School of Chinese Materia Medica, Beijing University of Chinese Medicine, NO. 11 Beisanhuandong Road, Chaoyang District, Beijing, 100029, China.
J Ethnopharmacol. 2019 Oct 28;243:112101. doi: 10.1016/j.jep.2019.112101. Epub 2019 Jul 22.
Earthworms, a type of animal drugs from traditional Chinese medicine, have been used to treat coagulation for many years with less adverse effects and similar anticoagulant effects compared to the commonly used anticoagulants. There are four species of earthworms recorded in Chinese Pharmacopoeia, while few of them were studied and deficient information were involved in the NCBI and UniProt earthworm protein database. We have adopted a transcriptomic-proteomics-anticoagulant bioactivity integrated approach to investigate a seldom-studied Chinese Pharmacopoeia recorded species, Pheretima guillelmi.
In the present study, we aimed to reveal the anticoagulant bioactivity of Pheretima guillelmi, and identify its functional proteins via LC-MS/MS-transcriptome cross identification.
With the aid of fibrinogen-thrombin time assay, Pheretima guillelmi was found to possess strong anticoagulant activity, and the bioactivity was quite stable under 30-50 °C and near-neutral conditions. A comprehensive non-reference transcriptome assembly of P. guillelmi was first established to supplement the currently inadequate earthworm protein database and to illustrate the active proteins. Illumina RNA sequencing generated 25,931,175 of clean reads with over 97% high-quality clean reads (Q20) and assembled an average of 133,228 of transcript and 106,717 of unigenes. A total of 11,259 coding sequences were predicted via ESTScan (3.0.3). The P. guillelmi unigenes were searched and annotated against public database. The bioactive proteins in P. guillelmi were with broad distribution of molecular weight. With bottom-up proteomics analysis, ten proteins were identified against UniProt and NCBI earthworm database; and 31 proteins with high-confidence were matched against transcriptomic established P. guillelmi database.
This study illuminated the therapeutic potency of P. guillelmi for antithrombus and provide a new strategy to investigate animal drugs of Chinese materia medica.
地龙，一种中药动物药，多年来一直用于治疗凝血，与常用的抗凝剂相比，副作用较小，抗凝效果相似。《中国药典》中记载了四种地龙，而对其中几种的研究较少，NCBI 和 UniProt 地龙蛋白数据库中涉及的信息也较少。我们采用转录组-蛋白质组-抗凝生物活性综合方法，研究了一种研究较少的《中国药典》记录物种——威廉环毛蚓。
本研究旨在揭示威廉环毛蚓的抗凝生物活性，并通过 LC-MS/MS-转录组交叉鉴定鉴定其功能蛋白。
借助纤维蛋白原-凝血酶时间测定法，发现威廉环毛蚓具有较强的抗凝活性，在 30-50°C 和近中性条件下，生物活性相当稳定。首次建立了威廉环毛蚓的综合非参考转录组组装，以补充目前不足的地龙蛋白数据库，并阐明活性蛋白。Illumina RNA 测序生成了 25931175 条清洁读数，高质量清洁读数（Q20）超过 97%，平均组装了 133228 条转录本和 106717 条 unigenes。通过 ESTScan（3.0.3）共预测了 11259 个编码序列。威廉环毛蚓的 unigenes被搜索并注释到公共数据库中。威廉环毛蚓中的生物活性蛋白分子量分布广泛。通过自上而下的蛋白质组学分析，从 UniProt 和 NCBI 地龙数据库中鉴定出 10 种蛋白；并从转录组建立的威廉环毛蚓数据库中匹配了 31 种具有高置信度的蛋白。
本研究阐明了威廉环毛蚓治疗血栓的功效，并为研究中药动物药提供了新的策略。
