In Silico Identification of Protein Disulfide Isomerase Gene Families in the De Novo Assembled Transcriptomes of Four Different Species of the Genus Conus

Small peptides isolated from the venom of the marine snails belonging to the genus Conus have been largely studied because of their therapeutic value. These peptides can be classified in two groups. The largest one is composed by peptides rich in disulfide bonds, and referred to as conotoxins. Despite the importance of conotoxins given their pharmacology value, little is known about the protein disulfide isomerase (PDI) enzymes that are required to catalyze their correct folding. To discover the PDIs that may participate in the folding and structural maturation of conotoxins, the transcriptomes of the venom duct of four different species of Conus from the peninsula of Baja California (Mexico) were assembled. Complementary DNA (cDNA) libraries were constructed for each species and sequenced using a Genome Analyzer Illumina platform. The raw RNA-seq data was converted into transcript sequences using Trinity, a de novo assembler that allows the grouping of reads into contigs without a reference genome. An N50 value of 605 was established as a reference for future assemblies of Conus transcriptomes using this software. Transdecoder was used to extract likely coding sequences from Trinity transcripts, and PDI-specific sequence motif “APWCGHCK” was used to capture potential PDIs. An in silico analysis was performed to characterize the group of PDI protein sequences encoded by the duct-transcriptome of each species. The computational approach entailed a structural homology characterization, based on the presence of functional Thioredoxin-like domains. Four different PDI families were characterized, which are constituted by a total of 41 different gene sequences. The sequences had an average of 65% identity with other PDIs. Using MODELLER 9.14, the homology-based three-dimensional structure prediction of a subset of the sequences reported, showed the expected thioredoxin fold which was confirmed by a “simulated annealing” method.

从芋螺属（Conus）海洋蜗牛的毒液中分离得到的小分子肽，因其治疗价值而被广泛研究。此类肽可分为两类，其中占比最大的一类富含二硫键，被称为芋螺毒素（conotoxins）。尽管芋螺毒素因其药理学价值而具有重要意义，但目前对催化其正确折叠所需的蛋白质二硫键异构酶（protein disulfide isomerase, PDI）的相关认知仍较为有限。为发掘可能参与芋螺毒素折叠与结构成熟过程的PDI，研究人员对采自墨西哥下加利福尼亚半岛的4种不同芋螺物种的毒管转录组进行了组装。研究为每个物种构建了互补DNA（complementary DNA, cDNA）文库，并采用Illumina基因组分析仪平台完成测序。原始RNA-seq数据通过Trinity（一款无需参考基因组即可将测序读段聚类为重叠群的从头组装软件）转换为转录本序列。研究设定N50值为605，作为后续使用该软件组装芋螺转录组的参考标准。研究使用Transdecoder从Trinity生成的转录本中提取潜在编码序列，并通过PDI特异性序列基序"APWCGHCK"捕获潜在的PDI。随后开展了计算机模拟分析，以表征各物种毒管转录组所编码的PDI蛋白序列集。该计算分析基于功能型硫氧还蛋白样结构域的存在，进行结构同源性表征。最终共鉴定出4个不同的PDI家族，共计包含41种不同的基因序列。这些序列与其他PDI的平均序列一致性为65%。研究使用MODELLER 9.14对部分已报道的序列开展基于同源建模的三维结构预测，结果显示其具有预期的硫氧还蛋白折叠构象，该结论通过"模拟退火"方法得到了验证。