Efficient oxidative folding of conotoxins and the radiation of venomous cone snails

Abstract

The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.

Fig. 1.

Shells of cone snails. Seven different Conus species (of the ≈500 total) are illustrated: the examples shown represent the three major feeding types of Conus. Experimental data that are described in the text were obtained from these species. (Top) The glory-of-the-sea cone, C. gloriamaris (Left); the cloth-of-gold cone, C. textile (Right). (Middle) C. omaria (Left); C. consors (Center); C. aurisiacus (Right). (Bottom) The fly-speck cone, C. stercusmuscarum (Left); C. betulinus (Right). C. betulinus is worm-hunting, whereas C. consors, C. aurisiacus, and C. stercusmuscarum are piscivorous (fish hunting). C. textile, C. gloriamaris, and C. omaria are molluscivorous (mollusc hunting). A PCR screen of PDI was carried out on most of these species (see text). A comparison of the spasmodic peptide of C. textile and C. gloriamaris provided the basis for the work on the role of posttranslational modification in Conus peptide folding. C. textile was the species used for most of the experimental studies described in this article.

Fig. 2.

PDI from C. textile venom duct. (A) SDS/PAGE of extracts from different parts of a venom duct. Lanes 1–4 represent equal fragments from distal to proximal parts of the venom duct. Lane 5 is a bovine PDI. The ≈55-kDa band from lane 3 was extracted, and its N-terminal sequence was determined (see text). (B) Structures of PDI-1 and PDI-2 from C. textile. The signal sequence, thioredoxin active sites (Trx), and the ER retention signal are marked. Domains a, b, b′,a′, and c are based on the assignments for human PDI (9, 10). The shaded residues represent differences between PDI-1 and PDI-2.

Fig. 3.

Comparison of Gla-containing peptides from Conus species. The posttranslational modification of glutamate to γ-carboxyglutamate (Gla or γ) is shown, and a comparison of the sequences of four Gla-containing Conus venom peptides to the N-terminal sequence of human blood clotting factor IX is shown. Note that one Conus peptide, conantokin-G from C. geographus, has a Gla domain motif that has similar spacing to factor IX (see boxed sequences), whereas the three other Gla-containing peptides do not have an arrangement of Gla residues characteristic of a Gla domain. #, amidated C terminus; W, 6-bromotryptophan; T′, glycosylated threonine; γ, γ-carboxyglutmate; h-FIX, human factor IX.

Fig. 4.

Effect of Ca²⁺ on the oxidative folding of peptides from C. textile and C. gloriamaris. (a and b) Sequences of peptides from C. textile and C. gloriamaris.(c–f) HPLC analysis of oxidative folding of the peptides. The folding mixtures contained reduced and oxidized glutathione, and the reactions were carried out in the presence of 10 mM CaCl₂ or 1 mM EDTA. Experimental details are described in Materials and Methods. The correctly folded species (*) has the shortest retention time. (c) Folding of C. textile peptide in the absence of Ca. (d) Folding of C. gloriamaris peptide in the absence of Ca. (e) Folding of C. textile peptide in the presence of Ca. (f) Folding of C. gloriamaris peptide in the presence of Ca. (g and h) Kinetics of forming the native peptides from C. textile (g) and C. gloriamaris (h). The folding reactions were performed as described in Materials and Methods. Dashed lines, +10 mM Ca²⁺; solid lines +1 mM EDTA. (i and j) Accumulation of the native peptides from C. textile (i) and C. gloriamaris (j) in the folding reactions at 24 h, with increasing concentration of calcium ions. Two other bars represent accumulation of the native peptides in the presence of 1 mM EDTA or 100 mM Mg²⁺. All experimental points are averages from duplicate experiments.

Fig. 5.

Ca²⁺-assisted oxidation of the Gla-containing model peptide. The peptide (a) was designed based on the sequence of peptide from C. textile. Cys-2, Cys-12, and Cys-16 were replaced by Ala residues, and Thr-19 had an amidated C terminus (labeled #). The disulfide bond between Cys-6 and Cys-18 is also shown. A hypothetical model illustrating how a coordination of Ca²⁺ by the two Gla residues in the peptide may favor formation of a disulfide bridge (-SS-) by bringing two Cys thiols in close proximity. (b and c) HPLC analysis of oxidation of the model peptide with a mixture of oxidized (1 mM) and reduced (2 mM) glutathione and in the presence of either 10 mM CaCl₂ or 1 mM EDTA. The oxidation mixtures were quenched by acidification after 2 or 10 min and analyzed by reversed-phase C₁₈ analytical HPLC. L, linear (reduced) peptide; Ox, oxidized form.