residues occurring in Ov20, and, even more dramatically, for hydrophobic sites conserved across the entire array. This is exemplified by the regions of highest probability helix, from Pro27 to Thr62, from Asp78 to Arg117, and from Met126 to Ala153.Figure 6Structural predictions for Ov20 and its homologs. A, alignment of Ov20 (GenBankTMaccession no. L27686) with putative homologs from the human lymphatic filariasis parasite B. malayi (Bm20, GenBank accession no. U69169), the potato cyst nematode Globodera pallida (PCN, GenBankTM accession no.Y09293), and two sequences from C. elegans (Ce1and Ce2, sequences F02A9.2 and F02A9.3, clone CEL F02A9, GenBankTM accession no. Z19555). The amino acids of the presumptive hydrophobic leader/signal peptide are shown inlowercase letters. The alignment was generated using the MultAlin program through SwissProt set for the Dayhoff comparison matrix. In the consensus line, uppercase letters refer to conservation of amino acid position across the entire array, andlowercase letters refer to cases where that amino acid occurs in that position in more than half of the sequences. Other symbols are as follows: % is for either F or Y, and # is for anyone of NDQEBZ. The PHD line shows the secondary structure prediction from submission of the multiple alignment to the PHD secondary structure prediction program; H = helix, L = loop, · = no prediction; no extended/β structure was predicted), and the final line indicates conserved hydrophilic (+) and hydrophobic (•) positions. ConsensusN-glycosylation sites are underscored and the consensus casein kinase phosphorylation site is in boldface type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Amphipathic helices of Ov20. Helical wheels from Pro27 to Thr62 of Ov20 (top) and helix 2 from Asp78 to Val113 (bottom). The hydrophobic amino acids of Ov20 are circled, and those hydrophobic positions conserved across the entire array are marked by boldface circles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Particularly surprising was a strong prediction for coiled-coil structure in Ov20 (Fig. 8). The strongest predictions are for positions 61–89 and 119–155 (probabilities 0.78 and 1.00, respectively), and there is also a possibility of coiled coil from positions 90 to 118 (probability 0.46). All the programs used predicted similar regions of coiled coil in Ov20, and, albeit at slightly lesser probability, in its homologs. Interactions between helices in a coiled-coil conformation might explain the extremely high stability of Ov20 to both heat and chemical denaturation. The coiled-coil interactions could presumably act between the two stretches of predicted coiled coil within a single molecule of Ov20, or be involved in intramolecular interactions between other Ov20 molecules, such as in leucine zippers (33Branden C. Tooze J. Introduction to Protein Structure. Garland Publishing, New York1991Google Scholar, 34Szilak L. Moitra J. Krylov D. Vinson C. Nat. Struct. Biol. 1997; 4: 112-114Crossref PubMed Scopus (78) Google Scholar). The highly helical ABA-1-type of fatty acid- binding protein of nematodes, for instance, are known to form dimers (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar, 32Kennedy M.W. Britton C. Price N.C. Kelly S.M. Cooper A. J. Biol. Chem. 1995; 270: 19277-19281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 35Kennedy M.W. Allen J.E. Wright A.S. McCruden A.B. Cooper A. Mol. Biochem. Parasitol. 1995; 71: 41-50Crossref PubMed Scopus (39) Google Scholar).Figure 8High probability prediction of coiled coil in Ov20. Output from "Coils" program for the Ov20 sequence with the following windows; 14 residues (·····), 21 residues (- - - - -), 28 residues (⎻⎻). The residues are numbered from the beginning of the coding sequence including the hydrophobic leader sequence. Other coiled-coil prediction algorithms gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Another unusual feature is a consensus casein kinase II phosphorylation site that appears in Ov20 and all its homologs (see Fig. 6). Phosphorylation has been found to control the biological activity of certain proteins, including those involved in gene regulation, controlling homodimerization, and even the stability of α-helices (34Szilak L. Moitra J. Krylov D. Vinson C. Nat. Struct. Biol. 1997; 4: 112-114Crossref PubMed Scopus (78) Google Scholar, 36Ganjeizadeh M. Zolotarjova N. Huang W.-H. Askari A. J. Biol. Chem. 1995; 270: 15707-15710Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 37Olson J.K. Bishop G.A. Grose C. J. Virol. 1997; 71: 110-119Crossref PubMed Google Scholar, 38Brownlie P. Ceska T.A. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 39Surette M.G. Levit M. Liu Y. Lukat G. Ninfa E.G. Ninfa A. Stock J.B. J. Biol. Chem. 1996; 271: 939-945Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 40Buelt M.K. Xu Z. Banaszak L.J. Bernlohr D.A. Biochemistry. 1992; 31: 3493-3499Crossref PubMed Scopus (56) Google Scholar). The phosphorylation site of Ov20 is, therefore, potentially involved in control processes, and its localization within the cells of developing parasite embryos might therefore be pertinent. It is, however, also secreted by the parasites, and the PSORT program predicts sorting of the protein into a secretory compartment in the synthesizing cell. It is possible, therefore, that that Ov20 has functions intracellularly in nematodes and that its secretion might be an adaptation for parasitism.The predominance of helical structures within Ov20 clearly sets it apart from the small RBPs of the β-barrel hydrophobic ligand-binding proteins. Acyl coenzyme A-binding protein is known from crystal studies also to be predominately helical, but it does not bind free fatty acids, the nucleotide group of the coenzyme being required to shield the aliphatic tail from the solvent environment (41Kragelund B.B. Andersen K.V. Madsen J.C. Knudsen J. Poulsen F.M. J. Mol. Biol. 1993; 230: 1260-1277Crossref PubMed Scopus (122) Google Scholar). The only other family of retinoid-binding proteins of similar size and helicity that has been reported are individual units of the polyprotein allergens of nematodes (NPAs), such as ABA-1 (26Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar, 32Kennedy M.W. Britton C. Price N.C. Kelly S.M. Cooper A. J. Biol. Chem. 1995; 270: 19277-19281Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 35Kennedy M.W. Allen J.E. Wright A.S. McCruden A.B. Cooper A. Mol. Biochem. Parasitol. 1995; 71: 41-50Crossref PubMed Scopus (39) Google Scholar). These are predicted to be four-helix proteins with no β content, but Ov20 is distinct from the NPAs in that it is not produced as a polyprotein and its primary structure has none of the characteristic features of the NPAs, such as the conserved Trp and pair of Cys residues. This absence of tryptophan residues is another distinguishing feature of Ov20 and its homologs; in all the β-barrel RBPs, and in serum albumin, there is a Trp residue in or near the ligand-binding site (4Papi
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