Abstract
Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.
Highlights
Hirudo nipponia, which feeds mainly on the blood of mammals or frogs, is widely distributed in China (Yang, 1996), and this leech is listed as an animal raw material for Chinese medicinal materials (Commission, 2015)
Unigene5370 and Unigene3091 were annotated as thrombin-inhibiting hirudins; Unigene12407 and Unigene19293 were annotated as an elastase-specific inhibitor, called guamerin, which was previously identified in the leeches H. nipponia (Jung et al, 1995) and Poecilobdella manillensis; and saratin, as a pharmacological component associated with the inhibition of platelet aggregation, was identified from the Unigene2825 transcript in Haementeria officinalis (2e−42) (Gronwald et al, 2008)
The salivary gland transcriptome of the leech H. nipponia was sequenced by the BGIseq-500 platform to obtain 6.5 Gb of data
Summary
Hirudo nipponia, which feeds mainly on the blood of mammals or frogs, is widely distributed in China (Yang, 1996), and this leech is listed as an animal raw material for Chinese medicinal materials (Commission, 2015). In recent years, transcriptome sequencing analysis has been carried out on the salivary glands of leeches, and the pharmacological components have been screened and subjected to evolutionary analysis (Min, Sarkar & Siddall, 2010; Kvist et al, 2014; Kvist et al, 2016; Siddall, Brugler & Kvist, 2016). These components are divided into four groups based on their different pharmacological effects. The fourth group is the antimicrobial peptide (AMP) group, which includes destabilase I, destabilase II and hirudomacin (Ding et al, 2019); members of this group can inhibit microbial growth
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