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. 2015 Sep:103:60-4.
doi: 10.1016/j.toxicon.2015.06.018. Epub 2015 Jun 19.

Production of (15)N-labeled α-amanitin in Galerina marginata

Hong Luo  1 , Brandon DuBois  2 , R Michael Sgambelluri  3 , Evan R Angelos  2 , Xuan Li  4 , Daniel Holmes  5 , Jonathan D Walton  6
Affiliations

Production of (15)N-labeled α-amanitin in Galerina marginata

Hong Luo et al. Toxicon. 2015 Sep.

Abstract

α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.

Keywords: Amanita; Galerina; Isotope labeling; LC-MS; NMR; α-Amanitin.

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Figures

Fig. 1
Fig. 1
Liquid culture of G. marginata. A. Shake culture. B. Close-up of flasks showing mycelial balls.
Fig. 2
Fig. 2
Time course of 15N-labeled α-amanitin accumulation in G. marginata grown in shake culture. Each data point is the average of two biological replicates. Error bars indicate the range.
Fig. 3
Fig. 3
HPLC chromatograms of the (A) first and (B) third separation of 15N-labeled α-amanitin. The y-axis is OD280 (mAU).
Fig. 4
Fig. 4
Mass spectrometric analysis of (A) native and (B) 15N-labeled α-amanitin. A. [M+H+] = 919.4, [M+Na+] = 941.4, and [M+K+] = 957.4; B. [M+H+] = 929.4, [M+Na+] = 951.4, and [M+K+] = 967.4. (C) Overlaid spectra of 14N-amanitin and 15N-amanitin, shown in expanded scale.
Fig. 5
Fig. 5
1H-15N HSQC spectrum of 15N-labeled α-amanitin. The spectrum shows ten signals corresponding to the ten N atoms in α-amanitin.
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