Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant.
After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR. Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg.L-1 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD. We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44-C48 and C63-C155), a pairing close to that of hamster aphrodisin. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6 +/- 0. 3 microM. Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide. Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatile-odorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3-methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate). Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells.
Briand
L
L
Biochimie et Structure des Protéines, Unité de recherches INRA, Jouy-en-Josas, France.
Nespoulous
C
C
Perez
V
V
Rémy
J J
JJ
Huet
J C
JC
Pernollet
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004220:Disulfides; D000431:Ethanol / Q000494:pharmacology; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009831:Olfactory Mucosa / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D011719:Pyrazines / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000873:Anthracenes; D018076:DNA, Complementary; D004220:Disulfides; D008024:Ligands; D011719:Pyrazines; D018035:Receptors, Odorant; D011994:Recombinant Proteins; D012997:Solvents; C045648:odorant-binding protein; D000431:Ethanol; C049062:1-aminoanthracene; C034376:2-isobutyl-3-methoxypyrazine; D004121:Dimethyl Sulfoxide