SUBTYPES OF ODORANT-BINDING PROTEINS - HETEROLOGOUS EXPRESSION AND LIGAND-BINDING

Odorant-binding proteins (OBP) in the mucus of the olfactory epitheliu m are thought to transfer the hydrophobic odorous compounds through th e aqueous barrier towards the chemo-sensory cells. To evaluate their b inding properties, two distinct OBP subtypes of the rat were expressed as N-terminal His-tagged fusion proteins in Escherichia coli, thus al lowing an efficient purification. Based on gel chromatography and CD s pectroscopy analysis the recombinant OBP subtypes seem to share severa l structural features with other members of the lipocalin family. Appr oaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2-[H-3]-isobut yl-3-methoxypyrazine whereas OBP2 did not shown any specific binding t o this compound. In contrast, the chromophore 1-anilinonaphthalene 8-s ulfonic 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-te rt-butyl-alpha-methyl dihydrocinnamic aldehyde) and citralva (3,7-dime thyl-2,6-octadienenitrile), also displaced efficiently the chromophore , whereas pyrazine derivatives including 2-isobutyl-3-methoxy-pyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities.