Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis.

Ruo-Mei Cheng,James Mwangi,Rui-Pu Sun,Xiao-Peng Tang,Ren Lai,Zhen-Qing Zhang,Cheng-Bo Long,Ai-Lin Long

doi:10.24272/j.issn.2095-8137.2019.037

Ruo-Mei Cheng, James Mwangi + Show 6 more

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https://doi.org/10.24272/j.issn.2095-8137.2019.037

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Abstract

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor (bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4x104, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at the C-terminus. cDNA cloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame (ORF). Sequence alignment showed that bdellin-HM-2 shared similarity with the “non-classical” Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa (FXIIa), factor XIa (FXIa), factor Xa (FXa), thrombin, or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.

Highlights

Protease inhibitors occur naturally in living organisms, including animals (Shadrin et al, 2015; Vicuna et al, 2015; Wang et al, 2005; Zhang, 2006), plants (Kim et al, 2009; Ryan, 1990), fungi (Sabotic & Kos, 2012), and bacteria (Supuran et al, 2002)
Purification of bdellin-HM-2 The crude extracts from H. manillensis were resolved into several fractions by DEAE Sephadex A-50 column
The cDNA had an open reading frame (ORF) of 396 nucleotides coding a pro-protein of 132 amino acids, including a signal peptide of 18 residues and mature bdellin-HM-2 of 114 residues (Figure 2A)

Summary

Introduction

Protease inhibitors occur naturally in living organisms, including animals (Shadrin et al, 2015; Vicuna et al, 2015; Wang et al, 2005; Zhang, 2006), plants (Kim et al, 2009; Ryan, 1990), fungi (Sabotic & Kos, 2012), and bacteria (Supuran et al, 2002). They have multifunctional roles in many physiological processes and play an important role in biological functions of venomous animals, such as in predation (Birrell et al, 2007) and defense (Ali et al, 2002). The classical Kazal domain has two residues between cys and cys, whereas the non-classical Kazal inhibitor has a spacer region between cys and cys, ranging from three to seven residues

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