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

Huang, Kangning; Strynadka, N.C.J.; Bernard, Vincent; Peanasky, Robert J.; James, Michael N.G.

doi:10.1016/s0969-2126(00)00068-x

Cited by 75 publications

(65 citation statements)

References 60 publications

(77 reference statements)

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“…Here we present our analysis of one of the two main components of this fraction. As we have shown, this is a trypsin inhibitor related to the protease inhibitors from Ascaris (Grasberger et al, 1994;Huang et al, 1994).…”

mentioning

confidence: 76%

“…The scissile peptide bond in these inhibitors is between residues 31 and 32. These are in the sequence Cys-ProLeu/Met-Cys in the chymotrypsin/elastase and Cys-Thr-Arg/ Glu-Cys in the trypsin inhibitor (Peanasky et al, 1984;Grasberger et al, 1994;Huang et al, 1994). At the homologous positions of the BSTI peptide, the sequence Cys-Asp-Lys-Lys-Cys is present, which in fact suggested that this acts as an inhibitor of trypsin-like enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…The 3D structure for the Ascaris trypsin inhibitor was determined by NMR spectroscopy (Grasberger et al, 1994); moreover, the molecular structure of the complex of porcine elastase with a chymotrypsin/elastase inhibitor from A . suum has been solved to 2.4 A by X-ray crystallography (Huang et al, 1994). The sequence and pattern similarities detected with the databank searches suggested that the BSTI sequence could be modeled on the template of the A .…”

Section: Isolation and Amino Acid Sequence Of Bstimentioning

confidence: 99%

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BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes

et al. 1996

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From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascarissuum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 pM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor. With few exceptions, these were rather small molecules containing less than 30 amino acids. As for their biological function, two main groups can be discerned. One comprises a multitude of peptides interacting with specific receptors present in the nervous system, the gastrointestinal tract and other organs of mammals. Many of these peptides are in fact similar or even identical to hormones and neurotransmitters. The second group encompasses antimicrobial peptides, at least some of which exert their function through direct binding to the phospholipid bilayer of cell membranes.Recently, a new family of peptides, the xenoxins, was isolated and characterized from skin secretions of Xenopus laevis (Kolbe et al., 1993). These 66-amino-acid peptides are related to neurotoxins and cytotoxins from snake venoms, but they are apparently devoid of toxic activity. In the course of our analysis of the skin secretion of Bombina bombina, a main fraction containing peptides with an apparent molecular mass of 8-10 kDa was isolated. In view of the earlier findings on xenoxins, we Reprint requests to: G. Kreil, Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria; e-mail: gkreil@oeaw.ac.at.wanted to test whether similar constituents were also present in skin secretion of this species. Here we present our analysis of one of the two main components of this fraction. As we have shown, this is a trypsin inhibitor related to the protease inhibitors from Ascaris (Grasberger et al., 1994; Huang et al., 1994).The peptide has been termed Bombina skin trypsin inhibitor, abbreviated BSTI. Results Isolation and amino acid sequence of BSTISkin secretions from B. bombina were collected and extracted with n-butanol. The aqueous phase was chromatographed over ConA-Sepharose to remove glycoproteins, proteoglycans, mucins etc. The eluate was further fractionated by molecular sieve chromatography (see the Materials and methods). One of the main peaks contained polypeptides with an apparent molecular weight of 8-10 kDa, as shown by SDS-PAGE. Separation of this fraction by reverse-phase HPLC yielded three peaks (see Fig. 1). Of these, the main constituent was investigated f...

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confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Isolation and Amino Acid Sequence Of Bstimentioning

confidence: 99%

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BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes

et al. 1996

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“…comm.). Such a discrepancy was recently reported for the Ascaris chymotrypsin/elastase inhibitor, for which disulfide bridges found both by NMR and X-ray agree but disagree with biochemical studies (Grasberger et al, 1994;Huang et al. 1994).…”

Section: Discussionmentioning

confidence: 58%

Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase

Egloff

Sarda

Verger³

et al. 1995

Protein Science

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Colipase ( M , 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt Colipase lacks well-defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger-like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C-terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.

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“…By using RT-PCR, we successfully amplified a cDNA whose translated amino acid sequence showed limited homology to the anticoagulants from A. caninum, AcAP5 and AcAPc2. The 40 -44% amino acid sequence identity (52-55% similarity) to the two AcAP sequences, in addition to the conservation and alignment of the 10 cysteine residues, which are known to play a critical role in defining the tertiary molecular structure of the Ascaris type inhibitors (9,33,34), suggests that AceAP1 is, in fact, a member of this family of nematode proteins. In addition to those from Ancylostoma hookworms, Ascaris-type inhibitors have also been identified other nematode species, including A. suum (7,8), Anisakis simplex (35), and Trichuris muris (36).…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

Harrison

Nerlinger

Bungiro

et al. 2002

Journal of Biological Chemistry

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Bloodfeeding hookworms, which currently infect over a billion people in the developing world, are a leading cause of gastrointestinal hemorrhage and iron deficiency anemia. The major anticoagulant inhibitor of coagulation factor Xa has been identified from the hookworm parasite Ancylostoma ceylanicum using reverse transcription PCR and 3-rapid amplification of cDNA ends. This is the first anticoagulant cloned from a hookworm species for which humans are recognized permissive hosts. Despite ϳ50% amino acid similarity, A. ceylanicum anticoagulant peptide 1 (AceAP1) is both immunologically and mechanistically distinct from AcAP5, its homologue isolated from the dog hookworm Ancylostoma caninum. Studies using plasma clotting times and single stage chromogenic assays of factor Xa activity have demonstrated that the recombinant AceAP1 protein is substantially less potent than AcAP5 and that soluble whole worm protein extracts of adult A. ceylanicum possess less anticoagulant activity than extracts of A. caninum. These values correlate with previously reported differences in bloodfeeding capabilities between these two species of hookworm, suggesting that factor Xa inhibitory activity is predictive of hookworm bloodfeeding capabilities in vivo. These fundamental differences in the mechanism of action and immunoreactivity of the major anticoagulant virulence factors from related Ancylostoma hookworm species may have significant implications for human vaccine development.

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

Cited by 75 publications

References 60 publications

BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes

BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes

Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase

Molecular Characterization of Ancylostoma Inhibitors of Coagulation Factor Xa

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