Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Diversity of Odorant-binding Proteins and Chemosensory Proteins in Insects
1 University of Pisa, Pisa, Italy and 2 China Agricultural University, Beijing, China
Correspondence to be sent to: Paolo Pelosi, e-mail: ppelosi{at}agr.unipi.it
Key words: chemosensory proteins, insects, LUSH, messenger molecules, odorant-binding proteins, pheromones
Odour and pheromone perception occurs through a complex series of events, many aspects of which have been elucidated in the most recent years. The discovery of membrane-bound olfactory receptors, both in vertebrates and in insects, and their functional expression in heterologous systems have shown that they can be directly activated by odorant and pheromone molecules.
Although a pathway of signal transduction from the peripheral olfactory receptors to the areas of the central nervous system can explain how olfactory messages are translated into behavioural responses, the role of soluble proteins at the periphery of the olfactory system remains elusive.
On one hand, messenger molecules (odorants and pheromones) are able to directly activate olfactory receptors, but on the other hand, the very high concentration of soluble proteins around olfactory dendrites indicates an important, yet unknown, role. In fact, the great amount of energy involved in their synthesis and turnover cannot be justified without a great benefit for the individual or for the species. This is particularly true for insects, which often live on a very critical energy balance.
In vertebrates, odorant-binding proteins (OBPs) represent the soluble proteins
present in the perireceptor area (Pelosi et
al., 1982
;
Pelosi, 2001), in insects two classes
of soluble proteins have been so far identified in the lymph of chemosensilla,
odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) (Vogt and Riddiford, 1981;
Pelosi, 1998;
Steinbrecht, 1998;
Vogt et al., 1999;
Jacquin-Joly et al., 2001;
Calvello et al., 2003). These
three classes of polypeptides share a small size (12–18 kDa), very high solubility
and a capacity of reversibly binding small molecules, such as odorants and pheromones.
However, they are structurally very different. Vertebrates’ OBPs belong to the
lipocalin family and are folded in a ß-barrel motif, made mostly of antiparallel
ß-strands. By contrast, insects’ OBPs and CSPs mainly contain 
-helical
domains, but folded in two different patterns (Sandler et al., 2000;
Lartigue et al., 2002;
Tegoni et al., 2004). There
is no similarity also in their amino acid sequences. OBPs are generally divergent across
species and within the same species, with percent of conserved residues as low in some
cases as 8%. Their signature is represented by a pattern of six cysteines in
conserved positions, that are connected in the native protein by three interlocking
disulphide bridges. CSPs are better conserved with often >50% of identical
residues even between members of phylogenetically distant species. Their signature is
constituted by four cysteines connected by disulphide links between adjacent residues,
resulting in the formation of two small loops of eight and four amino acids.
Several pieces of evidence indicate that OBPs and CSPs of insects represent two
classes of proteins performing similar roles, despite their different structures. In
fact, both classes of proteins reversibly bind small ligands with dissociation constants
in the micromolar range. In some cases complexes with ligands have been crystallized and
their structures resolved. Also, both OBPs and CSPs are secreted in the lymph of
chemosensilla, where their concentration can reach millimolar levels. Depending on the
species, they can be expressed in one-pore contact sensilla as well as in multipore
olfactory hairs (Angeli et al.,
1999).
Originally OBPs were considered as the proteins active in peri-receptor events of
chemoreception, while CSPs were regarded as involved in general, yet undefined,
functions. This view was also supported by the observation that OBPs were antennal
specific, while CSPs had been identified in several chemosensory as well as
non-chemosensory tissues. However, the antennal specificity of OBPs, first observed in
Lepidoptera and other orders of insects, is not verified in other cases. In the Argentine
ant, for instance, a CSP has been recently reported as antennal specific (Ishida et al., 2002). At about the same time we
showed that in the paper wasp Polistes dominulus CSP is antenna-specific, while
OBP is also expressed in legs and wings (Calvello
et al., 2003). The same distribution has also been found in another
wasp, Vespa crabro, indicating that different species may have chosen OBPs or
CSPs as the ‘important proteins’ during evolution.
The presence of CSPs or OBPs in non-sensory organs, such as the wings, has suggested
an additional role for these soluble proteins, in analogy to what is known in some
vertebrates. The male mouse, for instance, secretes in the urine large amounts of
lipocalins, structurally very similar or even identical to their nasal OBPs. These
proteins, which are void of ligands when purified from the nasal mucosa, are associated
to specific pheromones in the urine. We have demonstrated that in some insect species
CSPs (in the locusts) or OBPs (in the wasps), when purified from the wings, are complexed
with different small compounds that may be involved in chemical communication (Ban et al., 2003;
Calvello et al., 2003).
Therefore, like lipocalins in vertebrates, both OBPs and CSPs in insects could be endowed
with a dual role, detecting as well as releasing chemical messengers.
In addition, the families of OBPs and CSPs could include members bearing no relationship to chemical communication, as in the case of lipocalins, that include proteins of passive transport across the blood stream and even enzymes. The genome projects have revealed the presence of 51 genes encoding OBP-like proteins in Drosophila melanogaster and 72 in Anopheles gambiae. It is likely that only a subgroup of these genes encode proteins relevant for olfaction or chemical communication.
Another element supporting an important role for OBPs and CSPs is their number in the same species. Generally, several proteins of these families are actually expressed in chemosensory organs, often structurally different from one another, so that subclasses can be identified. The diversity of soluble proteins within the same species suggests a discriminating role, although clear evidence supporting such hypothesis has not been provided.
Finally, the study of the three-dimensional structures of OBPs and CSPs in
association with their ligands have uncovered major conformational changes associated
with their binding. In the pheromone-binding protein of Bombyx mori the
C-terminal region, which has no definite structure at neutral pH, folds into an
-helical segment at low pH and fits into the pheromone-binding cavity (Horst et al., 2001). This mechanism has
been proposed to occur in the proximity of the dendritic membrane to release the
pheromone molecule and handle it to the receptor. The CSP of Mamestra brassicae
also changes its conformation in the presence of ligands (Campanacci et al., 2003), swelling its binding
pocket to accommodate up to three molecules of bromododecanol.
These pieces of information suggest that OBPs and CSPs perform similar roles in chemical communication of insects and that an important, although still undiscovered, function is associated with these soluble polypeptides.
To get insights into the function of these proteins, the study of mutants where their absence is associated with a modified behaviour is extremely informative. So far only two such phenotypes have been reported.
In D. melanogaster the knock-out of the gene encoding the odorant-binding
protein LUSH has been reported to modify the flies’ behaviour to ethanol
(Kim et al., 1998). In fire
ants it has been observed that colonies making more than one queen do not express one of
their OBPs (Krieger and Ross, 2002).
In the latter case, being such OBP normally produced in the thorax, it is difficult to
conceive that this protein could mediate perception of pheromonal compounds. More likely,
this OBP could be a pheromone carrier, as in the case of wasps, and therefore its absence
might prevent the pheromone being released in the environment.
Recently we investigated the role of LUSH in more detail by measuring the binding of
bacterial expressed protein to several compounds. Unexpectedly, we were unable to detect
any binding to ethanol or lower alcohols. On the contrary, the best ligands of LUSH were
aromatic molecules of medium size, such as the fluorescent probe used in our assay
(N-phenyl-1-naphthylamine) and some dialkyl phthalates (Zhou et al., 2004).
In the crystal structure of LUSH, the C-terminus is folded back into the core of the
protein, in a fashion similar to what observed in the acidic form of B. mori
pheromone-binding protein (Kruse et al.,
2003). Such structure would not allow the entrance of a large ligand into the
binding cavity without a major conformational change, involving flipping of the
C-terminal region outside the protein. This hypothesis is supported by the fact that in
our experiments the binding of aromatic molecules is not associated with quenching of
fluorescence of the only tryptophan residue, located in the C-terminal segment and well
inside the binding pocket in the published structure.
Conformational changes associated with ligand binding could be a general phenomenon for OBPs and CSPs and suggest some further interactions of these proteins with other elements of the signal transduction chain.
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