A novel lipid-binding protein from the cestode Moniezia expansa.
A lipid-binding protein (LBP) has been purified from the cytosol of the cestode Moniezia expansa. The native LBP was found to be an oligomer of approx. 250 kDa, consisting of 11 kDa monomers. The LBP bound saturated and unsaturated fatty acids, but not their CoA derivatives, with KD values in the range 0.68-7.8 microM. Cholesterol, dihydroergosterol, bilirubin and retinoids were also bound, but alpha-tocopherol, bile acids, alk-2-enals and alka-2,4-dienals were not. Evidence suggests that there are two binding sites per subunit, each with different specificities. The fluorescent fatty acid 11-[(5-dimethylaminonaphthalene-1-sulphonyl)amino]undecanoic acid (DAUDA) and retinol both showed an additional high-affinity binding site with a density of approximately 0.1 per subunit, suggesting specific binding to the oligomer. The amino acid composition of Moniezia LBP was distinct from that of previously characterized fatty acid-binding proteins (FABPs). The protein was not N-terminally blocked and yielded a unique amino acid sequence, unrelated to that of any known FABP; there was also evidence of microheterogeneity. Polyclonal antibodies raised to the Moniezia protein did not cross-react with mammalian, nematode or digenean FABP. The Gibbs free energy for protein folding (13.02 kJ/mol; 3.1 kcal/mol), determined by urea denaturation, was identical for both the native and ligand-bound Moniezia LBP. CD spectra showed that the Moniezia protein contained 36% alpha-helix and that the secondary structure underwent only minor changes on ligand binding. Moniezia LBP binds a range of anthelmintics, with KD values again in the range 0.66-7.3 microM. It is possible that, in helminths, binding proteins may play a role in determining the specificity and site of action of anthelmintics.
Janssen
D
D
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, U.K.
Barrett
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000871:Anthelmintics / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002589:Cestoda / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002942:Circular Dichroism; D003600:Cytosol / Q000737:chemistry; D003619:Dansyl Compounds / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005456:Fluorescent Dyes; D005590:Fractional Precipitation; D015801:Helminth Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D013816:Thermodynamics
D000871:Anthelmintics; D002352:Carrier Proteins; D003619:Dansyl Compounds; D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D015801:Helminth Proteins; D046911:Macromolecular Substances; C054400:11-(dansylamino)undecanoic acid