A Kazal-Type Inhibitor of Human Mast Cell Tryptase: Isolation from the Medical LeechHirudo medicinalis, Characterization, and Sequence Analysis

Abstract

Human tryptase, a tetrameric proteinase expressed by mast cells, is virtually unique among the serine proteinases as it is not inhibited by any proteinaceous inhibitor tested so far. We have now isolated, sequenced, and characterized an inhibitor of human tryptase from the medical leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) was purified to apparent homogeneity by cation exchange and affinity chromatography. Amino acid sequencing of the protein consisting of 46 residues (M(r) 4738) revealed a high degree of similarity to the non-classical Kazal-type inhibitors bdellin B-3 and rhodniin, inhibitors isolated from the medical leech and the insect Rhodnius prolixus, respectively. LDTI is a tight-binding and relatively specific inhibitor of human tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with similar affinities. Inhibition studies using small chromogenic substrates revealed that LDTI inhibits the amidolytic activity of tryptase by approximately 50%, suggesting that most likely due to steric hindrance LDTI binds to and inhibits only 2 of 4 active sites of tryptase. LDTI appears useful as a prototype of inhibitors of human tryptase and as a pharmacological tool for the investigation of the role of tryptase in health and disease.

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

“…The culture conditions for rLDTI secretion by strain H449 in shake flasks were adapted to 5 1 in a laboratory fermenter and the cell supernatant was used for purification and characterization of the expressed polypeptide(s). The isolation procedure for rLDTI is based on the basic nature of the leech protein for which an isoelectric point of > 10 was suggested by Sommerhoff et al (1994). Thus, rLDTI could be bound under rather selective conditions to a cation-exchange resin directly from the crude yeast cell supernatant.…”

“…A synthetic gene encoding LDTI in preferred yeast codon usage (Bennetzen and Hall, 1982) was assembled from three synthetic oligonucleotides in a PCR reaction. The coding sequence was deduced from the published amino acid sequence of the 46-amino-acid species of LDTI purified from the leech H. medicinalis as described in Sommerhoff et al (1994). In addition, the gene was extended at its 5' end by a Kex2p cleavage site and a BglII site to provide for in-frame fusion to the a-factor leader in plasmid pFBY166.…”

“…Equilibrium dissociation constants (K,) for the complexes of leech LDTI and rLDTI with human lung tryptase, bovine pancreatic trypsin and chymotrypsin were determined essentially as described in Sommerhoff et al (1994), using the strategies suggested by Bieth (1980) and the equation for tight-binding inhibitors (Morrison, 1969).…”

“…A molecular mass of 4737.6 Da was determined by ESI-MS, which is identical to the theoretical mass of full-length LDTI assuming three disulfide bridges. An isoelectric point of 10.0 was measured by Sommerhoff et al (1994). In case of human mast cell tryptase, the K, vaIue is caIculated for the interaction of LDTI with two of four active sites of the tryptase tetramer.…”

“…LDTI shows ~4 5 % sequence similarity to the non-classical Kazal-type inhibitors bdellin B3 (Fink et al, 1986;Friedrich et al, 1993). The binding loop responsible for protease inhibition is proposed to be Cys6-Lysl1, with Lys8 as the primary determinant of inhibitor specificity (Miihlhahn et al, 1994;Sommerhoff et al, 1994). The identity of the purified leech protein as a tryptase inhibitor was verified expressing a synthetic gene based on the amino acid sequence of LDTI in yeast, yielding biologically active protein .…”