WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

Single-Molecule Long-Read Sequencing Facilitates Transcriptomic Research For Whitmania Pigra, The Most Utilized Medical Leech In Chinese Traditional Medicine For Anticoagulant Therapy

Although leeches are of great medical and economic value in anticoagulant therapy, full-length transcriptomes for leeches remain scarce. Here, we generated the first full-length transcriptome for the paddy leech Whitmania pigra (the most widely utilized medical leech in Chinese traditional medicine) through Pacific Biosciences (Pacbio) single-molecule long-read sequencing. A total of 191,676 full-length non-chimeric (FLNC) reads were obtained, 30,660 were high-quality unique full-length transcripts. The BUSCO (Bench-marking Universal Single-Copy Orthologues) accession of completeness demonstrated that 74.8% of BUSCOs were complete. We functionally annotated 28,144 transcripts were in public databases, including NR, gene ontology (GO), Pfam, etc. Furthermore, 1,314 long non-coding RNAs (LncRNAs), 2,574 alternative splicing (AS) events, 932 transcript factors (TFs), and 33,258 simple sequence repeats (SSRs) we identified across all transcripts. From the generated data, a total of 426 anticoagulant genes, including 122 Antistasins, 124 with the Fibrinogen beta and gamma chains, and 62 Kazal-type serine protease inhibitors were screened out. Twenty-five novel proteins were revealed following the evaluation of the annotations and products of these anticoagulant transcripts. The regulation network between LncRNAs and corresponding coding transcripts was found with the typical mang-to-many pattern, especially obvious in a specific type of protein, Guamerin. Collectively, the present findings provide a rich set of full-length cDNA sequences for W. pigra, which will greatly facilitate research on transcriptomic genetic for this species and leeches.

The Genome of Medicinal Leech (Whitmania pigra) and comparative genomic study for Exploration of Bioactive Ingredients

BackgroundLeeches are classic annelids that have a huge diversity and closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients.ResultsIn this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20% and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine pattern. Finally, we systematically explored and compared the active substances in the genomes of three leeches. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules.ConclusionsThis study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.