Submitted to: Molecular and Biochemical Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 22, 1999
Publication Date: N/A
Interpretive Summary: The overall purpose of our study was to investigate the role of the serine protease inhibitors in the host-parasite relationship of Anisakis simplex. To initiate our investigations, we purified and characterized two major forms of elastase isoinhibitors: the Anisakis serine protease inhibitors, ASPI-1 and ASPI-2 and compared them to the Ascaris chymotrypsin/elastase inhibitor-1 (AsC/E-1).We found that although both ascarid inhibitors share high amino acid sequence homology, the Anisakis inhibitor specifically inhibits elastase and only weakly inhibits chymotrypsin, whereas the Ascaris inhibitor inhibits both elastase and chymotrypsin. We propose that the specificities of the ascarid inhibitors for elastase and chymotrypsin reside in their amino acid sequence around the reactive site. Recently, the crystal and NMR structures of the Ascaris chymotrypsin/elastase isoform-1 and the trypsin inhibitor, respectively, have been resolved. The molecular structures revealed that both proteins lack the typical hydrophobic core seen in other canonical protease inhibitors. Unlike other serine protease inhibitors, both Ascaris inhibitors fold into similar structures with a novel topology which consists of two small beta sheets held together by five disulfide bridges and a reactive loop which is exposed on the surface. Because these inhibitors have structures unlike any other canonical protease inhibitors, they form a novel class of small serine protease inhibitors.
Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized.Because these inhibitors are similar in size (60 a.a.) and primary sequence (52% and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors.To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that 1)the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (Ka = 4.5 x 109 M-1 and Ka = 6.4 x 109 M-1, respectively), 2)rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (HLE) (1.6 x 109 M-1), and 3) it is a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 106 M-1). In contrast to the Anisakis inhibitor however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (Ka = 3.5 x 1010 M-1) and CHYM (Ka= 3.6 x 101 M-1).We also found that the determined reactive sites (P1-P1') of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P1' residues of AsC/E-1 constitute the same reactive site that was recognized by CHYM.