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 biochemical function for gp15/400. A single 14.5-kDa unit of gp15/400 has been expressed in Escherichia coli, and found to dimerise spontaneously. This protein (designated P-RUNG) has high-affinity fatty acid and retinoid binding activity, suggesting that the parent polypeptide itself has these properties. Fluorescent fatty acid probes show significant enhancement of fluorescence intensity and shifts in emission wavelength in the presence of P-RUNG, which can be reversed by competing non-fluorescent fatty acids (oleic, palmitic, steric, arachidonic), retinoids (retinol and retinoic acid) and oleoyl Coenzyme A, but not by tryptophan, cholesterol, caproic acid, squalene, tocopherol, tocopherol acetate, succinyl CoA, 2-methylbutyric acid and 2-methylvaleric acid. Changes in intrinsic fluorescence of retinol or retinoic acid confirmed the retinoid binding function. The results of fluorescence titration experiments are consistent with stoichiometric binding to a single protein site per monomer unit with affinities (Kd) 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 tryptophan residue of P-RUNG indicates that it also is deeply buried in a non-polar environment, but is probably not involved in ligand binding. Gp15/400, therefore, represents a new class of lipid binding protein which is possibly restricted to nematodes.
Kennedy
M W
MW
Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Bearsden, UK.
Allen
J E
JE
Wright
A S
AS
McCruden
A B
AB
Cooper
A
A
D000818:Animals; D000947:Antigens, Helminth; D001483:Base Sequence; D017178:Brugia malayi / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014212:Tretinoin / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
D000947:Antigens, Helminth; D005227:Fatty Acids; D015801:Helminth Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C094735:gp15-400 protein, Brugia pahangi; D014801:Vitamin A; D014212:Tretinoin