The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterization
1995
Kennedy, M.W. | Brass, A. | McCruden, A.B. | Price, N.C. | Kelly, S.M. | Copper, A.
We report here on the structure and function of the ABA-1 allergen protein of the parasitic nematode Ascaris, the first nematode allergen to be characterized in detail. Using the fluorescent fatty acid analog 11-(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino) undecanoic acid (DAUDA), it was demonstrated that ABA-1 is a fatty acid binding protein (FABP) with a high affinity for the fluorescent analog (8.8 X 10(-8) M) and for oleic acid in competition experiments (1.3 X 10(-8) M), with a single binding site for ligand per monomer unit. Blue-shifting of fluorescence emission of DAUDA upon binding was unprecedented in degree among FABPs, being equivalent to that occurring in cyclohexane. A similarly blue-shifted spectrum was obtained with a probe in which the fluorophore was bound to the alpha carbon of a fatty acid, indicating that the carboxylate group of bound fatty acids is probably not exposed to solvent. In competition experiments and by observation of changes in their intrinsic fluorescence, retinol and retinoic acid were also found to bind in the fatty acid binding site. Circular dichroism (CD) of the ABA-1 protein revealed a high alpha-helix content (59%) which was consistent with the four-helix structure for the protein predicted from sequence algorithms. Fluorescence measurements showed that the single, highly conserved tryptophan residue is deeply buried in an unusually apolar environment and that this was unaffected by ligand binding. DSC studies of thermal stability indicate that unfolding of the ABA-1 dimer is cooperative and biphasic (Tm approximately 71 and 89 degrees C), suggesting a two-domain thermal unfolding process, again consistent with the predicted structure. Only the folding of the high-temperature domain is reversible on cooling. DSC confirmed the gel filtration analysis, which indicated that ABA-1 forms a dimer. Aside from being the first nematode allergen for which structure or function has been elucidated, ABA-1 provides a highly manipulable model for investigation of the interaction between hydrophobic ligands and alpha-helical proteins.
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