Robert J. Peanasky

The molecular structure of the complex of Ascaris chymotrypsin/elastase inhibitor with porcine elastase

BACKGROUND The intestinal parasitic worm, Ascaris suum, produces a variety of protein inhibitors that defend the organism against the hosts proteinases. Eight different proteins from Ascaris suum have been identified as inhibitors of serine proteinases, targeting chymotrypsin, elastase and trypsin. These inhibitors share 30-40% sequence identity with one another, but have virtually no sequence identity with members of any of the other families of serine proteinase inhibitors. RESULTS The crystal structure of the complex of porcine pancreatic elastase with a chymotrypsin/elastase inhibitor from Ascaris suum (the C/E-1 inhibitor) has been solved to 2.4 A resolution by the molecular replacement method. The C/E-1 inhibitor exhibits a novel folding motif. There are only two small beta-sheets and two single-turn 3(10)-helices in this inhibitor. Unlike the majority of proteins, the C/E-1 inhibitor does not have a hydrophobic core. The presence and unique topography of the five disulfide bridges suggests that they play important roles in maintaining the tertiary structure of the inhibitor. In addition, the side chains of several charged residues from electrostatic and hydrogen-bonding cascades, which also probably compensate for the lack of extensive secondary structures and a hydrophobic core. The reactive-site loop of this inhibitor displays a conformation that is characteristic of most serine proteinase inhibitors. CONCLUSIONS The structure of the C/E-1 inhibitor confirms that inhibitors from Ascaris suum belong to a novel family of proteinase inhibitors. It also provides conclusive evidence for the correct disulfide bridge connections. The C/E-1 inhibitor probably acts by a common inhibitory mechanism proposed for other substrate-like protein inhibitors of serine proteinases. The unusual molecular scaffolding presents a challenge to current folding algorithms. Proteins like the C/E-1 inhibitor may provide a valuable model system to study how the primary sequence of a protein dictates its three-dimensional structure.

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3.

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1.75 Å and 2.45 Å resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel β-sheet flanked by an α-helix. In the enzyme–inhibitor complex, the N-terminal β-strand of PI-3 pairs with one strand of the ‘active site flap’ (residues 70–82) of pepsin, thus forming an eight-stranded β-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1′–S3′). The molecular structure of the pepsin–PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: The primary structure☆☆☆

The complete primary structure of five chymotrypsin/elastase isoinhibitors isolated from Ascaris lumbricoides was determined by conventional methods. These structures represent the first sequence set for the Ascaris inhibitor family. All five isoinhibitors are single-chain polypeptides crosslinked by five disulfide bridges. Isoinhibitor 1 consists of 63 amino acid residues and has glycine at the N-terminal and histidine at the C-terminal. Isoinhibitors 2-5 all have arginine at the N-terminal, differ at positions 25 and 40, and have different C-terminal regions. Isoinhibitors 2 and 4 have asparagine at positions 25 and serine at position 40, whereas isoinhibitors 3 and 5 have lysine and threonine at these positions, respectively. The different C-terminal regions of isoinhibitors 2-5 account for their varying lengths. Isoinhibitor 1 has no sequence heterogeneity. Frequent repetitions of various dipeptides and one tripeptide are evident along the peptide chain of isoinhibitors 2-5. None of the isoinhibitors contains the aromatic amino acids phenylalanine or tyrosine. Comparison of the amino acid sequence of isoinhibitor 1 with the sequence of isoinhibitors 2-5 shows that they differ at a minimum of 16 positions. The primary structures of isoinhibitors 1-5 from Ascaris do not demonstrate a great degree of homology when compared with the sequence of presently known proteinase inhibitors. However, these isoinhibitors share with a very large number of inhibitor families the presence of half-cystine in the P3 position.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzymes.

Five isoinhibitors, proteins that inactivate chymotrypsin and elastase, were isolated from aqueous extracts of the intestinal parasite Ascaris lumbricoides var. suum by affinity chromatography. They were named in the order that they eluted from a CM-Sephadex C-25 column at pH 8.6 using a salt gradient. Isoinhibitor 1, first reported in this paper, is anionic on polyacrylamide gel electrophoresis at pH 9.3. The other four isoinhibitors are cationic on electrophoresis at pH 9.3, separable from each other, and identical with those reported previously [R.J. Peanasky and G. M. Abu-Erreish (1971) in Proceedings International Research Conference on Proteinase Inhibitors (Fritz, H., and Tschesche, H., eds.), pp. 281-293, de Gruyter, New York]. Amino acid compositions show differences between the isoinhibitors. Antibody to isoinhibitor 1 reacts with its self-antigen only. Antibody to isoinhibitor 5 reacts with isoinhibitors 2-5 but not with isoinhibitor 1. Association equilibrium constants show that each of the isoinhibitors interacts most avidly with alpha-chymotrypsin. For isoinhibitor 1, the K alpha for alpha-chymotrypsin was 2.6 X 10(11) M-1, for porcine elastase I 1.6 X 10(10) M-1, and for Subtilisin Carlsberg 3.3 X 10(7) M-1. For isoinhibitors 2-5, the K alpha ranges were 7.1 X 10(10) to 1.3 X 10(11) M-1 for alpha-chymotrypsin, 1.0 X 10(9) to 5.6 X 10(9) M-1 for porcine elastase I, and 6.0 X 10(8) to 1.3 X 10(9) M-1 for subtilisin Carlsberg. Because of the strong affinity of these inhibitors for alpha-chymotrypsin and elastase, two proteins in the normal environment of the nematode, the name isoinhibitors of chymotrypsin/elastase is suggested for these proteins.

Ascaris suum: localization by immunochemical and fluorescent probes of host proteases and parasite proteinase inhibitors in cross-sections.

Cross-sections of muscle, intestine, and genital tract fluoresced in defined locations when live Ascaris suum adults were incubated in medium containing chymotrypsin liganded with fluorescein-5-isothiocyanate. This suggests that the protease, or portions of it, are assimilated by A. suum. A. suum chymotrypsin/elastase isoinhibitors were found in muscle sarcolemma, eggs, sperm, and intestine, and host chymotrypsin was localized in the same regions of these tissues by immunofluorescence and immunoperoxidase techniques. These experiments demonstrate that host chymotrypsin enters the parasite, that it is present in specific regions of Ascaris, and that it probably exists as an enzyme-inhibitor complex.

Ascaris suum: Are trypsin inhibitors involved in species specificity of Ascarid nematodes?

Inhibitors of porcine trypsin were prepared from aqueous extracts of the parasitic nematodes Ascaris suum (hogs) and Ascaris lumbricoides (human). In this study three experiments were performed. (1) Polyclonal antibodies were prepared against one isoform of trypsin inhibitor from each parasitic nematode. Each antibody reacted with all isoforms from itself as well as all isoforms from the other parasite. (2) Association equilibrium constants were measured by titrating host trypsins (porcine or human) with the isoforms of trypsin inhibitors from A. suum and A. lumbricoides. While three of the combinations formed tight complexes that can be precipitated, the fourth complex, A. suum trypsin inhibitor-human trypsin has a Ka that is a 300 to 1000 times weaker interaction than the three other titration pairs. (3) Live A. suum worms were incubated in isosmotic media that contained either porcine trypsin or human trypsin. A suum worms survived in porcine trypsin and in the controls but were killed and digested after exposure for 5 days in human trypsin. The first experiment suggests that the trypsin inhibitors from A. suum and A. lumbricoides have similar epitopes, while the second experiment suggests that there are differences near the reactive site of the inhibitors. The consequences of these differences are dramatically demonstrated by the third experiment in which live A. suum worms in the presence of human trypsin die and are digested but those in porcine trypsin survive. These experiments suggest that in order to parasitize a host, a nematode requires a complement of protease inhibitors that interact strongly with those host proteases that are in their environment.

Isolation of the trypsin inhibitors in Ascaris lumbricoides var. suum using affinity chromatography

A method for isolating three water-soluble trypsin inhibitors from Ascaris lumbricoides var. suum by affinity chromatography is described. The trypsin inhibitors captured by affinity chromatography are resolved into three species by chromatography on CM-Sephadex at pH 8.1. The inhibitors are named in the order that they are released from the CM-Sephadex column. Ascaris Trypsin Inhibitor 1 is the same as inhibitor CM-1 described by P. Portman and W. Fraefel (1967, Helv. Chim. Acta50, 2078–2086) and inhibitor Peak I of U. Kucich and R. J. Peanasky (1970, Biochim. Biophys. Acta200, 47–57). Ascaris Trypsin Inhibitor 2 is the inhibitor described by J. Pudles, R. H. Rola, and A. K. Matida (1967, Arch. Biochem. Biophys.120, 594–601) and inhibitor CM-2 of Portman and Fraefel (1967). Ascaris Trypsin Inhibitor 3 is the same as inhibitor Peak II of Kucich and Peanasky (1970). Ascaris Trypsin Inhibitor 1 is 80%, Ascaris Trypsin Inhibitor 2 is 8%, and Ascaris Trypsin Inhibitor 3 is 12% of the water-soluble trypsin inhibitors present in Ascaris. With this procedure all of the Ascaris trypsin inhibitors can be isolated in a few days. This shortens the exposure of personnel to crude extracts of Ascaris and diminishes the biological hazard of working with them. Frequent exposure to Ascaris extracts may evoke an anaphylactic response in personnel.

Carboxypeptidase inhibitors from Ascaris suum: the primary structure

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT10DCTNGEKCVQ20KNKICSTIVE30IQRCEKEHFT40IPCKSNNDCQ50VWAHEKICN K60LPWGL65 . The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a 9-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition.

Trypsin inhibitors from Ascaris lumbricoides var. Suis

Abstract The body walls of Ascaris lumbricoides contain two trypsin inhibiting fractions which differ with respect to Ki. The Ki values are 90 nM for Peak I and 13 nM for Peak II inhibitors. In the presence of 10 mM p- toluenesulfonyl- l -arginine methyl ester, the complex formed between Peak I inhibitor and pork trypsin is dissociated to the extent of 65–75%; the complex with the Peak II inhibitor dissociates to the extent of 19–22%. Peak I inhibitor was obtained in a highly purified form. It chromatographs as a symmetrical peak on an Amberlite IRC-50 column with constant specific activity across the peak. It is homogeneous in the ultracentrifuge with an s20, w value of 1.00 S. It has a molecular weight of 5520, and is composed of 50 amino acids. Leucine, histidine, methionine and tyrosine are absent. Spectral analysis confirms the presence of one equivalent of tryptophan, two of phenylalanine and four of cystine.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: The reactive site☆☆☆

Five isoinhibitors of chymotrypsin/elastase present in aqueous extracts of Ascaris were isolated. The reactive site in each isoinhibitor, the peptide bond that during encounter is positioned over the catalytic site in chymotrypsin, is Leu-Met. This bond was hydrolyzed by incubating intact isoinhibitors with 5-25 mol% chymotrypsin at pH 3.2 for 4-6 days (isoinhibitor 1) or 2.5-5 weeks (isoinhibitors 2-5). The reaction under these conditions did not proceed beyond 60% modified isoinhibitor (peptide bond hydrolyzed) and 40% intact inhibitor. The Leu-Met bond, hydrolyzed in modified isoinhibitor, can be resynthesized at pH 7.6 by incubating modified inhibitor with a stoichiometric amount of chymotrypsin bound to Sepharose CL-4B and then dissociating the complex in a kinetically controlled fashion with 5% trichloroacetic acid. The product, intact inhibitor, was obtained in greater than 80% yield. The site in the isoinhibitor that is positioned over the catalytic site in elastase during encounter is the same as for encounter with chymotrypsin. The Leu-Met bond hydrolyzed during encounter with elastase can be resynthesized by chymotrypsin. Chymotrypsin and elastase bind to the inhibitor at the same site.