Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum

Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Ani sakis simplex, have been isolated and characterized. Because these inhibito rs are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (A sC/E-1), we suggest that these Anisakis elastase inhibitors belong to the s ame unique class of canonical inhibitors formed by the family of Ascaris in hibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Struct ure 1994,2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhi bitors in Pichia pastoris and found that: (1) the association constant of r ASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibito r (K-a = 4.5 x 10(9) and 6.4 x 10(9) M-1, respectively); (2) rASPI-1 is a p otent inhibitor of PPE and human leukocyte elastase (K-a = 1.6 x 109 M-1); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (K-a = 1.2 x 10(6) M-1). In contrast to the Anisakis inhibitor, however, rAsC/E inhibi tor-1 is a very strong inhibitor of both PPE (K-a = 3.5 x 10(10) M-1) and C HYM (K-a = 3.6 x 10(12) M-1). We also found that the determined reactive si tes (P-1-P-1') of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Me t 29 and Leu 31-Met 32, respectively. These P-1-P-1' residues of AsC/E-1 co nstitute the same reactive site as that also recognized by CHYM (Peanasky R J, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;23 2:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward th eir cognate serine proteases may be attributed to the P-1 and P-3' residues in the inhibitors. Elastase, which recognizes both alanine and leucine, ca n accommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1. ( C) 1999 Elsevier Science B.V. All rights reserved.