Amino-acid-sequence determination and biological activity of tessulin, a naturally occurring trypsin-chymotrypsin inhibitor isolated from the leech Theromyzon tessulatum

We purified a new trypsin-chymotrypsin inhibitor, designated tessulin, from the rhynchobdellid leech Theromyzon tessulatum. This 9-kDa peptide was pur ified to apparent homogeneity by gel-permeation and anion-exchange chromato graphies followed by reverse-phase HPLC. The structure of tessulin was dete rmined by reduction, S-beta-pyridylethylation, trypsin digestion, automated Edman degradation and matrix-assisted laser desorption mass spectrometry ( m/z 8985 Da). The 81-amino-acid peptide possesses 16 cysteines and exhibits a 16% sequence similarity with antistain-type inhibitors. Tessulin inhibit s trypsin (K-i 1 pM) and chymotrypsin (K-i 150 pM) and exhibits no activity with thrombin, factor Xa, cathepsin G and elastase. This is the first tryp sin-chymotrypsin inhibitor isolated from leeches that does not inhibit elas tase or cathepsin G, except for cytin and therin. Furthermore, tessulin, in conjunction with other serine-protease inhibitors isolated from Theromyzon (therin, theromin), significantly diminishes the level of human granulocyt e and monocyte activation induced by lipopolysaccharides (10 mu g) The comb ined level of inhibition is higher than that of aprotinin, another serine-p rotease inhibitor used biomedically. Thus, tessulin may be clinically signi ficant in reducing inflammatory events.