α-Conotoxin MI

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab')2 displayed highly potent neutralization and detoxification efficacy. 12.5 μL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 μg, 1.7 × LD50), while 6.25 μL (mouse) or 10.41 μL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins.

Conotoxins (CTXs) are a variety of mixed polypeptide toxins, among which α-conotoxin MI (CTX-MI) is the most toxic. Serious toxic symptoms, a lack of counteracting drugs, and cumbersome detection processes have made CTX-MI a hidden danger for humans. One of the obstacles to resolving this problem is the absence of specific recognition elements. Aptamers have shown great advantages in the fields of molecule detection, drug development, etc. In this study, we screened and characterized aptamers for CTX-MI through a programmed process. MBMI-01c, the isolated aptamer, showed great affinity, with an affinity constant (KD) of 0.524 μM, and it formed an antiparallel G-quadruplet (GQ) structure for the specific recognition of CTX-MI. Additionally, an aptasensor based on the biolayer interferometry (BLI) platform was developed and displayed high precision, specificity, and repeatability with a limit of detection (LOD) of 0.26 μM. This aptasensor provides a potential tool for the rapid detection of CTX-MI in 10 min. The aptamer can be further developed for the enrichment, detoxification, and biological studies of CTX-MI. Additionally, the programmed process is applicable to screening and characterizing aptamers for other CTXs.

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.