Characterization of novel M‐superfamily conotoxins with new disulfide linkage

Abstract

The M‐superfamily with the typical Cys framework (–CC–C–C–CC–) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M‐superfamily conotoxins can be provisionally categorized into four branches (M‐1, M‐2, M‐3, M‐4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176–8186]. Here we report the purification of seven M‐superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M‐1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M‐superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M‐1 and M‐2 and only one to M‐3. The disulfide analyses of two M‐1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1–C5, C2–C4, C3–C6) which is different from those of the M‐4 branch (C1–C4, C2–C5, C3–C6) and M‐2 branch (C1–C6, C2–C4, C3–C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom

“…From the eight full-length precursors belonging to the m-2 branch, four have mature peptides that were reported previously: Mr3.3, MrIIIB, MrIIIG, and MrIIID (43)(44)(45). Among this group, MrIIIG precursor has the highest expression level with 230 matching reads.…”

“…Twelve precursors that belong to the m-1 branch were identified (Mr039-Mr050), including the previously characterized MrIIIE, MrIIIF, Mr3.8 and Mr1e precursors (43)(44)(45). All precursors in this branch had a pre-sequence cleavage site LGQR or KR, yielding predicted mature peptides with 11 to 16 amino acids and three disulfide bonds, except Mr1e, which has only four cysteines.…”

α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors

“…The AChBP binding activity of ImI is also dramatically affected by the substitution and the consequent conformational change [19]. It has been reported that the same cysteine pattern may correlate with a different connectivity of disulfide bridges [14,48]. Thus it would be very intriguing to see, without the conserved Pro residue, how the native Lp1.1 toxin folds and acts on AChR.…”

Conotoxins and Other Conopeptides

“…The sequences of the amplified transcripts are then typically determined by Sanger sequencing. A few gene superfamilies, including A (Puillandre, Watkins, and Olivera, 2010;Santos et al, 2004), I1 (Jimenez et al, 2003), I2 (Kauferstein et al, 2004;Liu et al, 2009), O1 (Conticello et al, 2001;Duda and Palumbi, 1999;Kauferstein, Melaun, and Mebs, 2005;Luo et al, 2006), O2 (Conticello et al, 2001), T (Conticello et al, 2001;Walker et al, 1999) and M (Conticello et al, 2001;Corpuz et al, 2005;Han et al, 2006;Wang et al, 2008), have been extensively studied using this approach. Recent second generation sequencing transcriptomics studies were all carried out using the 454 GS-FLX Titanium technology, generating 200 000-900 000 reads with an average read length of 200-400 bp (Dutertre et al, 2013;Hu et al, 2011Hu et al, , 2012Lluisma et al, 2012;Terrat et al, 2012).…”

“…However, the connectivity between disulfide bonds is an important type of post-translational modification that cannot be fully determined using mass spectrometry. In some cases, disulfide connectivities can be determined by analyzing the mass of nonreduced conotoxin fragments (Dy et al, 2006;M€ oller et al, 2005), but often more labor-intensive methods must be used such as the chromatographic coelution of wild-type peptides and synthetic disulfide isomers (Han et al, 2006;Hidaka et al, 1990), or even directly by structural characterization of the peptides either by X-ray crystallography (Hu et al, 1997) or nuclear magnetic resonance (NMR) spectroscopy (Hill, Alewood, and Craik, 2000;Kang, Jois, and Kini, 2006).…”