Wu Y et al. (2014), Influence of disulfide connectivity on structur...

XB-ART-49844

Molecules 2014 Jan 15;191:966-79. doi: 10.3390/molecules19010966.

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Influence of disulfide connectivity on structure and bioactivity of α-conotoxin TxIA.

Wu Y , Wu X , Yu J , Zhu X , Zhangsun D , Luo S .

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Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3β2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3β2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3β2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3β2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

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Species referenced: Xenopus laevis
Genes referenced: acin1 snai1 vsig1

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	Figure 1. A schematic representation of globular (native), ribbon and beads isomers’ disulfide connectivities of α-CTx TxIA. #, amidated COOH terminal.
	Figure 2. RP-HPLC and ESI-MS analysis of α-CTx TxIA. (A) HPLC chromatography of Hainan C. textile crude venom using Vydac C18 semi-preparative column (10 μm, 10 mm × 250 mm). The arrow denotes α-CTx TxIA. The linear gradient was a 5%–35% solvent B gradient in 75 min, then 35%–65% gradient in 35 min. (B) Analytical HPLC profile of native α-CTx TxIA from C. textile venom using a linear gradient of a 10%–30% eluate B, and 90%–70% eluate A over 30 min, In panel A & B, eluate B is 0.05% TFA in 90% ACN, remainder water; eluate A is 0.075% TFA in water. Absorbance was monitored at 214 nm; (C) ESI-MS analysis of native α-CTx TxIA with calculated mass of 1,656.67 Da.
	Figure 3. (A) Reduced native TxIA MS/MS spectrum of the precursor ion of m/z 831.356 [M+2H]2+ with the assignment of a series of b-ions obtained under collision-induced dissociation (CID) conditions; (B) Synthetic linear TxIA MS/MS spectrum of the precursor ion m/z 831.356 [M+2H]2+, obtained under CID conditions.
	Figure 4. Three synthetic α-CTx TxIA isomers’ mass analyzed by ESI-MS. (A) Globular isomer; (B) Ribbon isomer; (C) Beads isomer.
	Figure 5. HPLC analysis of native and synthetic α-CTx TxIA isomers co-injection. (A) Co-injection traces of three α-CTx TxIA isomers synthesized by two-step oxidation method. (B) Co-injection traces of synthetic globular TxIA and native peptide. (C) Co-injection traces of synthetic ribbon and native TxIA. Peptides were analyzed on a reversed-phase analytical Vydac C18 (5 μm, 4.6 mm × 250 mm) HPLC column using a linear gradient of a 10%–30% eluate B, and 90%–70% eluate A over 30 min, where B = 0.05% TFA in 90% ACN, remainder water; A = 0.075% TFA in water. Absorbance was monitored at 214 nm.
	Figure 6. Air oxidation random folding of α-CTx TxIA linear peptide in 0.1 M NH4HCO3 buffer. (A) Co-injection trace of synthetic linear peptide and reduced native α-CTx TxIA; (B) RP-HPLC analysis after random folding. Peptides were analyzed on a reversed phase analytical Vydac C18 (5 μm, 4.6 mm × 250 mm) HPLC column using a linear gradient of a 10%–30% eluate B, and 90%–70% eluate A over 30 min, where B = 0.05% TFA in 90% ACN; A = 0.075% TFA in water. Absorbance was monitored at 214 nm.
	Figure 7. Representative ACh-evoked currents of rat α3β2 nAChRs expressed in Xenopus oocytes obtained in the absence (Control) and presence of 3 isomers of α-CTx TxIA. (A) Globular, (B) Ribbon, and (C) Beads.
	Figure 8. Inhibition concentration-response curves for the isomers of α-CTx TxIA. Values are mean ± SEM from 6 to 10 separate oocytes. Globular, Ribbon and Beads isomers were tested on rat α3β2 nAChRs expressed in Xenopus oocytes.
	Figure 9. Circular dichroism (CD) spectra of native α-CTx TxIA and its isomers.

References [+] :

Armishaw, Conotoxins as research tools and drug leads. 2005, Pubmed