Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Sheng-Yu Huang,Bing-Shin Liu,Sung-Fang Chen,Min-Han Lin,Tin-Yu Wei,Sheng-Kuo Chiang,Wang-Chou Sung

doi:10.3390/toxins11010052

Abstract

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide dimethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a β-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Highlights

The linkage of disulfide bonds is one of the post-translational modifications involved in protein structure construction and stabilization [1]
These results demonstrate that (1) both the syn-Cardiotoxin A3 (CTX A3) folded protein and the non-native isomer could induce the antibody to recognize the native CTX A3 toxin, but (2) only the syn-CTX A3 folded protein can effectively induce the neutralization antibodies through immunization, which indicates the disulfide bond pattern plays a critical role in the immunogenicity and antigenicity of the disulfide-rich immunogen
A total of 13 disulfide linked peptides containing different cysteine pairings were repeatedly detected from the folding intermediates

Summary

Introduction

The linkage of disulfide bonds is one of the post-translational modifications involved in protein structure construction and stabilization [1]. Multiple disulfide bonds commonly occur on membrane and secretary proteins, maintaining their bioactive structures and allowing them to perform their designed functions in the extracellular environment. Advances in genomic technology have allowed the expression of disulfide-rich proteins in in vitro and in vivo folding systems [6,7,8], but the lack of specific enzymes usually leads to the generation of diverse isomers with non-native disulfide bonds, which impacts the product quality [9,10]. Given that the disulfide bond pattern is highly correlated with the bioactive structure, direct identification of the disulfide bonds of folding intermediates could allow the evaluation of folding conditions as a first step towards manufacturing a bioactive product

Methods

Results

Conclusion