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

Biochem J. 2000 Sep 1;350 Pt 2(Pt 2):369-79.

Abstract

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 Escherichia coli 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Blotting, Southern
  • Blotting, Western
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Circular Dichroism
  • Cloning, Molecular
  • Cysteine / chemistry
  • DNA, Complementary / metabolism
  • Disulfides
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Female
  • Glycosylation
  • Lectins / metabolism
  • Ligands
  • Lipocalins
  • Male
  • Mass Spectrometry
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Plasmids / metabolism
  • Protein Binding
  • Protein Isoforms
  • Protein Processing, Post-Translational*
  • Protein Structure, Secondary
  • RNA / metabolism
  • Recombinant Proteins / metabolism
  • Saliva / chemistry*
  • Salivary Proteins and Peptides / chemistry
  • Salivary Proteins and Peptides / genetics*
  • Salivary Proteins and Peptides / metabolism*
  • Sequence Analysis, Protein
  • Sequence Homology, Amino Acid
  • Spectrometry, Fluorescence
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Swine

Substances

  • Carrier Proteins
  • DNA, Complementary
  • Disulfides
  • Lectins
  • Ligands
  • Lipocalins
  • Protein Isoforms
  • Recombinant Proteins
  • Salivary Proteins and Peptides
  • salivary lipocalin protein, Sus scrofa
  • RNA
  • Cysteine

Associated data

  • GENBANK/AJ249974