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

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.

There are at least four types of protease inhibitors, including serine, cysteine, aspartic, and metalloprotease inhibitors (Leung et al., 2000). The Kazal family is one of the best-known groups of serine protease inhibitors (Rimphanitchayakit & Tassanakajon, 2010). Kazal-type inhibitory can be sorted into classical and non-classical Kazal domains. The classical Kazal domain has two residues between cys4 and cys5, whereas the non-classical Kazal inhibitor has a spacer region between cys4 and cys5, ranging from three to seven residues (Moser et al., 1998). There are highly homologous threedimensional structures in the Kazal-type serine proteinase inhibitors regardless of length of amino acid sequences between the cysteines and amino acid sequence variation (Eigenbrot et al., 2012). The P1 residue, located in the second amino acid downstream of the second conserved cysteine residue, is inserted into the S1 specificity pocket of the protease in a substrate-like way (Bode & Huber, 1992;Laskowski & Kato, 1980). Several Kazal-type serine protease inhibitors have been characterized from leeches. A few "non-classical" Kazal inhibitors have been reported from different leeches, including bdellin-B-3 (Fink et al., 1986), bdellin-KL (Kim et al., 2001), and bdellin-HM (Lai et al., 2016). In this study, bdellin-HM-2 was purified and characterized from the leech H. manillensis. To the best of our knowledge, bdellin-HM-2 is the first Kazaltype serine protease inhibitor displaying anticoagulant properties identified from H. manillensis.

Collection of crude extracts
The H. manillensis leeches were purchased from Jinbian aquafarm, Qinzhou City, Guangxi Province in China. The leeches were still alive when transported to the laboratory. We prepared the crude extracts from the leech heads as described previously (Lai et al., 2016). In short, leech heads were separated from the bodies, washed in 0.9% saline, quickly frozen, and then ground in liquid nitrogen.

Purification of bdellin-HM-2
Purification of bdellin-HM2 followed similar methods described in our previous published article (Lai et al., 2016). Briefly, crude extracts were dissolved in 50 mmol / L Tris-HCl buffer (pH 8.9) and subsequently separated by a DEAE Sephadex A-50 column (5 cm diameter, 60 cm length, GE, USA). Elution was performed at a flow rate of 15 mL/h at 4°C and 3.0 mL fractions were collected in separate tubes. The absorbance of the fractions was monitored at both 215 and 280 nm. Fractions that could prolong the activated partial thromboplastin time (aPTT) were pooled and lyophilized prior to further purification. The powder from the previous step was dissolved and loaded for reverse-phase high-performance liquid chromatography (RP-HPLC) on a C 18 column (Waters, Milford, MA, USA, 5 μm particle size, 250 mm×4.6 mm). Elution was carried out with a linear gradient of 10% -60% solution B (99.9% acetonitrile, 0.1% TFA) for 60 min at a flow rate of 1 mL / min. The eluted fraction that prolonged aPTT was collected.

Mass spectrometric analysis and peptide sequencing
The molecular weight of the collected fraction was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Autoflex speed TOF/ TOF, Bruker Daltonik GmbH, Bruker Corporation, Germany) using positive ion and linear mode, with specific operating parameters including a 20 kV ion acceleration voltage, 50time accumulation for single scanning, and 0.1% accuracy of mass determinations. The partial peptide sequence of the N-terminal was determined by automatic Edman degradation on a pulsed liquid-phase sequencer (PPSQ-31A, Shimadzu Corporation, Japan).

RNA extraction and cDNA library construction
Total RNA from the head of H. manillensis was extracted using Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions and was dissolved in RNase-Free water. A SMART ™ PCR cDNA construction kit (Clontech, Palo Alto, CA, USA) was used for synthesizing cDNA, as described previously (Lai et al., 2016).

Screening of cDNA encoding bdellin-HM-2
To screen the cDNA encoding the precursor of bdellin-HM-2, synthesized cDNA was used as the template for PCR, following previously described methods (Lai et al., 2016). Briefly, two pairs of oligonucleotide primers (Table 1) were used in PCR reactions, where primers 1 and 3 were designed according to the partial N-terminal sequence of bdellin-HM-2 and primers 2 and 4 were from the SMART ™ PCR cDNA construction kit. The PCR conditions were as described previously (Lai et al., 2016).

Purification of bdellin-HM-2
The crude extracts from H. manillensis were resolved into several fractions by DEAE Sephadex A-50 column. The fraction that prolonged the aPTT was indicated by a bar ( Figure 1A). We then obtained the purified peptide exerting an aPTT inhibitory effect, named bdellin-HM-2 (indicated by an arrow in Figure 1B), using a C 18 RP-HPLC column. MALDI-TOF-MS showed that bdellin-HM-2 had a molecular weight (MW) of 14141.5 ( Figure 1C).

Primary structure of bdellin-HM-2
Based on automatic Edman degradation, the partial Nterminal sequence of bdellin-HM-2 was determined to be ETECVCTLELKQVCGS. According to the N-terminal sequence, degenerate primers were designed (Table 1) to clone the cDNA encoding the precursor of bdellin-HM-2 from the cDNA library. A 399 bp cDNA encoding the precursor of bdellin-HM-2 was obtained. 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 (indicated by box) and mature bdellin-HM-2 of 114 residues (Figure 2A). The theoretical MW of mature bdellin-HM-2 was 13144.78, which was not consistent with the observed mass by mass spectrometry analysis ( Figure 1C). This inconsistency may be due to post-translational modification of the protein. Sequence alignment showed similarity to bdellin-KL (Kim et al., 2001), bdellin-B-3 (Fink et al., 1986), and bdellin-HM (Lai et al., 2016), which are"non-classical "Kazal serine protease inhibitors ( Figure 2B). Multiple sequence alignment showed that the six cysteine residues and threonine-tyrosine residues were highly conserved among different species ( Figure 2C). There were multiple HHXDD and HXDD motifs at the C-terminus of bdellin-HM-2.

Figure 1 Purification of bdellin-HM-2 from H. manillensis
A: Crude extracts were fractionated using DEAE Sephadex A-50 ion exchange. Fraction exerting inhibitory activity on aPTT is indicated by a bar ( -). B: Fraction exerting inhibitory activity on aPTT was further purified by C 18 reverse-phase high-performance liquid chromatography (RP-HPLC). Protein peak exerting inhibitory activity on the aPTT is marked by an arrow. C: Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of purified native bdellin-HM-2.

DISCUSSION
Several protease inhibitors exerting anticoagulant effects have been found from leeches (Hong & Kang, 1999;Markwardt, 2002;Salzet et al., 2000;Salzet, 2001;Strube et al., 1993;Tuszynski et al., 1987). In this report, a novel protease inhibitor (bdellin-HM-2) with anticoagulant effects was purified and further characterized from H. manillensis for the first time. The cDNA encoding bdellin-HM-2 precursor was cloned from the cDNA library, and the mature bdellin-HM-2 consisted of 114 amino acid residues. MALDI-TOF-MS showed that the MW of bdellin-HM-2 was 14141.5, compared to the theoretical molecular weight of 13144.78, a difference of 996.72, which is not consistent with the theoretical value. Research shows glycosylation influences the function of protein, governs physiology, and contributes to disease (Ohtsubo & Marth, 2006). We speculated that bdellin-HM-2 was O-glycosylated at Thr-20, Thr-25, Ser-34, Thr-38, and Thr-46. (Gupta & Brunak, 2002), although further research on these O-glycosylation sites should be performed in the future.
Kazal-type inhibitors with one or more Kazal domains are characterized by multiple HHXDD and HXDD motifs in their amino acid sequences (Laskowski & Kato, 1980) and by their highly homologous three-dimensional structures (Van et al., 1995). Each Kazal domain usually contains six conserved cysteine residues forming three intra-molecular disulfide  bridges (Laskowski & Kato, 1980;Magert et al., 1999). P1 residue, which contributes to the inhibitory specificity, is located at the second position after the second cysteine residue of the Kazal domain (Bode & Huber, 1992). Although bdellin-HM-2 showed high similarity to bdellin-HM and bdellin-KL by sequence analysis and belongs to the family of nonclassical Kazal domains, enzyme activity tests showed that bdellin-HM-2 had no inhibitory effects on trypsin, elastase, chymotrypsin, kallikrein, FXIIa, FXIa, FXa, thrombin, or plasmin under the assay conditions. Sequence alignment showed that the P1 residue of bdellin-HM-2 was different from bdellin-HM, bdellin-KL, and bdellin-B-3. The difference in P1 residue was considered the cause of the enzyme activity test results.
Bdellin-HM-2 prolonged the aPTT, implying that bdellin-HM-2 functioned to help H. manillensis obtain a blood meal by inhibiting blood coagulation. Results showed that the activity was dose-independent. Further work to identify the target of bdellin-HM-2 in blood is necessary. Blood-sucking animals obtain a blood meal by overcoming the host' s natural blood coagulation (De Marco et al., 2010;Dodt, 1995;Markwardt, 1996;Mende et al., 1999). The anticoagulant peptide obtained from H. manillensis not only facilitates our understanding of the mechanism of blood feeding for H. manillensis, but also provides a new candidate for the development of clinical anticoagulant drugs.
In conclusion, bdellin-HM-2 identified from H. manillensis prolonged the aPTT but exhibited no influence on PT and no inhibitory activity on trypsin, elastase, chymotrypsin, kallikrein, FXIIa, FXIa, FXa, thrombin, or plasmin under the assay conditions. Further research on O-glycosylation sites will be performed in the future. Bdellin-HM-2 is the first identified Kazal-type serine protease inhibitor from H. manillensis that shows a potent anticoagulant effect.

COMPETING INTERESTS
The authors declare that they have no competing interests.  Chinese Academy of Sciences) for help in the analysis of mass spectrometry results.