A 'conovenomic' analysis of the milked venom from the mollusk-hunting cone snail Conus textile--the pharmacological importance of post-translational modifications

Abstract

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai'i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50 = 34.2 nMol kg(-1). The non-post-translationally modified form, [Pro](2,8)[Glu](16)α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 μM. Interestingly its comparative PD50 (3.6 μMol kg(-1)) in invertebrates was ~100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.

Keywords: (Gla); 1H-Benzotriazolium-1-[bis(Dimethylamino)Methylene]-5-Chloro Hexafluorophosphate-(1-),3-Oxide; 9-Fluorenylmethoxycarbonyl-L-Aspartic Acid-beta-t-Butyl Ester; 9-Fluorenylmethoxycarbonyl-O-t-Butyl-L-Hydroxyproline; 9-Fluorenylmethoxycarbonyl-S-Acetamidomethyl-L-Cysteine; 9-Fluorenylmethoxycarbonyl-S-Trityl-L-Cysteine; 9-fluorenylmethyloxycarbonyl; API; Acm; CH(3)CN; CHCA; CID; Conopeptides; Conus textile; DCM; DHB; DIEA; DMF; DV; ESMS; FDA; Fmoc; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Cys(Acm)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Gla(otBu)(2)-OH; Fmoc-Gln(Trt)-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Food and Drug Administration; GBR; Great Barrier Reef; HCTU; IND; M(r); MALDI-MS; MALDI-TOF-(TOF)-MS; MS; MV; Mass spectrometry; Milked venom; N,N-diisopropylethylamine; N-Fmoc-L-γ-Carboxyglutamic Acid γ,γ-Di-t-Butyl Ester; N-alpha-9-Fluorenylmethoxycarbonyl-N-beta-Trityl-L-Asparagine; N-alpha-9-Fluorenylmethoxycarbonyl-N-g-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-Sulfonyl-L-Arginine; N-alpha-9-Fluorenylmethoxycarbonyl-N-gamma-Trityl-L-Glutamine; N-alpha-9-Fluorenylmethoxycarbonyl-Nim-Trityl-L-Histidine; PCR; PSD; PTM; Post-translational modifications; R(t); RE; RP-HPLC; RV; Radula Venom; Radula tooth; Retention time; SEM; SPPS; TCEP; TFA; TIPS; Trifluoroacetic acid; Triisopropylsilane; Tris(2-carboxyethyl)phosphine; UV; Ultra-violet; acetamidomethyl; acetonitrile; amino acid; atmospheric pressure ionization; average molecular mass; cDNA; collision induced dissociation; complimentary DNA; dichloromethane; dihydroxybenzoic acid; dimethylformamide; duct venom; electro spray mass spectrometry; investigational new drug; m/z; mRNA; mass spectrometry; mass to charge ratio; matrix assisted desorption/ionization mass spectrometry; matrix assisted desorption/ionization-time of light-(time of flight)-mass spectrometry; messenger RNA; nAChR; nicotinic acetylcholine receptor; polymerase chain reaction; post-source decay; post-translational modification; radula extract; reverse phase - high performance liquid chromatography; scanning electron micrograph; solid phase peptide synthesis; α-Conotoxin; α-Cyano-4-hydroxycinnamic acid; αα; γ-gammacarboxyglutamic acid.