A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

Abstract

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.

Fig. 1

A. Shells of Conus, Terebra, Turrids. Top, Terebra subulata (Terebridae); Middle row: left, Hastula hectica (Terebridae); right, Conus marmoreus; Bottom row: left, Gemmula speciosa; right, Lophiotoma olangoensis (both Turridae). B. Left to right: Gemmula speciosa, G. diomedea, G. sogodensis, and G. kieneri.

Fig. 2

Purification of gsp9a and gsp9b. The components of the G. speciosa venom duct extract prepared as described under Methods were separated by sequential elution with gradients of 4.5% to 54% acetonitrile in 55 min and 54% to 90% acetonitrile in 8 min in the presence of 0.1% TFA. The absorbance profile at 220 nm is shown in Fig. 2, Panel A. An aliquot of Peak 1 in Panel A was reduced and pyridylethylated. The pyridylethylated mixture was fractionated on an analytical C18 column employing a gradient of 0.45% acetonitrile/min in 0/1% TFA, (Panel B). The more hydrophobic peak was determined as described in Methods to be gsp9a and the sequence is shown in Table 2. Peak 2 in Panel A was subfractionated on an analytical C₁₈ HPLC column with a gradient of 0.18% acetonitrile/min in 0.1% TFA (Panel C). The components of the more hydrophobic peak were reduced, pyridylethylated, and then fractionated with a gradient of 0.45% acetonitrile/min in 0.1% TFA on an analytical C₁₈ HPLC column (Panel D). The main peak in Panel D was determined by MALDI and amino acid sequencing to be gsp9b (Table 2).