9.3 KDa components of the injected venom of Conus purpurascens define a new five-disulfide conotoxin framework

Abstract

The 83-residue conopeptide (p21a) and its corresponding nonhydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions, p21a has a unique, 10-cystine/5-disulfide 7-loop framework with extended 10-residue N-terminus and a 5-residue C-terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13-cystine conopeptide, con-ikot-ikot, isolated from the dissected venom of the fish-hunting snail Conus striatus. However, unlike con-ikot-ikot, p21a does not form a dimer of dimers. MALDI-TOF mass spectrometry suggests that p21a may form a noncovalent dimer. p21a is an unusually large conotoxin and in so far is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilize toxins that are comparable in size to the ones commonly found in other venomous animals.

Fig. 1

Extraction of injected venom from C. purpurascens. (A) A fish is placed in front of the snail for stimulation. The stimulated snail extends its proboscis seeking to strike prey (B) The snail strikes the vial with the trap, where the injected venom is collected.

Fig. 1

Extraction of injected venom from C. purpurascens. (A) A fish is placed in front of the snail for stimulation. The stimulated snail extends its proboscis seeking to strike prey (B) The snail strikes the vial with the trap, where the injected venom is collected.

Fig. 2

Purification of p21a. The fraction that corresponds to p21a is marked with an arrow. Three squirts of injected venom were applied to an analytical Vydac C18 column and eluted with a linear gradient of 1 % B/min over 100 min at a flow rate of 1 ml/min. The buffers were 0.1 % TFA (Buffer A) and 0.1% TFA in 60% acetonitrile (Buffer B). The fraction that corresponded to p21a was re-purified under the same conditions (inset).

Fig. 3

MALDI-TOF mass spectra (linear mode) of: (A) fraction corresponded to p21a. (B) p21a after reduction/alkylation with DTT/Iodoacetamide, indicating the presence of ten cysteines.

Fig. 4

SDS-PAGE and MALDI-TOF MS of the injected venom of C. purpurascens (A) native (B) reducing/alkylating conditions.