Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation 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 displaced by the steroidal sex pheromone, as well as by several odorants.
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September 2000
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Research Article|
August 23 2000
Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin
Dietrich LOEBEL;
Dietrich LOEBEL
1
*Institut fur Physiologie, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
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Andrea SCALONI;
Andrea SCALONI
1
†Centro Internazionale Servizi di Spettrometria di Massa, National Research Council, via Pansini 5, 80131 Napoli, Italy
‡I.A.B.B.A.M., National Research Council, via Argine 1085, 80100 Napoli, Italy
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Sara PAOLINI;
Sara PAOLINI
§Dipartimento di Medicina Interna and Sezione INFM, Università di Perugia, via del Giochetto 6, 06126 Perugia, Italy
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Carlo FINI;
Carlo FINI
§Dipartimento di Medicina Interna and Sezione INFM, Università di Perugia, via del Giochetto 6, 06126 Perugia, Italy
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Lino FERRARA;
Lino FERRARA
‡I.A.B.B.A.M., National Research Council, via Argine 1085, 80100 Napoli, Italy
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Heinz BREER;
Heinz BREER
*Institut fur Physiologie, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
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Paolo PELOSI
Paolo PELOSI
2
‖Dipartimento di Chimica e Biotecnologie Agrarie, University of Pisa, via S. Michele 4, 56124 Pisa, Italy
2To whom correspondence should be addressed (e-mail ppelosi@agr.unipi.it).
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Publisher: Portland Press Ltd
Received:
February 24 2000
Revision Received:
May 18 2000
Accepted:
June 26 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 369–379.
Article history
Received:
February 24 2000
Revision Received:
May 18 2000
Accepted:
June 26 2000
Citation
Dietrich LOEBEL, Andrea SCALONI, Sara PAOLINI, Carlo FINI, Lino FERRARA, Heinz BREER, Paolo PELOSI; Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin. Biochem J 1 September 2000; 350 (2): 369–379. doi: https://doi.org/10.1042/bj3500369
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