Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encod ing SAL was cloned and sequenced. From a single individual, two protein iso forms, differing in three amino acid residues, were purified and structural ly characterized by a combined Edman degradation/MS approach. These experim ents ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-transl ationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, wherea s the remaining two occur as free thiols, SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein h as been built. A SAL isoform was expressed in Escherichia coli in good yiel ds. Protein chemistry and CD experiments verified that the recombinant prod uct shows the same redox state at the cysteine residues and that the same c onformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displac ed by the steroidal sex pheromone, as well as by several odorants.