ABSTRACT
We had previously reported the purification and partial characterisation of four distinct odorant-binding proteins from male mouse nasal epithelium. One of these, named OBP-I appeared to be a heterodimer, whose subunits, Ia and Ib showed significant similarity in their N-terminal amino acid sequences with hamster aphrodisin. In this paper, we report the complete amino acid sequences of these two polypeptide chains, as deduced from nucleotide sequences of their relative cDNA. These data confirm the high similarity of both proteins with hamster aphrodisin. A comparison with the sequences of other known OBPs indicate that they are more closely related to members of class I, including bovine OBP, rat OBP-I and pig OBP-I. A putative odorant-binding site is indicated by the presence of amino acid residues conserved with respect to the bovine protein, whose three-dimensional structure has been recently resolved. In-situ hybridisation has revealed identical expression patterns for the two proteins, further supporting the heterodimeric structure of these proteins in the nasal mucus.
Subject(s)
Odorants , Olfactory Mucosa/metabolism , Receptors, Odorant/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Cattle , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , Dimerization , Gene Expression , In Situ Hybridization , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pheromones/genetics , Polymerase Chain Reaction , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Odorant/chemistry , Sequence Homology, Amino Acid , SwineABSTRACT
Large amounts of an odorant-binding protein have been isolated from submaxillary glands of mature male pig. This polypeptide molecule is sex-specific, being absent in females. On electrophoretic gels under denaturing conditions it migrated as a broad band with an apparent molecular mass of around 20 kDa. Electrospray mass spectrometry revealed the presence of three main components, whose mass differences are not interpretable as result of any common post-translational modifications, indicating the presence of distinct polypeptide chains. N-terminal Edman degradation yielded a single sequence of 29 amino acids. It includes the lipocalin signature (-G-X-W-) and shows clear homology with a subclass of odorant-binding proteins present in mouse saliva, nasal mucus and urine. The purified protein still retained small ligands tightly bound; among them 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol, both known sex pheromones for the pig, were identified. The protein also binds 2-isobutyl-3-methoxypyrazine, a good ligand for most odorant-binding proteins, with a dissociation constant of 5 microM.
Subject(s)
Carrier Proteins/chemistry , Odorants , Pheromones/metabolism , Submandibular Gland/physiology , Amino Acid Sequence , Animals , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Female , Gas Chromatography-Mass Spectrometry , Kinetics , Male , Mass Spectrometry , Mice , Molecular Sequence Data , Nasal Mucosa/physiology , Peptide Fragments/chemistry , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Sex Characteristics , SwineABSTRACT
After the isolation of two odorant-binding proteins (OBP-I and OBP-II) from mouse nasal tissue, we have purified two additional OBPs, which bind tritiated 2-isobutyl-3-methoxypyrazine. OBP-III is a homodimer with subunits of M(r) 22,000 and pI 4.2. OBP-IV is a homodimer with subunits of M(r) 21,000 and pI 4.85. N-terminal amino acid sequences indicate that OBP-III is identical in its first 40 amino acids to the mouse urinary protein, MUP-5, (ii) OBP-IV is > 90% identical in its first 30 amino acids to the MUP-4, OBP-II is nearly 80% similar in its first 40 amino acids to OBP-I of the rat, and both subunits of OBP-I are > 50% identical with hamster aphrodisin.
Subject(s)
Receptors, Odorant/isolation & purification , Animals , Cricetinae , Electrophoresis, Polyacrylamide Gel , Macromolecular Substances , Mice , Molecular Weight , Olfactory Mucosa/chemistry , Pyrazines/metabolism , Rats , Receptors, Odorant/chemistry , Receptors, Odorant/metabolism , Sequence Homology , TritiumABSTRACT
1. Two soluble proteins, with good affinity to tritiated 2-isobutyl-3-methoxypyrazine, have been purified from mouse nasal mucosa. 2. The first protein is a heterodimer with subunits of apparent M(r) 18 and 19 kDa and isoelectric point of 4.9; the second is a monomer of M(r) 21 kDa and isoelectric point of 4.8. 3. The characteristics of these binding proteins are compared with those of the other known OBPs and urinary proteins and their putative role is discussed.
