Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577, Japan.
Int J Mol Sci. 2018 Oct 31;19(11):3418. doi: 10.3390/ijms19113418.
Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 ( Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.
富含二硫键的肽在自然界中含量丰富，它们的研究为蛋白质折叠、结构和功能提供了引人入胜的见解。毒蛇芋螺属于一类表达自然界中发现的最大的富含二硫键肽（芋螺毒素）之一的生物。芋螺毒素的结构支架多样性表明，已经进化出了专门的分子适应机制，以确保其高效折叠和分泌。我们最近表明，经典的蛋白二硫键异构酶（PDI）和一种芋螺毒素特异性的 PDI（csPDI）在芋螺毒液腺中广泛表达，在芋螺毒素折叠中发挥主要作用。在这里，我们鉴定了毒蛇芋螺内质网氧化还原酶-1（Ero1），并通过重新氧化芋螺 PDI 和 csPDI 来研究其在芋螺毒素氧化折叠中的作用。我们表明，Ero1 优先将 PDI 氧化再氧化为 csPDI，这表明 csPDI 的再氧化可能依赖于一种 Ero1 独立的分子途径。尽管 PDI 优先被 Ero1 氧化再氧化，但 Ero1 和 csPDI 的组合效应提供的折叠产率高于 Ero1 和 PDI。我们进一步证明，当所有三种酶都存在时，两种模型芋螺毒素的体外折叠速率最高，表明这些酶可能协同作用。我们的研究结果为一类最具多样性的富含二硫键肽的产生提供了新的见解，并可能改进当前用于药理学研究的毒液肽体外生产的方法。
