http://orcid.org/0000-0002-3570-8928
Figures
Abstract
Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism’s medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia.
Citation: Lu Z, Shi P, You H, Liu Y, Chen S (2018) Transcriptomic analysis of the salivary gland of medicinal leech Hirudo nipponia. PLoS ONE 13(10): e0205875. https://doi.org/10.1371/journal.pone.0205875
Editor: Gabriel Agbor, Institute of Medical Research and Medicinal Plant Studies, CAMEROON
Received: January 15, 2018; Accepted: October 3, 2018; Published: October 19, 2018
Copyright: © 2018 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Illumina sequencing data from salivary glands of H. nipponia were deposited to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) under accession number of SRP126617.
Funding: This study was founded by the Traditional Chinese Medicine Science and Technology Project of Chongqing Health and Family Planning Commission in China (zy201602097 to PS), Natural Science Foundation of Chongqing (cstc2018jcyjAX0674 to PS), Science and Technology Innovation Funds by the Chongqing Science and Technology Commission of China (cstc2016shmszx1247 to SC), and the Fundamental Research Funds for the Chongqing Academy of Chinese Material Medica (2016csts-jbky-01910 to ZL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Hirudo nipponia Whitman (Shui Zhi in Chinese) is a traditional Chinese medicine with significant medicinal values. It has been widely used to treat cardiovascular and cerebrovascular diseases, hyperlipidemia, thrombosis, inflammation, and tumor diseases in China and was first recorded in the classic book on Chinese Materia Medica, Shen-Nong-Ben-Cao-Jing (ca. 100 AD) [1–3]. Now, this leech is mainly used in cerebral thrombosis and cerebral apoplexy in clinical treatment and is listed in the Pharmacopoeia of the People’s Republic of China [4,5]. To date, over 34 active ingredients, including peptides, phosphatidylcholines, pteridines, and other components, have been isolated and/or structurally identified in H. nipponia [1,6,7,8]. More than 300 Shui Zhi-containing prescriptions are produced by many pharmaceutical manufacturers, including the Maixuekang capsule, Tongxinluo capsule and Da Huang Zhe Chong pill [9–11].
Although this medicinal leech has been used as treatment for various ailments in China, Korea and Japan for a long time, neither its pharmacologically active compounds nor its molecular information has been thoroughly investigated. Next generation sequencing platforms based on the RNA-Seq technique are a powerful and efficient tool to detect novel transcripts.
Therefore, we utilized Illumina de novo sequencing technology to detect the transcriptional profiles of H. nipponia. The sialotranscriptome described here provides a significant amount of gene resources for a comprehensive understanding of the pharmacologically active compounds produced by this leech. This study also provides useful information on leading compounds with pharmaceutical potential in H. nipponia.
Materials and methods
Animals and RNA extraction
All leeches were obtained from an adult Hirudo nipponia colony grown in a medical leech breeding base at the Chongqing Academy of Chinese Materia Medica (Nan’an, China). The 50 leeches were maintained in glass container filled with 15 L dechlorinated tap water at 25°C and 12 h/12 h day/night cycles prior to dissection. Every 5 days, half of the water was removed and replaced with fresh water. Prior to RNA extraction, leeches were washed in 0.5% bleach for 1 min and subsequently rinsed in deionized water for 30 seconds to minimize contamination with bacteria. Salivary tissue masses lying posterior to the 3 muscular jaws from twenty leeches were removed aseptically by sterilized dissecting tool and subsequently rinsed in 0.5% bleach for 1 min followed by rinsing in deionized water for 1 min [12–14].Total RNA was then extracted from the abovementioned salivary tissues using a RNAprep Pure Tissue Kit (Tiangen, China). RNA quality was monitored on 1% agarose gels, and the concentration was determined using a Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA).
Library preparation for transcriptome sequencing
Construction of the cDNA libraries and RNA-Seq were performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). First, mRNA was purified from 1.5 μg of total RNA from salivary gland tissue using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in NEB Next First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments that were preferentially 150~200 bp in length. Then, 3 μL of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.
De novo transcriptome assembly and functional annotation
The clean reads were obtained by removing reads with adapter sequences, reads containing ploy-N (N>0.1%), and low-quality reads from the raw data. After filtering, the high-quality clean data were de novo assembled by a Trinity RNA-Seq Assembler [15]. Unigenes were matched to publicly accessible databases using Blastx (E-value ≤ 10−5), including NCBI redundant protein sequences (Nr), NCBI non-redundant nucleotide sequences (Nt), protein family (Pfam), EuKaryotic Orthologous Groups (KOG), Swiss-Prot Protein Sequence Database (Swiss-Prot), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases.
Multiple sequences alignments and analysis
All unigenes were converted to their corresponding predicted amino acid sequences with Virtual Ribosome [16]. These putative polypeptide sequences then were used to retrieve orthologous sequences from GenBank nr with blastp and from GenBank EST with tblastn. In addition to global annotations predicted against GenBank nr databases, blastx comparisons were made against a locally compiled sequence database of the following accessions: Q07558 hirudin from H. medicinalis, P81492 hirudin from Hirudinaria manillensis, P15358 antistasin from Haementeria officinalis, AAA65645 ghilanten from H. ghilianii, P80302 hirustasin from H. medicinalis, AAD09442 guamerin from H.nipponia, 2K13 saratin from H. officinalis, AAA96144 destabilase from H. medicinalis, AAF73890 bdellin-kl from H. nipponia, P82107 bdellin A from H. medicinalis, P09865 bdellin B from H. medicinalis, and etc. Comparative amino acid sequence alignment was accomplished with CLUSTAL W and BoxShade server at heep://www.ch.embnet.org/software/BOX_form.html. Signal peptide prediction was accomplished with SignalP 4.1 server at http://www.cbs.dtu.dk/services/SignalP/.
Results and discussion
Transcriptome sequencing and assembly
Illumina sequencing data from the salivary glands of H. nipponia were deposited to NCBI SRA database under accession number SRP126617. A total of 89,867,368 Illumina paired-end raw reads were identified (Table 1). In total, 84,657,362 clean reads were obtained by filtering out adaptor sequences, ambiguous nucleotides, and low-quality sequences. The assembly of clean reads using Trinity software resulted in 50,535 unigenes that ranged from 201 bp to 28,648 bp with a N50 length of 1,878 bp (Table 1). The length distribution of all unigenes and transcripts is shown in Fig 1.
Gene annotation of salivary gland
The 50,535 unigene sequences from the salivary gland of H. nipponia were functionally annotated by searching diverse public databases, including Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO. Overall, a total of 23,490 unigenes (46.48%) were successfully annotated with this strategy. The percentage of unique sequences annotated based on Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG and GO were 35.99%, 13.54%, 32.91%, 22.56%, 30.66%, 18.74% and 33.08%, respectively (S1 Table).
The E-value distribution of best hits according to the Nr database revealed that 51.50% of the annotated sequences have strong homology (E-value < 1e-45) and that 48.50% of the homology sequences ranged from 1e-5 to 1e-45 (Fig 2A). The similarity distribution indicated that 72.60% of the annotated sequences had a similarity greater than 60% (Fig 2B). For species classification, the highest percentage of unique sequences matched with leech Helobdella robusta (49.5%), followed by the polychaete worm Capitella teleta (10.2%) (Fig 2C).
(A) E-value distribution of Blastx hits for unigenes with a cut-off E-value of 1.0e-5. (B) Similarity distribution of Blastx hits for unigene. (C) Species classification is shown as a percentage of the total homologous sequences with an E-value of at least 1e-5.
Functional annotation and pathway assignment
Based on GO, an international standardized gene functional classification system, 16,718 non-redundant unigenes were assigned to 54 level 2 GO terms, which were classified into three main functional categories, i.e., biological process, cellular component and molecular function (Fig 3).
GO functional annotations are summarized in three main categories: biological process, cellular component and molecular function. Each category represents a GO term assigned by Blast2GO analysis.
In the category of biological process function, the terms cellular process (9,617, 20.67%) and metabolic process (8,064, 17.33%) were the dominant subcategories, followed by single-organism process (7,550, 16.23%). In the category of cellular components, a high percentage of genes were assigned to cell (5,224, 18.28%) and cell part (5,224, 18.28%). For molecular function, the two most abundant categories were binding (9,452, 47.25%) and catalytic activity (6,417, 32.08%). (S2 Table).
Non-redundant unigenes were compared with the KOG database for the analysis of orthologous gene products. A total of 11,404 unigenes with significant homology were assigned to appropriate KOG clusters. These KOG classifications were divided into 26 functional categories. Among them, the cluster of ‘Signal transduction mechanisms’ (19.46%) represented the largest group, followed by ‘General function prediction only’ (16.61%), ‘Posttranslational modification, protein turnover, chaperones’(10.69%) and ‘transcription’ (6.28%) (Fig 4).
The annotated sequences were mapped to the KEGG database to identify the main biological pathways in the salivary gland. In total, 9,471 assembled unigenes in the sialotranscriptome of H. nipponia were assigned to 230 KEGG pathways. Among the pathways, ribosome, cAMP signaling pathway, MAPK signaling pathway and a few others were highly represented (S1 Fig). The top 20 KEGG pathways are shown in Table 2.
Alignments and genes of interest
The H. nipponia sialotranscriptome results with high scoring matches revealed a large number of medicinally useful bioactive molecules. Among them, we focused on several proteins potentially related to therapeutic effects, including anticoagulant, thrombolytic, anti-inflammatory, antitumor, anesthetic and vasodilator effects. Anticoagulants included in the locally compiled data set used for the BLASTx comparisons were listed in Table 3.
Anticoagulants Leech salivary glands can produce a diverse pharmacological cocktail of a wide variety of anticoagulants [17]. In the current transcriptome database of H. nipponia, nine non-redundant unigene sequences involved in anticoagulation and antithrombotic processes showed a high similarity to known leech species. Some of their active ingredients have been isolated and characterized. Hirudin is the most potent natural direct thrombin inhibitor known to date with 65 amino acids, and it can form an irreversible, tight bond to thrombin’s active site [18–20]. Two putative transcripts averaged 52% amino acid identity (E-value = 8.08e-12) and 45% identity (E-value = 8.12e-6) to hirudin from Hirudinaria manillensis [21,22]. Several properties of the hirudin “core” motifs associated with hirudin’s binding to the thrombin catalytic pocket are conserved in the putative H. nipponia sequence, including: CLC, as well as a GSNV region conservatively replaced by chemically similar NSNL in H.nipponia. All 6 cysteines, presumably involved in 3 disulfide bonds, are evolutionary conserved as well. (Fig 5). Another thrombin inhibitor named theromin was also found in the H. nipponia sialotranscriptome. The thrombin inhibitor exhibited a high value (Ki = 12 fmol/L) for theromin compared with the value for hirudin (Ki = 21 fmol/L) [23].
Similar residues are shaded, with the highlighted homology level ranging from dark black (100% identity), black (75–100% identity), grey (50–75% identity), to light grey (33–50% identity). Red boxes correspond to predicted secretory signal peptides. Green boxes outline conserved cysteines. Hnip, Hirudo nipponia; Hmed, Hirudo medicinalis; Pvir, Poecilobdella viridis; Hman, Hirudinaria manillensis; Hoff, Haementeria officinalis; Hghil, Haementeria ghilianii. *The sequences of H. nipponia described in this study.
Several serine protease inhibitors, including hirustasin, antistasin, ghilanten, guamerin and piguamerin, were found in the salivary gland. Hirustasin can inhibit trypsin, chymotrypsin, cathepsin G and tissue kallikrein, but it does not inhibit blood coagulation factor Xa activity [24]. In contrast, antistasin and ghilanten are potent inhibitors of factor Xa and are highly homologous to each other [25–27]. Guamerin and piguamerin, which have been purified from H. nipponia [28,29], were also found in the present sialotranscriptome. Guamerin has a stronger and more specific effect on the inhibition of neutrophilic and pancreatic elastase than piguamerin [28,30]. Furthermore, guamerin can inhibit the release of proinflammatory cytokines (IL-6 & TNF-α) and neutrophil infiltration in cerulein-induced acute pancreatitis [31]. The longest string of conserved amino acid residues across the 9 antistasin (Fig 5) was CxxGLKxDxNGCEY. All 8 cysteines, presumably involved in 4 disulfide bonds, are evolutionary conserved as well (Fig 5). Piguamerin potently inhibits plasma kallikrein, tissue kallikrein and trypsin, but it does not affect the activity of factor Xa, elastase or thrombin [29]. Bdellin-inhibitors, including bdellin A and bdellin-KL, were also blasted successfully according to previously published data [32,33]. They are both non-classical Kazal-type cysteine proteases inhibitors [34,35]. As an inhibitor of trypsin and plasmin, bdellin can exert an anti-inflammatory influence by inhibiting proteases involved in the spread of inflammation [17,36,37]. The longest string of conserved amino acid residues across the 5 bdellins was VCGxDGxTY (Fig 5).
A putative H. nipponia transcript averaged 75% amino acid identity (E-value = 3.70e-39) with saratin from Hirudo medicinalis. The leech protein saratin can prevent thrombocyte aggregation by interfering with the first binding step of thrombocytes to collagen by binding to collagen [38]. The longest strings of conserved residues were EEREDCWTFYANRKYT, and DLDECxKT55CFKTEYCYIVFEDTVN (Fig 5). Of these, only Thr55 in the alignment corresponds to a residue hypothesized to be involved in this protein’s binding functionality [38]. Destabilase is an isopeptidase that plays a major role in fibrinolytic activity [38] and inhibition of platelet aggregation [41]. Meanwhile, destabilase is also a lysozyme with combined enzymatic and non-enzymatic antibacterial activity [42]. In addition to evolutionary conservation of 14 cysteines, presumably involved in 7 disulfide bonds, there was a 36-residue string of high evolutionary conservation: CSETCVRAYMxRYGTxCTGGRTPTCxDYARIHxGGP (Fig 5). Leech carboxypeptidase inhibitor (LCI) is a potent inhibitor of pancreatic and plasma metallocarboxypeptidases that can be used to treat thrombotic disorders and other cardiovascular diseases [43,44].
Antibacterial Hyaluronidases, which are glycosidases that predominately degrade hyaluronic acid [45], have a beneficial antimicrobial effect [36]. The hyaluronidases were found in the current H. nipponia transcriptomic database [46]. It was reported that hyaluronan can facilitate the penetration or diffusion of pharmacologically active substances into body tissues [36,47] and can also enable thrombosis and cancer therapy [46]. Lysozyme is another antimicrobial substance in H. nipponia, and it had high identity (72%) to a lysozyme in Eisenia andrei [48]. In addition, we found two transcripts with similarity to antimicrobial peptides named theromacin and theromyzin. Both transcripts exhibited activity against gram-positive bacteria [49]. A homologue of translationally controlled tumor protein (TCTP) from the salivary gland of H. nipponia was highly similar to TCTP from Salmo salara [50]. TCTP is likely involved in the inhibition of inflammation, immunoregulation and virus prevention by modulating or suppressing cytokines and gene transcription [50,51]. Blastx analysis results showed that a transcript shares 67% sequence identity to lumbricin from Hirudo medicinalis and 60% sequence identity to lumbricin-1 from earthworm Lumbricus rubellus [52,53]. Lumbricin exhibits strong activity against fungi, gram-positive and gram-negative bacteria, but it does not have hemolytic activity [53]. Another substance, neuromacin, also has antimicrobial activity like lumbricin and theromyzin [53]. C-type lectin is a well-known pattern recognition receptor that can recognize and bind to terminal sugars in microorganisms and participates in immune defense [54,55]. Moreover, both destabilase and bdellin (mentioned above) also have great antibacterial and anti-inflammatory effects.
Antitumor In clinical practice, Shui Zhi and its compound medicines are commonly used to treat a wide range of cancers in China. Previously, published data provided evidence that Shui Zhi could benefit cancer therapy by inhibiting the proliferation of human HepG2 and DNA methylation [1]. Other reports clearly showed that peptides from anticoagulant proteins, such as hirudin and antistasin aforementioned, were metastatic inhibitors against various cancers [56,57]. Since coagulation is related to proliferation and metastasis, blocking the cascade can have an antitumor effect [58,59]. However, such studies are at only a preliminary stage. In addition, there were also over 75 hits with methyltransferases, implying that a potential DNA methylation mechanism probably exists in H. nipponia [60].
Altogether, the results described above provide direct evidence of the existence of therapeutically active compounds related to anticoagulant, antithrombotic, antibacterial, anti-inflammatory and antitumor effects. Among them, anticoagulatory, antithrombotic and antibacterial substances are the most widely studied, whereas the others are less well-known. Many active ingredients have not been discovered yet due to the absence of molecular evidence in public databases.
Conclusions
Although H. nipponia (Shui Zhi) is a potential animal-sourced traditional Chinese medicine with important pharmaceutical value in China, research in this species has been hindered by limited molecular information on its genetic background. In the present study, we sequenced the transcriptome of the primary salivary glands of H. nipponia using Illumina sequencing technology. The assembled sequence data, comprising 50,535 unique transcripts, provide a value resource for understanding genomic data and the biosynthesis of key bioactive metabolites in H. nipponia. The present transcriptomic analysis also revealed a series of candidate genes encoding bioactive proteins related to medical treatment. This study has provided a reference for the synthesis of active substances and a valuable resource to further our understanding of the pharmacological mechanisms in H. nipponia.
Supporting information
S1 Fig. Pathway assignment based on KEGG.
(A) Cellular processes categories, (B) Environmental information processing categories, (C) Genetic information processing categories, (D) Cellular processes categories, and (E) Organismal systems categories.
https://doi.org/10.1371/journal.pone.0205875.s001
(TIF)
S1 Table. Summary statistics for the functional annotation of Hirudo nipponica sequences in public databases.
https://doi.org/10.1371/journal.pone.0205875.s002
(DOCX)
References
- 1. Dong H, Ren JX, Wang JJ, Ding LS, Zhao JJ, Liu SY, et al. (2016) Chinese medicinal leech: ethnopharmacology, phytochemistry, and pharmacological activities. Evid-Based Compl Alt 12: 1–11. PubMed: 27274755.
- 2. Lu YX, Cheng BX, Guo QS, Liu F, Shi HZ, Guo L. (2017) Studies on effects of water temperature, stocking density and feeding cycle on growth and feeding in Hirudo nipponia. Chin J Chin Mate Med 42: 2443–2448. pmid:28840681.
- 3. Shi P, Lu ZH, He YC, Li L, Chen SJ, Shi R. (2015) Study on reproductive performance of Hirudio nipponia. Chin Med Mat 38: 1144–1147. pmid:26762051.
- 4. Liu F, Guo QS, Shi HZ, Wang T, Zhu ZB. (2013) Genetic diversity and phylogenetic relationships among and within populations of Whitmania pigra and Hirudo nipponica based on ISSR and SRAP markers. Biochem Syst Eco 51: 215–223. doi.org/10.1016/j.bse.2013.08.020.
- 5. Xiao L, Nie J, Li D, Chen K. (2015) Peptides from two sanguinovorous leeches analyzed by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometric detector. Pharmacogn Mag 11: 32–37. pmid:25709207.
- 6. Li YB, Huang WH, Xiang Y. (2008) Three new pteridines, hirudinoidines A-C, from Hirudo nipponica Whitman. Helvetica Chimica Acta 91: 303–307.
- 7. Noda N, Tanaka R, Nishi M, Inoue S, Miyahara K. (1993) Isolation and characterization of seven lyso platelet activating factors and two lyso phosphatidylcholines from the crude drug “Suitetsu” (the leech, Hirudo nipponica). Chem Pharm Bull 41: 1366–1368. pmid:8403084.
- 8. Noda N, Tanaka R, Tsujino K, Miura M, Miyahara K, Hayakawa J. (1995) Two amphoteric galactocerebrosides possessing a tri-unsaturated long-chain base from the leech (Hirudo nipponica). Chem Pharm Bull 43: 567–570.
- 9. Ge CJ, Yuan F, Feng LX, Lv SZ, Liu H, Song XT. (2014) Clinical effect of Maixuekang Capsule (脉血康胶囊) on long-term prognosis in patients with acute coronary syndrome after percutaneous coronary intervention. Chin J Integr Med 20: 88–93. pmid:24338186.
- 10. Karalliedde LD, Kappagoda CT. (2009) The challenge of traditional Chinese medicines for allopathic practitioners. Am J Physiol Heart Circ Physiol 297: H1967–H1969. pmid:19855052.
- 11. Zhang YH, Liu JT, Wen BY, Liu N. (2009) Mechanisms of inhibiting proliferation of vascular smooth muscle cells by serum of rats treated with Dahuang Zhechong pill. J Ethnopharmacol 124:125–129. pmid:19527826.
- 12. Siddall ME, Brugler MR, Kvist S. (2016) Comparative transcriptomic analyses of three species of Placobdella (Rhynchobdellida: Glossiphoniidae) confirms a single origin of blood feeding in leeches. J Parasitol 102: 143–150. pmid:26535976.
- 13. Kvist S, Brugler MR, Goh TG, Giribet G, Siddall ME.(2013) Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilitiew in leeches. Invert Bio 133 (1): 74–98.
- 14. Min GS, Sarkar IN, Siddall ME. (2010) Salivary transcriptome of the North American medicinal leech, Macrobdella decora. J Parasitol 96: 1211–1221. pmid:21158638.
- 15. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29: 644–652. pmid:21572440.
- 16. Wernersson R. (2006) Virtual ribosome-A comprehensive DNA translation tool with support for integration of sequence feature annotation. Nucleic Acids Research 34: W385–W388. pmid:16845033.
- 17. Zaidi SM, Jameel SS, Zaman F, Jilani S, Sultana A, Khan SA, et al. (2011) A systematic overview of the medicinal importance of sanguivorous leeches. Altern Med Rev 16: 59–65. pmid:21438647.
- 18. Markwardt F. (1989) Development of hirudin as an antithrombotic agent. Semin Thromb Hemost 15: 269–282. pmid:2688099.
- 19. Stone SR, Hofsteenge J. (1986) Kinetics of the inhibition of thrombin by hirudin. Biochemistry 25: 4622–4628. pmid:3768302.
- 20. Greinacher A, Warkentin TE. (2008) The direct thrombin inhibitor hirudin. Thromb Haemost 99: 819–829. pmid:18449411 10.1160/TH07-11-0693.
- 21. Scacheri E, Nitti G, Valsasina B, Orsini G, Visco C, Ferrera M, et al. (1993) Novel hirudin variants from the leech Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization. Eur J Biochem 214: 295–304. pmid:7685281.
- 22. Electricwala A, Hartwell R, Scawen MD, Atkinson T. (1993) The complete amino acid sequence of a hirudin variant from the leech Hirudinaria manillensis. J Protein Chem 12: 365–370. pmid:8397794.
- 23. Salzet M, Chopin V, Baert J, Matias I, Malecha J. (2000) Theromin, a novel leech thrombin inhibitor. J Biol Chem 275: 30774–30780. pmid:10837466.
- 24. Söllner C, Mentele R, Eckerskorn C, Fritz H, Sommerhoff CP. (1994) Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis. Eur J Biochem 219: 937–943. pmid:8112345.
- 25. Brankamp RG, Sreekrishna K, Smith PL, Blankenship DT, Cardin AD. (1995) Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris. Protein Expr Purif 6: 813–820. pmid:8746634.
- 26. Dunwiddie CT, Waxman L, Vlasuk GP, Friedman PA. (1993) Purification and characterization of inhibitors of blood coagulation factor Xa from hematophagous organisms. Methods Enzymol 223: 291–312. pmid:8271959.
- 27. Han JH, Law SW, Keller PM, Kniskern PJ, Silberklang M, Tung JS, et al. (1989) Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties. Gene 75: 47–57. pmid:2470652.
- 28. Jung HI, Kim SI, Ha KS, Joe CO, Kang KW. (1995) Isolation and characterization of guamerin, a new human leukocyte elastase inhibitor from Hirudo nipponia. J Biol Chem 270: 13879–13884. pmid:7775446.
- 29. Kim DR, Kang KW. (1998) Amino acid sequence of piguamerin, an antistasin-type protease inhibitor from the blood sucking leech Hirudo nipponia. Eur J Biochem 254: 692–697. pmid:9688284.
- 30. Kim H, Chu TT, Kim DY, Kim DR, Nguyen CM, Choi J, et al. (2008) The crystal structure of guamerin in complex with chymotrypsin and the development of an elastase-specific inhibitor. J Mol Biol 376: 184–192. pmid:18155725.
- 31. Jo YJ, Choi HS, Jun DW, Lee OY, Kang JS, Park IG, et al. (2008) The effects of a new human leukocyte elastase inhibitor (recombinant guamerin) on cerulein-induced pancreatitis in rats. Int Immunopharmacol 8: 959–966. pmid:18486906.
- 32. Moser M, Auerswald E, Mentele R, Eckerskorn C, Fritz H, Fink E. (1998) Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)-primary structure, expression in yeast, and characterisation of native and recombinant inhibitor. Eur J Biochem 253: 212–220. pmid:9578479.
- 33. Kim YH, Choi JG, Lee GM, Kang KW. (2001) Domain and genomic sequence analysis of bdellin-KL, a leech-derived trypsin-plasmin inhibitor. J Biochem 130: 431–438. pmid:11530020.
- 34. Min GS, Sarkar IN, Siddall ME. (2010) Salivary transcriptome of the North American medicinal leech, Macrobdella decora. J Parasitol 96: 1211–1221. pmid:21158638.
- 35. Fink E, Rehm H, Gippner C, Bode W, Eulitz M, Machleidt W, et al. (1986) The primary structure of bdellin B-3 from the leech Hirudo medicinalis. Bdellin B-3 is a compact proteinase inhibitor of a "non-classical" Kazal type. It is present in the leech in a high molecular mass form. Biol Chem Hoppe Seyler 367: 1235–1242. https://doi.org/10.1515/bchm3.1986.367.2.1235. pmid:3828073
- 36. Sobczak N, Kantyka M. (2014) Hirudotherapy in veterinary medicine. Ann Parasitol 60: 89–92. pmid:25115059.
- 37. Sommerhoff CP, Söllner C, Mentele R, Piechottka GP, Auerswald EA, Fritz H. (1994) A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis. Biol Chem Hoppe Seyler 375: 685–694. pmid:7888081.
- 38. Gronwald W, Bomke J, Maurer T, Domogalla B, Huber F, Schumann F, et al. (2008) Structure of the leech protein saratin and characterization of its binding to collagen. J Mol Biol 381: 913–927. pmid:18585393.
- 39. Zavalova L, Lukyanov S, Baskova I, Snezhkov E, Akopov S, Berezhnoy S, et al. (1996) Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots. Mol Gen Genet 253: 20–25. pmid:9003282.
- 40. Reverter D, Vendrell J, Canals F, Horstmann J, Aviles FX, Fritz H, et al. (1998) A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis. Isolation, sequence analysis, cDNA cloning,recombinant expression, and characterization. J Biol Chem 273(49): 32927–32933. pmid:9830043.
- 41. Baskova I, Zavalova L, Berezhnoy S, Avdonin P, Afanasjeva G, Popov E, et al. (2000) Inhibition of induced and spontaneous platelet aggregation by destabilase from medicinal leech. Platelets 11: 83–86. pmid:10938885.
- 42. Zavalova LL, Yudina TG, Artamonova II, Baskova IP. (2006) Antibacterial non-glycosidase activity of invertebrate destabilase-lysozyme and of its helical amphipathic peptides. Chemotherapy 52: 158–160. pmid:16636539.
- 43. Sanglas L, Valnickova Z, Arolas JL, Pallarés I, Guevara T, Solà M, et al. (2008) Structure of activated thrombin-activatable fibrinolysis inhibitor, a molecular link between coagulation and fibrinolysis. Mol Cell 31: 598–606. pmid:18722183.
- 44. Arolas JL, Popowicz GM, Bronsoms S, Aviles FX, Huber R, Holak TA, et al. (2005) Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond. J Mol Biol 352: 961–975. pmid:16126224.
- 45. EI-Safory NS, Fazary AE, Lee CK. (2010) Hyaluronidases, a group of glycosidases: Current and future perspectives. Carbohyd Polym 81: 165–181.
- 46. Jin P, Kang Z, Zhang N, Du G, Chen J. (2014) High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers. Sci Rep 4: 4471 Published online. pmid:24667183.
- 47. Linker A, Hoffman P, Meyer K. (1957) The hyaluronidase of the leech: an endoglucuronidase. Nature 180: 810–811. pmid:13483529.
- 48. Josková R, Silerová M, Procházková P, Bilej M. (2009) Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei. Dev Comp Immunol 33: 932–938. pmid:19454335.
- 49. Tasiemski A, Vandenbulcke F, Mitta G, Lemoine J, Lefebvre C, Sautière PE, et al. (2004) Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum. J Biol Chem 279: 30973–30982. pmid:15102860.
- 50. Leong JS, Jantzen SG, von Schalburg KR, Cooper GA, Messmer AM, Liao NY, et al. (2010) Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome. BMC Genomics 11: 279. pmid:20433749.
- 51. Wei J, Guo M, Ji H, Yan Y, Ouyang Z, Huang X, et al. (2012) Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV). Fish Shellfish Immunol 33: 916–925. pmid:22986590.
- 52. Cho JH, Park CB, Yoon YG, Kim SC. (1998) Lumbricin I, a novel proline-rich antimicrobial peptide from the earthworm: purification, cDNA cloning and molecular characterization. Biochim Biophys Acta. 1408: 67–76. pmid:9784609.
- 53. Schikorski D, Cuvillier-Hot V, Leippe M, Boidin-Wichlacz C, Slomianny C, Macagno E, et al. (2008) Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia. J Immunol 181: 1083–1095. pmid:18606660.
- 54. Yu XQ, Kanost MR. (2003) Manduca sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. Dev Comp Immunol 27: 189–196. pmid:12590970.
- 55. Yang J, Wang L, Zhang H, Qiu L, Wang H, Song L. (2011) C-type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization. PLoS One 6: e17089. pmid:21347232.
- 56. Gasic GJ, Iwakawa A, Gasic TB, Viner ED, Milas L. (1984) Leech salivary gland extract from Haementeria officinalis, a potent inhibitor of cyclophosphamide- and radiation-induced artificial metastasis enhancement. Cancer Res 44: 5670–5676. pmid:6498828.
- 57. Merzouk A, Ghawi AM, Abdualkader AM, Abdullahi AD, Alaama M. (2012) Anticancer effects of medical malaysian leech saliva extract (LSE). Pharm Anal Acta 15: 1–5.
- 58. Gil-Bernabé AM, Lucotti S, Muschel RJ. (2013) Coagulation and metastasis: what does the experimental literature tell us?. Br J Haematol 162: 433–441. pmid:23691951.
- 59. Sig AK, Guney M, Guclu AU, Ozmen E. (2017) Medicinal leech therapy-an overall perspective. Integr Med Res 6: 337–343. http://dx.doi.org/10.1016/j.imr.2017.08.001. pmid:29296560
- 60. Zhong X. (2016) Comparative epigenomics: a powerful tool to understand the evolution of DNA methylation. New Phytol 210: 76–80. pmid:26137858.