Transcriptomic-proteomics-anticoagulant bioactivity integrated study of Pheretima guillemi

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.