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R J Peanasky

Publications and source records attributed to R J Peanasky.

At least 19 recordsLinked to original sources
PubMed2000

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 A and 2.45 A 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 beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-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.

Amino Acid Sequence↗
PubMed1994

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 host's 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.

Amino Acid Sequence↗
PubMed1994

Proteinase inhibitors in Ascarida.

Ascaris suum and A. lumbricoides are intestinal parasites that survive in a hostile hydrolytic environment They contain low-molecular-weight proteins that can inactivate most of the proteinases present in the worm's surroundings. Jeffrey Hawley, Mark Martzen and Robert Peanasky suggest that host specificity of these nematodes may be associated with the efficiency of their inhibitors.

Journal Article↗
PubMed1993

The serine protease inhibitor family from Ascaris suum: chemical determination of the five disulfide bridges.

Chymotrypsin/elastase inhibitor-1 is a member of the Ascaris family of serine protease inhibitors. It is characterized by five disulfide bridges in a polypeptide chain of 63 amino acids. The disulfide bridge pairing was resolved by cleavage at methionyl residues with cyanogen bromide followed by a combination of proteolytic digestions with glycyl endopeptidase, Staphylococcal serine proteinase, and submandibular proteinase A. The peptides were separated on a reverse-phase HPLC column. Amino acid analyses and N-terminal microsequencing of the cystine containing peptides revealed the disulfide bridge pairing between residues 5-54, 15-29, 18-38, 22-33, and 40-60. The disulfide bridge pairing of other members of this unique family was also assigned. The major isoform, trypsin inhibitor-1, and chymotrypsin/elastase inhibitor-4 share the same disulfide bridge pattern. These results strongly suggest that all members of the Ascaris family of serine protease inhibitors have the same disulfide bridge pattern which represents a unique motif.

Amino Acid Sequence↗
PubMed1992

Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E.

Proteolytic degradation (processing) of antigen by antigen-presenting cells is a major regulatory step in the activation of a T lymphocyte immune response. However, the enzymes responsible for antigen processing remain largely undefined. In this study we show that cathepsin E, and not the ubiquitous lysosomal cathepsin D, is the major aspartic proteinase in a murine antigen-presenting cell line, A20. This enzyme is localized to a non-lysosomal compartment of the endosomal system in these cells. Functional studies using a highly specific inhibitor of cathepsin E show that this enzyme is essential for the processing of ovalbumin by this cell line. Thus, cathepsin E, whose function was hitherto unknown, may play a major role in antigen processing.

Animals↗
PubMed1992

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.

Animals↗
PubMed1990

Primary structure of the major pepsin inhibitor from the intestinal parasitic nematode Ascaris suum.

The major pepsin inhibitor from Ascaris suum was isolated by affinity chromatography and chromatofocusing. Its amino acid sequence was determined by automated Edman degradation of peptide fragments. Peptides were produced by chemical and enzymatic cleavage of pyridylethylated protein and were purified by reverse-phase high-performance liquid chromatography. The inhibitor consists of 149 residues with the following sequence: QFLFSMSTGP10FICTVKDNQV20FVANLPWTML30EGDDIQVGKE40 FAARVEDCTN50VKHDMAPTCT60KPPPFCGPQD70MKMFNFVGCS80VLGNKLFIDQ90KYVRDLTAK D100 HAEVQTFREK110IAAFEEQQEN120QPPSSGMPHG130AVPAGGLSPP140PPPSFCTVQ149. It has a molecular weight of 16,396. All cysteines are engaged as disulfide bonds: Cys(13)-Cys(59), Cys(48)-Cys(66), and Cys(79)-Cys(146). The protein is probably composed of two domains connected by a short hydrophobic region. This is the first aspartyl protease inhibitor of animal origin that has been sequenced. The sequence has no significant homology with any other known protein.

Amino Acid Sequence↗
PubMed1990

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors.

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1986

Ascaris suum: immunoperoxidase and fluorescent probe analysis of host proteases and parasite proteinase inhibitors in developing eggs and second stage larvae.

Live Ascaris suum females were incubated in medium containing chymotrypsin liganded to fluorescein-5-isothiocyanate, and eggs in the parasite's genital tract took up the probe and fluoresced. Eggs passed by these worms into the medium containing fluorescent probe retained their fluorescence in formaldehyde-saline and by 65 days had developed into second stage infective larvae. Eggs passed naturally by untreated worms were incubated in media containing fluorescent probes and all of the eggs exposed to chymotrypsin liganded to fluorescein-5-isothiocyanate were extensively labeled. Control eggs were labeled sporadically and less intensely, indicating specificity in the uptake of environmental proteins. Chymotrypsin from the parasite's environment can bind to A. suum eggs, and this occurs both inside the worm's genital tract and outside of the parasite. Immunoperoxidase studies showed that IgG developed against chymotrypsin or against A. suum chymotrypsin/elastase isoinhibitors A or C, binds to antigens in cross sections of second stage larvae and their egg shell coats. This suggests that host chymotrypsin is retained during development and may be complexed to A. suum isoinhibitors A and C.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Ascaris suum: biosynthesis and isoinhibitor profile of chymotrypsin/elastase isoinhibitors.

Chymotrypsin/elastase isoinhibitors were radiolabeled when live, adult Ascaris suum were incubated in tissue culture medium (NCTC-135) supplemented with L-[35S]cysteine. This is the first demonstration that the synthesis of these proteins occurs in A. suum; the isoinhibitors are not host products utilized by the parasite against its host. Of five chymotrypsin/elastase isoinhibitors demonstrable in A. suum, only isoinhibitors 1, 4, and 5 were found in each worm. The amino acid sequences of these three isoinhibitors indicate that they are gene products and are not obtained by modification after translation. The two inhibitors that are not observed could arise by limited proteolysis. The same chymotrypsin/elastase isoinhibitor profile present in each nematode eliminates any speculation that the multiple forms arise from an adaptation between A. suum and its host. A new chymotrypsin/elastase isoinhibitor nomenclature is proposed, so that isoinhibitor 1 is now Isoinhibitor A, and isoinhibitors 4 and 5 are now Isoinhibitors B and C, respectively.

Animals↗
PubMed1984

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.

Amino Acids↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Amino Acid Sequence↗
PubMed1983

Trypsin inhibitors from Ascaris: the reactive P1 site of the inhibitors (a correction) and location of the inhibitors and host trypsin in cross-sections of Ascaris.

Ascaris trypsin inhibitors 1, 2, and 3 have arginine at their reactive P1 site. This corrects an earlier report that lysine is the reactive P1 site residue in Ascaris trypsin inhibitor 1 (Peanasky et al., 1974, Bayer Symposium V: Proteinase Inhibitors, pp. 649-666). The present work illustrates that the residue modification method of Fritz et al. (1969, Z. Physiol. Chem., 350, 933-944) may not be reliably interpreted when trypsin inhibitors have an unusually high lysine content (greater than 12% of the molecular weight of the inhibitor). Thus the following procedure is recommended: treat the inhibitor with maleic anhydride first and second with butanedione reagent; then remove the maleyl groups in an acid environment and determine the activity of the inhibitor. Immunoperoxidase staining shows that antibody to Ascaris trypsin inhibitor 1 binds to body wall muscle, intestine, eggs and sperm in cross-sections of Ascaris. Antibody to TLCK-porcine trypsin binds to the same tissues and at the same sites as the antibody to Ascaris trypsin inhibitor 1. This is the first demonstration that a protein that originated in the host has been found in the parasite, Ascaris. Analyses of homogenates and of extracts of separated tissues always show an excess of free trypsin inhibitor and no evidence of active trypsin. The host protein is present inside the parasite, probably as the trypsin-inhibitor complex.

Animals↗