European Biophysics Journal

, Volume 32, Issue 5, pp 465–476 | Cite as

The polyprotein and FAR lipid binding proteins of nematodes: shape and monomer/dimer states in ligand-free and bound forms

  • Alexandra S. SolovyovaEmail author
  • Nicola Meenan
  • Lindsay McDermott
  • Antonio Garofalo
  • Jannette E. Bradley
  • Malcolm W. Kennedy
  • Olwyn Byron


Nematodes produce two classes of small, helix-rich fatty acid- and retinol-binding proteins whose structures and in vivo functions remain to be elucidated. These are the polyprotein allergens (NPA) and the FAR proteins. The solution properties of recombinant forms of these proteins from parasitic [Ascaris suum (As) and Onchocerca volvulus (Ov)] and free-living [Caenorhabditis elegans (Ce)] nematodes have been examined. Analytical ultracentrifugation (AUC) showed that, contrary to previous findings, the rAs-NPA-1A polyprotein unit (~15 kDa) is a monomer, and this stoichiometry is unaltered by ligand (oleic acid) binding. The rOv-FAR-1 and rCe-FAR-5 proteins differ in that the former forms a tight dimer and the latter a monomer, and these oligomeric states are also unaffected by ligand binding or protein concentration. Sedimentation equilibrium experiments showed that the partial specific volume v̄ of the unliganded proteins agree well with the value calculated from amino acid composition extrapolated to experimental temperature, and was unaffected upon ligand binding. Data from small-angle X-ray scattering (SAXS) indicated that both of the monomeric proteins rAs-NPA-1A and rCe-FAR-5 are globular, although slightly elongated and flattened. These data are in good agreement with shapes predicted from sedimentation velocity experiments and hydrodynamic bead modelling. On the basis of functional and secondary structural homology with the ligand-binding domain of the retinoic acid receptor RXRα, de novo atomic resolution structures for rAs-NPA-1A and rCe-FAR-5 have been constructed which are consistent with the SAXS and hydrodynamic data.


Analytical ultracentrifugation Hydrodynamic bead modelling Nematode fatty acid binding proteins Protein-ligand interaction Small-angle X-ray scattering 



A.S.S. is supported by a grant from the Wellcome Trust to O.B., M.W.K. and others (05606/Z/99/Z), and we are also indebted to the Wellcome Trust for their support of this project through other grants to M.W.K. The work was also supported by an ESRF beam time award (LS-1872) and by the Universities of Salford and Nottingham. Special thanks to the team of Dr. Wim Bras (ESRF, Grenoble, particularly Dr. Marc Malfois) for assistance in the SAXS experiments and Marcelo Nöllmann for help in SAXS data treatment.


  1. Ackerman CJ, Harnett MM, Harnett W, Svergun DI, Byron O (2003) 19 Å solution structure of the filarial nematode immunomodulatory protein, ES-62. Biophys J 84:489–500PubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  3. Aszódi A, Taylor WR (1994) Folding polypeptide alpha-carbon backbones by distance geometry methods. Biopolymers 34:498–505Google Scholar
  4. Aszódi A, Gradwell MJ, Taylor WR (1995) Global fold determination from a small number of distance restraints. J Mol Biol 251:308–326PubMedGoogle Scholar
  5. Barrett J, Saghir N, Timanova A, Clarke K, Brophy PM (1997) Characterisation and properties of an intracellular lipid-binding protein from the tapeworm Moniezia expansa. Eur J Biochem 250:269–275PubMedGoogle Scholar
  6. Berger B, Wilson DB, Wolf E, Tonchev T, Milla M, Kim PS (1995) Predicting coiled coils by use of pairwise residue correlations. Proc Nat Acad Sci USA 92:8259–8263PubMedGoogle Scholar
  7. Blaxter M (1998) Caenorhabditis elegans is a nematode. Science 282:2041–2046PubMedGoogle Scholar
  8. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D (1995) Crystal structure of the ligand binding domain of the human nuclear receptor RXRα. Nature 375:377–382Google Scholar
  9. Britton C, Moore C, Gilleard JS, Kennedy MW (1995) Extensive diversity in repeat unit sequences of the cDNA encoding the polyprotein antigen/allergen from the bovine lungworm Dictyocaulus viviparus. Mol Biochem Parasitol 72:77–88CrossRefPubMedGoogle Scholar
  10. Burkhard P, Stetefeld J, Strelkov SV (2001) Coiled coils: a highly versatile protein folding motif. Trends Cell Biol 11:82–88PubMedGoogle Scholar
  11. Christie JF, Dunbar B, Davidson I, Kennedy MW (1990) N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum, and MHC-restricted IgE responses to it. Immunology 69:596–602PubMedGoogle Scholar
  12. Coe NR, Bernlohr DA (1998) Physiological properties and functions of intracellular fatty acid-binding proteins. Biochim Biophys Acta 1391:287–306PubMedGoogle Scholar
  13. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890PubMedGoogle Scholar
  14. Cuff JA, Barton GJ (1999) Evaluation and improvement of multiple sequence methods for protein secondary structure prediction. Proteins Struct Funct Genet 34:508–519CrossRefPubMedGoogle Scholar
  15. Cuff JA, Clamp ME, Siddiqui AS, Finlay M, Barton GJ (1998) JPred: a consensus secondary structure prediction server. Bioinformatics 14:892–893CrossRefPubMedGoogle Scholar
  16. Duel HJ (1951) The lipids: their chemistry and biochemistry. Interscience, New YorkGoogle Scholar
  17. Durchschlag H (1986) Specific volumes of biological macromolecules and some other molecules of biological interest. In: Hinz H-J (ed) Thermodynamic data for biochemistry and biotechnology. Springer, Berlin Heidelberg New York, pp 45–128Google Scholar
  18. Edelstein SJ, Schachman HK (1973) Measurement of partial specific volume by sedimentation equilibrium in H2O-D2O solutions. Methods Enzymol 27:82–98PubMedGoogle Scholar
  19. Eftink MR, Ghiron CA (1976) Exposure of tryptophanyl residues in proteins: quantitative determination by fluorescence quenching studies. Biochemistry 15:672–679PubMedGoogle Scholar
  20. Eftink MR, Ghiron CA (1984) Indole fluorescence quenching studies on proteins and model systems: use of the efficient quencher succinimide. Biochemistry 23:3891–3899Google Scholar
  21. Flower DR (1996) The lipocalin protein family: structure and function. Biochem J 318:1–14PubMedGoogle Scholar
  22. García de la Torre J (2001) Hydration from hydrodynamics. General considerations and applications of bead modelling to globular proteins. Biophys Chem 93:159–170CrossRefPubMedGoogle Scholar
  23. García de la Torre J, Huertas ML, Carrasco B (2000) Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. Biophys J 78:719–730PubMedGoogle Scholar
  24. Garofalo A, Kläger SL, Rowlinson MC, Nirmalan N, Klion A, Allen JE, Kennedy MW, Bradley JE (2002) The FAR proteins of filarial nematodes: secretion, glycosylation and lipid binding characteristics. Mol Biochem Parasitol 122:161–170PubMedGoogle Scholar
  25. Garofalo A, Rowlinson M-C, Ngwa A, Hughes JM, Kelly SM, Price NC, Cooper A, Watson DG, Kennedy MW, Bradley JE (2003) The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties, structural characteristics and developmental regulation. J Biol Chem 278:8065–8074CrossRefPubMedGoogle Scholar
  26. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino-acid sequence data. Anal Biochem 182:319–326PubMedGoogle Scholar
  27. Guex N, Diemand A, Peitsch MC (1999) Protein modelling for all. Trends Biol Sci 24:364–367CrossRefGoogle Scholar
  28. Haughland RP (1996) Handbook of fluorescent probes and research chemicals, 6th edn. Molecular Probes, Eugene, Ore., USAGoogle Scholar
  29. Janssen D, Barrett J (1995) A novel lipid-binding protein from the cestode Moniezia expansa. Biochem J 311:49–57PubMedGoogle Scholar
  30. Kennedy MW (2000) The polyprotein lipid binding proteins of nematodes. Biochim Biophys Acta 1476:149–164PubMedGoogle Scholar
  31. Kennedy MW (2001) Structurally novel lipid-binding proteins. In: Kennedy MW, Harnett W (eds) Parasitic nematodes. Molecular biology, biochemistry, and immunology. CABI, Wallingford, UK, pp 309–330Google Scholar
  32. Kennedy MW, Qureshi F (1986) Stage-specific secreted antigens of the parasitic larval stages of the nematode Ascaris. Immunology 58:515–522PubMedGoogle Scholar
  33. Kennedy MW, Qureshi F, Fraser EM, Haswell-Elkins MR, Smith HV (1989) Antigenic relationships between the surface-exposed, secreted and somatic materials of the nematode parasites Ascaris lumbricoides, Ascaris suum, and Toxocara canis. Clin Exp Immunol 75:493–500PubMedGoogle Scholar
  34. Kennedy MW, Brass A, McCruden AB, Price NC, Kelly SM, Cooper A (1995a) The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterisation. Biochemistry 34:6700–6710PubMedGoogle Scholar
  35. Kennedy MW, Britton C, Price NC, Kelly SM, Cooper A (1995b) The DvA-1 polyprotein of the parasitic nematode Dictyocaulus viviparus: a small helix-rich lipid binding protein. J Biol Chem 270:19277–19281PubMedGoogle Scholar
  36. Kennedy MW, Garside LH, Goodrick LE, McDermott L, Brass A, Price NC, Kelly SM, Cooper A, Bradley JE (1997) The Ov20 protein of the parasitic nematode Onchocerca volvulus. A structurally novel class of small helix-rich retinol-binding proteins. J Biol Chem 272:29442–29448PubMedGoogle Scholar
  37. Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41CrossRefGoogle Scholar
  38. Lerche MH, Poulsen FM (1998) Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins. Protein Sci 7:2490–2498PubMedGoogle Scholar
  39. Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein sequences. Science 252:1162–1164PubMedGoogle Scholar
  40. MacGregor RB, Weber G (1986) Estimation of the polarity of the protein interior by optical spectroscopy. Nature 319:70–73PubMedGoogle Scholar
  41. McDermott L, Moore J, Brass A, Price NC, Kelly SM, Cooper A, Kennedy MW (2001) Mutagenic and chemical modification of the ABA-1 allergen of the nematode Ascaris: consequences for structure and lipid-binding properties. Biochemistry 41:9918–9926CrossRefGoogle Scholar
  42. Moore J, McDermott L, Price NC, Kelly SM, Cooper A, Kennedy MW (1999) Sequence-divergent units of the ABA-1 polyprotein array of the nematode Ascaris suum have similar fatty-acid- and retinol-binding properties but different binding-site environments. Biochem J 340:337–343PubMedGoogle Scholar
  43. Peitsch MC (1996) ProMod and Swiss-Model: internet-based tools for automated comparative protein modelling. Biochem Soc Trans 24:274–279PubMedGoogle Scholar
  44. Prior A, Jones JT, Blok VC, Beauchamp J, McDermott L, Cooper A, Kennedy MW (2001) A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern. Biochem J 356:387–394PubMedGoogle Scholar
  45. Rothnagel JA, Steinert PM (1990) The repeating strucure of the units for mouse filaggrin and a comparison of the repeating units. J Biol Chem 265:1862–1865PubMedGoogle Scholar
  46. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619PubMedGoogle Scholar
  47. Scott DJ, Grossmann JG, Tame JRH, Byron O, Wilson KS, Otto BR (2002) Low resolution structure of the apo form of Escherichia coli haemoglobin protease Hbp. J Mol Biol 315:1179–1187CrossRefPubMedGoogle Scholar
  48. Storch J, Thumser AE (2000) The fatty acid transport function of fatty acid-binding proteins. Biochim Biophys Acta 1486:28–44PubMedGoogle Scholar
  49. Svergun DI (1992) Determination of the regularisation parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503CrossRefGoogle Scholar
  50. Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76:2879–2886PubMedGoogle Scholar
  51. Svergun DI, Petoukhov MV, Koch MHJ (2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80:2946–2953PubMedGoogle Scholar
  52. Taylor WR, Aszódi A (1994) Building protein folds using distance geometry: towards a general modelling and prediction method. In: Merz JM Jr, LeGrand SM (eds) The protein folding problem and tertiary structure prediction. Birkhäuser, Boston, pp 165–192Google Scholar
  53. Xia Y, Spence HJ, Moore J, Heaney N, McDermott L, Cooper A, Watson DG, Mei B, Komuniecki R, Kennedy MW (2000) The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties. Parasitology 120:211–224PubMedGoogle Scholar
  54. Yphantis DA (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297–317Google Scholar
  55. Yphantis DA, Waugh DF (1956) Ultracentrifugal charactersiation by direct measurement of activity. I. Theoretical. J Phys Chem 60:623–635Google Scholar

Copyright information

© EBSA 2003

Authors and Affiliations

  • Alexandra S. Solovyova
    • 1
    • 2
    Email author
  • Nicola Meenan
    • 3
  • Lindsay McDermott
    • 3
    • 4
  • Antonio Garofalo
    • 5
  • Jannette E. Bradley
    • 5
  • Malcolm W. Kennedy
    • 4
  • Olwyn Byron
    • 1
  1. 1.Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black BuildingUniversity of GlasgowGlasgow UK
  2. 2.Institute for Problems of Cryobiology and CryomedicineNational Academy of Science of the UkraineKharkovUkraine
  3. 3.Department of Chemistry, Joseph Black BuildingUniversity of GlasgowGlasgow UK
  4. 4.Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr BuildingUniversity of GlasgowGlasgow UK
  5. 5.School of Life and Environmental ScienceUniversity of NottinghamNottinghamUK

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