Insect Biochemistry and Molecular Biology 30 2000 1091–1098 www.elsevier.comlocateibmb
Soluble proteins from chemosensory organs of Eurycantha calcarata Insects, Phasmatodea
Silvana Marchese
a
, Sergio Angeli
a, b
, Annapaola Andolfo
c
, Andrea Scaloni
c
, Anna Brandazza
a
, Mario Mazza
d
, Jean-Franc¸ois Picimbon
e
, Walter S. Leal
e
, Paolo Pelosi
a,
a
Dipartimento di Chimica e Biotecnologie Agrarie, University of Pisa, Via S. Michele 4, 56124 Pisa, Italy
b
Scuola Superiore di Studi Universitari e Perfezionamento “S. Anna”, Pisa, Italy
c
Centro Internazionale Servizi di Spettrometia di Massa — IABBAM, Consiglio Nazionale delle Ricerche, Naples, Italy
d
Dipartimento di Etologia, Ecologia ed Evoluzione, University of Pisa, Pisa, Italy
e
National Institute of Sericultural and Entomological Science, Tsukuba, Japan Received 25 January 2000; received in revised form 13 March 2000; accepted 11 April 2000
Abstract
Three related nucleotide sequences, encoding mature proteins of 108–113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and
mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All
three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS
measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post- translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class
expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Chemosensory proteins; Odorant-binding proteins; Phasmids; Eurycantha calcarata; sequence analysis; RACE
1. Introduction
Odorant-binding proteins OBP are small soluble polypeptides highly concentrated in the fluids nasal
mucus in vertebrates and sensillar lymph in insects bathing the dendrites of chemosensory neurons Pelosi
1996, 1998; Pelosi and Maida, 1995; Steinbrecht, 1998. In insects, they are classified as PBPs pheromone-bind-
ing proteins and GOBPs general odorant-binding proteins, depending on their physiological ligands. Both
PBPs and GOBPs have been identified in several species from different orders of insects Vogt and Riddiford,
Corresponding author. Tel.: +
39-050-571564; fax: +
39-050- 574235.
E-mail address: ppelosiagr.unipi.it P. Pelosi.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 8 4 - 9
1981; Maida et al., 1993; Krieger et al. 1991, 1996; Dickens et al., 1995; Vogt et al., 1999; Danty et al.,
1999; Nagnan-Le Meillour et al., 1996; Ozaki et al., 1995; Raming et al., 1990; Wojtasek et al. 1998, 1999.
They share six conserved cysteine residues, connected by three disulphide bridges, as demonstrated in the case
of Bombyx mori Scaloni et al., 1999; Leal et al., 1999, that contribute to a compact structure of the molecule.
A different class of soluble proteins, expressed in chemosensory organs of insects, has recently received
increasing interest. The first member called OS-D: olfactory specific-D, McKenna et al., 1994, or A-10:
Pikielny et al., 1994 was reported to be expressed in the antennae of Drosophila melanogaster, among other
putative odorant-binding proteins. This protein contains only four conserved cysteines and therefore was not
classified as a putative OBP. The recent isolation of pro-
1092 S. Marchese et al. Insect Biochemistry and Molecular Biology 30 2000 1091–1098
teins similar to OS-D from antennae and other chemo- sensory organs of insects of different orders, however,
supports the hypothesis that these could represent another class of proteins involved in chemoreception.
Our search for odorant-binding proteins in insect orders other than Lepidoptera first led to the isolation of
low molecular weight polypeptides highly expressed in the sensory organs antennae, tarsi and mouth structures
of several species of Phasmids Tuccini et al., 1996; Mameli et al., 1996 bearing a significant similarity to
Drosophila OS-D. Members of this class were later described in the Orthopteran species Schistocerca grega-
ria Angeli et al., 1999, in three species of Lepidoptera, Cactoblastis cactorum Maleszka and Stange, 1997,
Mamestra brassicae Bohbot et al., 1998 and Bombyx mori Picimbon et al., 2000, as well as in the honey bee
Danty et al., 1998 and in cockroaches Picimbon and Leal, 1999. Unlike lepidopteran OBPs and their putative
homologues of other orders, OS-D-like proteins are well conserved across evolution, with 40–50 of identical
residues even between most distant species.
The physiological function of these proteins is still to be identified, and even their role in olfaction has been
questioned. A proposed role in carbon dioxide sensing Maleszka and Stange, 1997 has not received experi-
mental evidence. On the other hand, their involvement in chemosensation is well supported by their specific
expression in chemosensory organs, such as antennae, tarsi and mouth apparatus Angeli et al., 1999. More-
over, electron microscopy experiments, have clearly shown that in S. gregaria these proteins are highly con-
centrated in the lymph of contact sensilla of antennae, tarsi and labial palpi, but are absent in olfactory sensilla
Angeli et al., 1999. A protein similar to Drosophila OS-D is expressed in the ejaculatory bulb of the same
species Dyanov and Dzitoeva, 1995. The presence in this organ of the sex pheromone suggested the idea that
such protein could be a carrier for the hydrophobic mol- ecule. This view was supported by recent experiments
demonstrating reversible binding of the Drosophila pheromone vaccenyl acetate to the OS-D-like protein of
the Lepidopteran species M. brassicae Bohbot et al., 1998. Another polypeptide of the same class, called
p10, has been isolated from the regenerating legs of the cockroach Periplaneta americana Kitabayashi et al.,
1998: the authors suggest a function in the regeneration of limbs during the larval stages.
To provide additional information on the structure of this class of proteins, we have cloned three members of
this family from the antennae of the Phasmid Eurycantha calcarata.
A comparison
with similar
sequences expressed in insects of different orders reveals highly
conserved regions, probably involved in a common func- tion.
2. Materials and methods
