1094 S. Marchese et al. Insect Biochemistry and Molecular Biology 30 2000 1091–1098
3. Results
3.1. Cloning and sequencing of CSP of E. calcarata On the basis of the N-terminal amino acid sequence
previously reported for protein p14 of E. calcarata Mameli et al., 1996, we designed a degenerated primer
to amplify specific nucleotide sequences encoding such a protein. In order to minimise the degeneracy of the
oligonucleotide, we selected the sequence corresponding to amino acids 7–11. This part of the sequence is also
conserved in similar proteins of other species of Phas- mids Mameli et al., 1996, Orthoptera Angeli et al.,
1999 and Blattoidea Picimbon and Leal, 1999. The amplification products of both antennal and tarsi cDNA
showed a major band of around 550 bp, that was purified and cloned, as described in Section 2.
Sequencing of a number of clones, obtained from the antennal sample, afforded three distinct sequences that
were each confirmed in at least five clones obtained from two different PCR experiments. In contrast, the sample
obtained from the tarsi afforded only one sequence, that was confirmed in eight different clones from three PCR
amplification products. This polynucleotide, identical to the one obtained from the antennae, encodes for a pro-
tein, whose N-terminus matches the amino acid sequence determined on the protein extracted from both parts of
the body Mameli et al., 1996.
While the complete sequence of one of the proteins expressed was reconstructed by combining the cDNA
derived sequence with information obtained by direct Edman degradation of the protein, the N-terminus of the
other two polypeptides expressed in the antennae remained unknown. To obtain such information, the
RACE technique was applied to a sample of antennal cDNA, using specific primers and the protocol reported
in Section 2. The full length cDNA and amino acid sequences are reported in Fig. 1. As reported in our pre-
vious paper Angeli et al., 1999, we adopted the general name of CSP ChemoSensory Protein for a protein of
this class, followed by the initials of the species and a serial number.
3.2. Post-translation modifications To verify the cDNA sequence of CSP-ec1 and obtain
information on post-translational modifications of the polypeptide, a sample of the protein, purified from the
tarsi, as previously reported Mameli et al., 1996, was subjected to electrospray mass spectrometry analysis
after fractionation by HPLC. Two chromatographic peaks were isolated data not shown relative to polypep-
tides with molecular masses of 12,757.2
± 0.9 Da and
11,893.7 ±
0.8 Da, respectively Fig. 2. These values were associated to truncated forms of the protein CSP-
ec1, namely fragments 1-109 and 9-109, respectively. In
Fig. 1. Nucleotide and derived amino acid sequences of clones: A
eury 5B encoding protein CSP-ec1, B eury 1B encoding protein CSP- ec2, and C eury 6B encoding protein CSP-ec3. The signal peptides
and the polyadenylation signals are underlined. The two stop triplets are indicated by asterisks.
fact, the theoretical values calculated on the basis of the encoded sequence corresponded to molecular mass
values of 12,757.2 Da and 11,892.8 Da, assuming all cysteine residues involved in disulfide bonds are as
described for the S. gregaria species Angeli et al., 1999. These results were confirmed by automated
Edman degradation of the two protein bands after HPLC separation on a reverse phase C-18 column. In fact, the
first peak gave the N-terminal sequence EGGKYT–, while the second peak started with YDNVNL–. Such
peptides may have been originated from the native pro- tein by the action of proteolytic agents during the purifi-
cation procedure. These data also ruled out the presence of additional post-translational modifications.
1095 S. Marchese et al. Insect Biochemistry and Molecular Biology 30 2000 1091–1098
Fig. 2. Transformed electrospray mass spectra of the two samples of p14 purified from the tarsi of E. calcarata. The molecular masses were
calculated from the multiply charged ion traces. The measured masses agree with those calculated for truncated forms of the protein CSP-ec1, corresponding to peptides 1-109 left and 9-109 right.
3.3. Western blot A sample of p14 protein, purified from the tarsi of E.
calcarata, was used to raise polyclonal antibodies in a rabbit, using the protocol reported in Section 2. The
crude antiserum
was partially
purified by
45 ammonium sulphate precipitation and used in Western
blot experiments. Fig. 3 reports the results of cross immunoreactivity
performed with proteins p14 from E. calcarata, p19 from C. morosus Tuccini et al., 1996, p14 from Schis-
tocerca gregaria Angeli et al., 1999 and their relative polyclonal antisera. Although the three proteins are simi-
Fig. 3. Western blot analysis of CSPs purified from S. gregaria S, E. calcarata E and C. morosus C, using the antisera raised against the
same proteins. Arrows indicate the proteins utilised for raising antibodies. Molecular weight markers M are, from the top: BSA 66 kDa, ovalbumin 45 kDa, carbonic anhydrase 29 kDa, trypsin inhibitor 20 kDa,
α -lactalbumin 14 kDa.
lar enough to be assigned to the same class, each of them was stained only by its own relative antiserum.
3.4. Isolation of a CSP from the cuticle of E. calcarata
A crude extract from the cellular layer underlying the cuticle showed, in SDS-PAGE, an intense band migrat-
ing with an apparent molecular weight of 14 kDa. This protein was purified applying the same protocol used for
the tarsi and subjected to N-terminal Edman degradation. The first 19 residues NH
2
–EGGKYTTKYDNVNLEE VFG– matched exactly those of CSP-ec1, while the
1096 S. Marchese et al. Insect Biochemistry and Molecular Biology 30 2000 1091–1098
other two sequences expressed in the antennae both showed differences in this region.
4. Discussion
