Cone snail milked venom dynamics--a quantitative study of Conus purpurascens

Abstract

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 μM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC₅₀ for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.

Fig. 1. Representative specimens of Conus purpurascens

Captive specimens 1–2 (representatives used in this study) and locality specimens 3–6. 1. Panama, long-term captive specimen - notice chalky appearance, loss of shell color and gloss; 2. Panama, long-term captive specimen - notice growth ridges and bands, shell was originally 23.9 mm in diameter and grew to 37.9 mm - at the broadest point; 3. Horseshoe Bay, Costa Rica, non-captive specimen; 4. Cocos Is. Costa Rica, non-captive specimen; 5. Gobernadora Is. Panama, non-captive specimen; 6. Jaco, Panama, non-captive specimen. (A) Scan Electron Microscopy of C. purpurascens radula harpoon showing apex, 1^st and 2^nd barbs – each radula is unique to each species of Conus; Insert (B) shows cross section of harpoon providing access to lumen and peptide venom remnants within.

Fig. 2. Representative RP-HPLC/UV profile of time differentiated milked venoms obtained from a single Conus purpurascens specimen (Panama, specimen No. 2 – Fig. 1)

Profiles A and B represent a time difference of approximately 8 months in captivity. Peaks labeled correspond to the individual conopeptides observed and their retentions (Rt), as listed in Table 2. Profiles demonstrate a level of chromatographic simplicity. Note: ψ-conotoxin PIIIF (Fig. 2B) is represented in two separate, closely eluting forms. Molecular mass analysis assigns these two peptides a PTM variants differing by a single trans-4-hydroxyproline modification.

Fig. 3. Maximal milked venom complexity observed a single captive specimen Conus purpurascens (Panama, specimen No. 1 – Fig. 1)

RP-HPLC/UV showing the dominant peptide peaks being expressed in separate captive specimen of C. purpurascens milked venom, MV #41. Six C. purpurascens conopeptides were observed by retention and MS analysis (see Table 4). This specific profile is used to illustrate quantitative amino acid analysis, as in reference to Table 2.

Fig. 4. MALDI-TOF mass spectra of radula harpoon (top), captive milked venom (middle), and crude duct venom (bottom) obtained from a single specimen of Conus purpurascens

Molecular Masses (m/z) common to all three are designated by a triangle (σ). An asterisk (M) indicates peaks that correspond to previously published conopeptides derived from C. purpurascens. 2,5-dihydroxybenzoic acid (DHB) was used as the matrix. One advantage of using DHB is that there is a low occurrence of matrix peaks and matrix adducts. To further ensure that common masses observed are not matrix peaks, mass spectra were compared to ‘matrix only’ mass spectra. No matrix peaks were detected.