Purification and Characterization of a Novel Kazal-Type Trypsin Inhibitor from the Leech of Hirudinaria manillensis

Abstract

Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of “non-classical” Kazal inhibitors according to its CysI-CysII disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F) XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (Ki) of (8.12 ± 0.18) × 10−9 M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin.

From structure to function – a family portrait of plant subtilases

“…The Kazal family consists of small (5 kDa) serine protease inhibitors classified as typical or atypical, based on the number of disulfide bonds. While typical Kazal inhibitors also inhibit other serine proteases, mainly trypsinlike enzymes (Morris et al, 2002;Lai et al, 2016;Pariani et al, 2016), atypical Kazals are more specific for SBTs . Kazal-like inhibitors secreted by the apicomplexan parasite Toxoplasma gondii inhibit host-derived SBTs, possibly contributing to colonization (Morris et al, 2002;Pszenny et al, 2002;Morris & Carruthers, 2003).…”

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

“…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-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).…”

“…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).…”

“…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 .…”