S-1-1
Possible Ligand-binding Proteins in the Olfactory Epithelium of the
Japanese Common Newt
Taro Mizumoto1, Jiro Muroran2 and Saburo Iburi
1
Department of Materials Science and Engineering, Muroran Institute of Technology,
27-1 Mizumoto, Muroran 050-8585, Japan
2
Applied Physics and Chemistry, The University of Electro-Communications,
Chofugaoka 1-5-1, Chofu, Tokyo 182-8585, Japan
Email: iwasat@mmm.muroran-it.ac.jp
1. Introduction.
Recently, the number of the chemicals has increased tremendously in our environment. Some of
these chemicals caused harmful effect to living organisms including humans. The mechanism causing
such toxic effects on the organisms are still not well-understood and possibly different from the each
chemical. However, the first step of the toxic effects should be an interaction between such
environmental chemicals and molecules such as proteins or other biological molecules of living
organisms. The large part of these proteins are embedded in the cytoplasmic membranes of cells
building living organisms and called “membrane receptors”. However, in the case of dioxin, it is very
hydrophobic, easily passes the cytoplasmic membrane, and binds aryl-hydrocarbon receptor (AhR) in
the cell. The complex of dioxin and AhR cause the change of gene expression pattern of the cell,
which results in harmful effects on organism. Therefore, to understand the mechanism of toxic effects
of a chemical it is very important to understand the interaction between the chemical and the target
protein, receptor. For the model system to study this interaction, an olfactory system of animal is very
suitable. We have been studying the olfactory system of the newt and found a protein (Cp-lipocalin)
that shows amino acid sequence homology to AhR.
2. Results and Discussion.
2.1 Several types of possible “ligand-binding protein”s were found in a cDNA library of the
olfactory epithelium of the newt.
We constructed a cDNA Library with messenger RNA obtained from the olfactory epithelium of
the newt, Cynopus pyrrhogaster. By a preliminary exhaustive analysis of the library, we found three
types of clones that are abundantly expressed in the olfactory epithelium of the newt. The deduced
amino acid sequence of two of them, clone #75 and #89, showed high sequence homology to each
other and to the members of lipocalin super-family, frog olfactory specific protein (OSP) and Hyla
japonica lipocalin-type protein, respectively. They were tentatively named as Cp-OSP and Cplipocalin. Lipocalins are a large family of small proteins that are characterized by the ability to bind
small hydrophobic molecules. Their primary sequences are diverged, but the 3D structure of the
lipocalins shows a conserved folding pattern. The molecular phylogenetic tree constructed by the
neighbor-joining methods confirmed that Cp-OSP and Cp-lipocalin are highly similar each other and
they belong to the lipocalin super-family (Fig. 1).
Another class of the possible lignad-binding protein was also found in the olfactry epithelium of
the newt. Analysis of the nucleotide sequence of the third clones revealed a high sequence similarity
to rat potential ligand-binding protein (RY2G5; PLBP). The deduced amino acid sequence showed
homology to rat ligand-binding protein and also to human BPI (BPI : Bactericidal/PermeabilityIncreasing protein) family proteins. Therefore, it was named as Cp-PLBP.
0.487
Gallus gallus CALⅡ
5
0.118
9
0.487
Homo sapiens MSFL2541
5
0.014
1
0.099
5
0.162
8
0.100
9
0.433
4
[GENETYX-MAC:Evolutionary Tree]
Method:UPGMA
0.443
6
No 75 : Cp-OSP
0.443
6
No. 89 : Cp-lipocalin
0.086
Hyla japonica lipocalin
2
0.086
Bufo marinus lipocalin
2
0.187
Xenopus laevis cpl-1
1
0.720
Rana nigromaculata osp
0
Fig. 1. Molecular phylogenetic
tree of Cp-OSP and Cp-lipocalin
constructed with Genetyx Mac
UPGMA program.
2.2 Cp-OSP and Cp-lipocalin were expressed in olfactory epithelium.
To elucidate tissue specificity of Cp-OSP and Cp-lipocalin, total RNA was isolated from several
tissues of the newt and the northern blotting analysis was performed with alkaline phosphatase-labeled
anti-sense probe of Cp-OSP or Cp-lipocalin. The results showed that the transcripts of the two clones
were highly expressed in the newt olfactory epithelium but not in brain, liver and intestine.
2.3 The transcripts of the three possible ligand-binding proteins were expressed in Bowman’s
gland.
The digoxygenin-labeled anti-sense riboprobe of each three possible lignad-binding proteins was
made and in situ hybridization was performed with a frozen section of the newt olfactory epithelium.
Based on the analysis, they were all expressed in cells composed of the Bowman’s grand, which
secrete mucus covering the olfactory epithelium. The results suggest that these possible ligandbinding proteins are produced in the Bowman’s grand and transferred to the mucus layer. The finding
that the deduced amino acid sequences of Cp-OSP and Cp-lipocalin have a possible signal sequence in
their N-terminal region is not contradictory to the suggestion. In the case of Cp-PLBP, because the
full length cDNA has not been unfortunately isolated yet, it is not sure that Cp-PLBP is really secreted
protein.
2.4 The distribution of the cells which express each possible ligand-binding protein showed
different pattern on the olfactory organ of the newt.
From the results of in situ hybridization with a frozen section of the olfactory epithelium, it is not
elucidated whether these proteins are produced in the same cells composed Bowman’s grand. To
clarify the question, we used serial frozen sections of the nasal cavity of C. pyrrohogaster, and studied
whether these proteins are produced in the same cells. The results of the in situ hybridization analysis
of the whole nasal cavity of the newt showed that the cells which express each possible ligand-binding
protein are different in their distribution pattern on the olfactory organ. Although the transcripts of
Cp-OSP were observed in almost all Bowman’s grand of the nasal cavity, those of Cp-lipocalin were
observed only in Bowman’s grand on central and dorsal part of the nasal cavity. The Bowman’s grand
on the ventral part of the cavity did not show the signal. The cells which express Cp-PLBP showed a
different distribution pattern from that of lipocalin-family proteins; only the Bowman’s grand on the
central part of the cavity expresses Cp-PLBP.
3. Concluding remarks.
Our results showed that the different types of possible ligand-binding proteins are expressed in the
different parts of the nasal organ of the newt and that possibly form heterogeneous mucous
environment on olfactory epithelium. Although the physiological role of the proteins and their
importance in olfactory sensation are remained unclear, their possibility of binding small molecules,
such as odorant, and the difference in distribution on the mucus layer suggest that a pre-discrimination
of odorant is performed by these proteins before odorants reach to olfactory receptors.