title: Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum.
abstract: 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 amino acids in length) 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 (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). 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 10(9) and 6.4 x 10(9) M(-1), respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (Ka = 1.6 x 10(9) M(-1)); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 10(6) 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 10(10) M(-1)) and CHYM (Ka = 3.6 x 10(12) M(-1)). We also found that the determined reactive sites (P1-P'1) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P'1 residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P'3 residues in the inhibitors. Elastase, which recognizes both alanine and leucine, canaccommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.
authors.affiliation: Department of Pathology, University of California, V.A. Medical Center, San Francisco, USA.
authors.forename: T T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: M A
authors.initials: MA
authors.lastname: Qasim
authors.forename: S
authors.initials: S
authors.lastname: Morris
authors.forename: C C
authors.initials: CC
authors.lastname: Lu
authors.forename: D
authors.initials: D
authors.lastname: Hill
authors.forename: M
authors.initials: M
authors.lastname: Laskowski
authors.forename: J A
authors.initials: JA
authors.lastname: Sakanari
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017163:Anisakis / Q000235:genetics / Q000378:metabolism*; D017165:Ascaris suum / Q000235:genetics / Q000378:metabolism*; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D018509:DNA, Helminth / Q000235:genetics; D005399:Fishes / Q000469:parasitology; D006790:Host-Parasite Interactions; D006801:Humans; D019476:Insect Proteins; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C401865:ASPI-1 protein, Anisakis simplex; C401866:ASPI-2 protein, Ascaris suum; D018509:DNA, Helminth; D019476:Insect Proteins; D007527:Isoenzymes; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D002918:Chymotrypsin; D010196:Pancreatic Elastase