Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia
Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia
复制标题
日本水蛭唾液腺中新型凝血酶抑制剂水蛭素-HN 的鉴定和表征
DOI：
10.7717/peerj.7716
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发表时间：
2019-09-30
期刊：
影响因子：
2.7
通讯作者：
Xu, Chengfeng
中科院分区：
文献类型：
--
作者：
Cheng, Boxing;Liu, Fei;Xu, Chengfeng
利用BGIseq-500平台获得吸血水蛭日本水蛭唾液腺转录组测序数据（6.5 Gb）。经鉴定分析，1个转录本（Unigene5370）标记为水蛭素HV3， e值为1e-29，命名为水蛭素hn。该转录本是一个新的凝血酶抑制剂基因，属于蛋白酶抑制剂I14（水蛭素）家族。水蛭素- hn全长270 bp，编码含有20-aa信号肽的89-aa蛋白。成熟的水蛭素- hn蛋白具有水蛭素的典型结构特征，如三个保守的二硫键和PKP和DFxxIP基序。通过原核表达获得水蛭素- hn蛋白（Hir和M-Hir），通过显色底物S2238和表面等离子体共振（SPR）相互作用分析，分别证明水蛭素- hn成熟蛋白具有抗凝血活性和凝血酶亲和力。通过比较Hir和M-Hir的活性和凝血酶亲和力，表明成熟水蛭素- hn蛋白的n端结构对抗凝血活性很重要。水蛭素- hn mRNA和蛋白的丰度在饥饿动物的唾液腺中高于喂食和喂食水蛭的唾液腺。这些结果为进一步研究水蛭素- hn与凝血酶的结构-功能关系奠定了基础。
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.