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.
Highlights
Protease inhibitors are ubiquitously distributed in living organisms such as plants, animals, fungi, and bacteria, which have the ability to target proteinase through different mechanisms resulting in either complete or partial inhibition
The crude extracts of H. manillensis were resolved into several fractions by DEAE Sephadex
The fraction with trypsin inhibitory activity is indicated by a bar (Figure 1A) and was applied to a C18 reverse-phase high-performance liquid chromatography (RP-HPLC) column for further purification
Summary
Protease inhibitors are ubiquitously distributed in living organisms such as plants, animals, fungi, and bacteria, which have the ability to target proteinase through different mechanisms resulting in either complete or partial inhibition. Typical Kazal domains have six cysteine residues forming a 1–5, 2–4, 3–6 disulfide bond pattern [14,15], while atypical Kazal domains only have two disulfide bonds [15,16,17]. When they react with proteases, the P1 residue described as residing at 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 manner [18]. The Kazal-type serine proteinase inhibitors possess highly homologous three-dimensional structures despite the differences in the length of amino acid sequences between the cysteines and amino acid sequence variation [20]
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