Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins

Abstract

An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.

Figure 1. The shell of Conus distans

Conus distans is a relatively large Conus species; the specimen shown, collected in the Philippines is 110mm in length.

Figure 2. Purification of di16a from the crude venom of C. distans

The arrow in each HPLC run indicates the location of fractions containing the peptide di16a. (A) The venom extract was chromatographed in a preparative Vydac C18 column eluted with the gradient described under the Materials and Methods. (B) The peak containing the fraction indicated by an arrow in (A) was subfractionated using an analytical Vydac C18 column under conditions described in Materials and Methods. (C) The peak indicated by an arrow in (B) was applied to an analytical Vydac C18 column and eluted using the gradient specified in Materials and Methods.

Figure 3. Mass spectrometry of the di16a peptide

The sequence of di16a (O= 4-transhydroxyproline; γ= carboxyglutamate) obtained by standard Edman methods is shown. The sequence obtained is consistent with the mass determined for the major peak. Measurements were carried out as described under Materials and Methods.

Figure 4. Co-elution of the native and synthetic di16a

(A) Native peptide (0.5 nmol) was applied on the analytical Vydac C18 column with a gradient of 4.5–22.5% ACN/40min at 1ml/min. The same conditions were used for the synthetic folded peptide (B). (C) Co-elution was carried out by mixing native and synthetic peptides on the analytical Vydac C18 column, using the same gradient and flow rate.

Figure 5. Sequence of di16a precursor

Top: the cDNA sequence and the corresponding predicted amino acid sequence encoded in the Di16.1 cDNA clone. The sequences used for 3′, 5′-RACE primers are underlined. Bottom: The peptide precursor sequence. The sequence in bold represents the mature toxin. The sequence in normal type is the signal sequence, with the pro region in italics. The amino acid residues underlined represent the posttranslational modifications (O= 4-transhydroxylated proline, γ=carboxyglutamate).