A KAZAL-TYPE INHIBITOR OF HUMAN MAST-CELL TRYPTASE - ISOLATION FROM THE MEDICAL LEECH HIRUDO-MEDICINALIS, CHARACTERIZATION, AND SEQUENCE-ANALYSIS

Human tryptase, a tetrameric proteinase expressed by mast cells, is vi rtually unique among the serine proteinases as it is not inhibited by any proteinaceous inhibitor tested so far. We have now isolated, seque nced, and characterized an inhibitor of human tryptase from the medica l leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) wa s purified to apparent homogeneity by cation exchange and affinity chr omatography. Amino acid sequencing of the protein consisting of 46 res idues (M(r) 4738) revealed a high degree of similarity to the non-clas sical Kazal-type inhibitors bdellin B-3 and rhodniin, inhibitors isola ted from the medical leech and the insect Rhodnius prolixus, respectiv ely. LDTI is a tight-binding and relatively specific inhibitor of huma n tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (E C 3.4.21.1) with similar affinities. Inhibition studies using small ch romogenic substrates revealed that LDTI inhibits the amidolytic activi ty of tryptase by similar to 50%, suggesting that most likely due to s teric hindrance LDTI binds to and inhibits only 2 of 4 active sites of tryptase. LDTI appears useful as a prototype of inhibitors of human t ryptase and as a pharmacological tool for the investigation of the rol e of tryptase in health and disease.