Subtypes of odorant-binding proteins--heterologous expression and ligand binding.
Odorant-binding proteins (OBP) in the mucus of the olfactory epithelium are thought to transfer the hydrophobic odorous compounds through the aqueous barrier towards the chemo-sensory cells. To evaluate their binding properties, two distinct OBP subtypes of the rat were expressed as N-terminal His-tagged fusion proteins in Escherichia coli, thus allowing an efficient purification. Based on gel chromatography and CD spectroscopy analysis the recombinant OBP subtypes seem to share several structural features with other members of the lipocalin family. Approaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2-[3H]-isobutyl-3-methoxypyrazine whereas OBP2 did not shown any specific binding to this compound. In contrast, the chromophore 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) specifically interacted with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity revealed that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p-tert-butyl-alpha-methyl dihydrocinnamic aldehyde) and citralva (3,7-dimethyl-2,6-octadienenitrile), also displaced efficiently the chromophore, whereas pyrazine derivatives including 2-isobutyl-3-methoxypyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities.
Löbel
D
D
University of Stuttgart-Hohenheim, Institute of Physiology, Stuttgart, Germany.
Marchese
S
S
Krieger
J
J
Pelosi
P
P
Breer
H
H
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001667:Binding, Competitive; D002942:Circular Dichroism; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes; D015870:Gene Expression; D066298:In Vitro Techniques; D008024:Ligands; D009812:Odorants; D009831:Olfactory Mucosa / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D018035:Receptors, Odorant / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008024:Ligands; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein; C027132:1-anilino-8-naphthalenesulfonate