THE GP15 400 POLYPROTEIN ANTIGEN OF BRUGIA-MALAYI BINDS FATTY-ACIDS AND RETINOIDS/

Gp15/400 is a surface-proximal antigen of the filarial nematode Brugia malayi, produced as a large polyprotein precursor comprising an array of polypeptide units of approx. 14.5 kDa. Here we describe a biochemi cal function for gp15/400. A single 14.5-kDa unit of gp15/400 has been expressed in Escherichia coli, and found to dimerise spontaneously. T his protein (designated P-RUNG) has high-affinity fatty acid and retin oid binding activity, suggesting that the parent polypeptide itself ha s these properties. Fluorescent fatty acid probes show significant enh ancement of fluorescence intensity and shifts in emission wavelength i n the presence of P-RUNG, which can be reversed by competing non-fluor escent fatty acids (oleic, palmitic, stearic, arachidonic), retinoids (retinol and retinoic acid) and oleoyl Coenzyme A, but not by tryptoph an, cholesterol, caproic acid, squalene, tocopherol, tocopherol acetat e, succinyl CoA, 2-methylbutyric acid and 2-methylvaleric acid. Change s in intrinsic fluorescence of retinol or retinoic acid confirmed the retinoid binding function. The results of fluorescence titration exper iments are consistent with stoichiometric binding to a single protein site per monomer unit with affinities (K-d) in the range 2 x 10(-6) M (for the fluorescent probe 11-((5-dansyl)amino)undecanoic acid) and 2 X 10(-7) M (for oleic acid). The extreme blue shift of the fluorescent fatty acid-protein complex suggests an unusually low polarity for the protein binding site. The intrinsic fluorescence of the single trypto phan residue of P-RUNG indicates that it also is deeply buried in a no n-polar environment, but is probably not involved in ligand binding. G p15/400, therefore, represents a new class of lipid binding protein wh ich is possibly restricted to nematodes.