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Jonathan D. Walton - One of the best experts on this subject based on the ideXlab platform.

  • peptide macrocyclization catalyzed by a prolyl oligopeptidase involved in α amanitin biosynthesis
    Chemistry & Biology, 2014
    Co-Authors: Hong Luo, Michael R. Sgambelluri, Evan R. Angelos, Sung Yong Hong, Jonathan D. Walton
    Abstract:

    Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. Here, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.

  • Profiling of Amatoxins and Phallotoxins in the Genus Lepiota by Liquid Chromatography Combined with UV Absorbance and Mass Spectrometry
    MDPI AG, 2014
    Co-Authors: Michael R. Sgambelluri, Sara Epis, Davide Sassera, Hong Luo, Evan R. Angelos, Jonathan D. Walton
    Abstract:

    Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of Amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled to UV absorbance and mass spectrometry, we analyzed the spectrum of peptide toxins present in six Italian species of Lepiota, including multiple samples of three of them collected in different locations. Field taxonomic identifications were confirmed by sequencing of the internal transcribed spacer (ITS) regions. For comparison, we also analyzed specimens of Amanita phalloides from Italy and California, a specimen of A. virosa from Italy, and a laboratory-grown sample of Galerina marginata. α-Amanitin, β-amanitin, amanin, and amaninamide were detected in all samples of L. brunneoincarnata, and α-amanitin and γ-amanitin were detected in all samples of L. josserandii. Phallotoxins were not detected in either species. No Amatoxins or phallotoxins were detected in L. clypeolaria, L. cristata, L. echinacea, or L. magnispora. The Italian and California isolates of A. phalloides had similar profiles of Amatoxins and phallotoxins, although the California isolate contained more β-amanitin relative to α-amanitin. Amaninamide was detected only in A. virosa

  • Colocalization of Amanitin and a Candidate Toxin-Processing Prolyl Oligopeptidase in Amanita Basidiocarps
    Eukaryotic Cell, 2010
    Co-Authors: Heather E. Hallen-adams, John S. Scott-craig, Jonathan D. Walton
    Abstract:

    Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide Amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the “MSDIN” gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.

  • Gene family encoding the major toxins of lethal Amanita mushrooms
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Heather E. Hallen, John S. Scott-craig, Jonathan D. Walton
    Abstract:

    Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable “toxin” region capable of encoding a wide variety of peptides of 7–10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

Heinz Faulstich - One of the best experts on this subject based on the ideXlab platform.

  • molecular characterization and inhibition of amanitin uptake into human hepatocytes
    Toxicological Sciences, 2006
    Co-Authors: Katrin Letschert, Heinz Faulstich, Daniela Keller
    Abstract:

    Amatoxins are the main poison of the green death cap (Amanita phalloides) and among the most dangerous natural toxins causing hepatic failure. A possible therapeutic approach is the inhibition of the transporting systems mediating the uptake of Amatoxins into human hepatocytes, which, however, have yet to be identified. In the current study we tested whether members of the organic anion‐transporting polypeptide (OATP) family, localized in the sinusoidal membranes of human hepatocytes, are involved in amatoxin uptake. For this, Madin Darby canine kidney strain II (MDCKII) cells stably expressing human OATP1B3, OATP2B1, or OATP1B1, were assayed for the uptake of 3 H-labeled O-methyldehydroxymethyl-a-amanitin. Under our conditions, only OATP1B3 was able to transport amanitin with a Km value of 3.7mM ± 0.6mM. Accordingly, toxin uptake was inhibited by OATP1B3 substrates and inhibitors (cyclosporin A, rifampicin, the quinoline derivatives MK571 ([(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid]) and montelukast, the cholecystokinin octapeptide (CCK-8), paclitaxel, and bromosulfophthalein), as well as by some antidotes used in the past for the treatment of human amatoxin poisoning (silibinin dihemisuccinate, penicillin G, prednisolone phosphate, and antamanide). These transport studies are in line with viability assays monitoring the toxic effect of amanitin on the transfected MDCKII cells. Further support for amatoxin transport was found in primary human hepatocytes, expressing OATP1B3, OATP2B1, and OATP1B1, where CCK-8, a substrate specific for OATP1B3, prevented the fragmentation of nucleoli, a lesion typical for amanitin action. In conclusion, we have identified OATP1B3 as the human hepatic uptake transporter for Amatoxins; moreover, substrates and inhibitors of OATP1B3, among others rifampicin, may be useful for

  • Molecular Characterization and Inhibition of Amanitin Uptake
    2005
    Co-Authors: Into Human Hepatocytes, Heinz Faulstich, Katrin Letschert, Daniela Keller
    Abstract:

    Amatoxins are the main poison of the green death cap (Amanita phalloides) and among the most dangerous natural toxins causing hepatic failure. A possible therapeutic approach is the inhibition of the transporting systems mediating the uptake of Amatoxins into human hepatocytes, which, however, have yet to be identified. In the current study we tested whether members of the organic anion–transporting polypeptide (OATP) family, localized in the sinusoidal membranes of human hepatocytes, are involved in amatoxin uptake. For this, Madin Darby canine kidney strain II (MDCKII) cells stably expressing human OATP1B3, OATP2B1, or OATP1B1, were assayed for the uptake of 3H-labeled O-methyl-dehydroxymethyl-a-amanitin. Under our conditions, only OATP1B3 was able to transport amanitin with a Km value of 3.7mM ± 0.6mM. Accordingly, toxin uptake was inhibited by OATP1B3 substrates and inhibitors (cyclosporin A, rifampicin, the quinoline deriva

  • Amatoxins and phallotoxins in amanita species high performance liquid chromatographic determination
    Mycologia, 1993
    Co-Authors: Françoise Enjalbert, C Gallion, F Jehl, H Monteil, Heinz Faulstich
    Abstract:

    (1993). Amatoxins and Phallotoxins in Amanita Species: High-Performance Liquid Chromatographic Determination. Mycologia: Vol. 85, No. 4, pp. 579-584.

  • simultaneous assay for Amatoxins and phallotoxins in amanita phalloides fr by high performance liquid chromatography
    Journal of Chromatography A, 1992
    Co-Authors: Françoise Enjalbert, C Gallion, F Jehl, H Monteil, Heinz Faulstich
    Abstract:

    Abstract A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight Amatoxins and phallotoxins. The method identifies both neutral toxins (α- and γ-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (β-amanitin, phallacidin, and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only Amatoxins. This relatively simple method should be suitable for the detection of Amatoxins and phallotoxins in almost any species of mushrooms.

Françoise Enjalbert - One of the best experts on this subject based on the ideXlab platform.

Zeki Severoglu - One of the best experts on this subject based on the ideXlab platform.

  • amanitin and phallotoxin concentration in amanita phalloides var alba mushroom
    Toxicon, 2013
    Co-Authors: Ertugrul Kaya, Recep Bayram, Kursat Oguz Yaykasli, Ayhan Saritas, Selim Karahan, Serdar Colakoglu, Ismail Yilmaz, Zeynep Aydin Sinirlioglu, Zeki Severoglu
    Abstract:

    Although rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of Amatoxins was measured in the gills. The pileus also contained a high amount of Amatoxins. Generally, Amatoxins and phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time.

Markus R Meyer - One of the best experts on this subject based on the ideXlab platform.

  • analysis of α and β amanitin in human plasma at subnanogram per milliliter levels by reversed phase ultra high performance liquid chromatography coupled to orbitrap mass spectrometry
    Toxins, 2020
    Co-Authors: Thomas P Bambauer, Lea Wagmann, Armin A Weber, Markus R Meyer
    Abstract:

    Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and β-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for Amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37-2890 pg/mL, and β-amanitin was found in seven plasma samples ranging from <20-7520 pg/mL. A LC-HRMS/MS method for the quantitation of Amatoxins in human blood plasma at subnanogram per milliliter levels was developed, validated, and used for the analysis of plasma samples. The method provides a valuable alternative to urine analysis, allowing thorough patient treatment but also further study the toxicokinetics of Amatoxins.