Identification of two metallothioneins in Agaricus crocodilinus reveals gene duplication and domain expansion, a pattern conserved across fungal species

Abstract

Agaricus crocodilinus (Agaricaceae), an edible saprotrophic mushroom, accumulates high concentrations of cadmium (Cd) in unpolluted environments. This study investigates whether this species has evolved mechanisms to store Cd complexed with metallothioneins (MTs), proteins that bind heavy metal ions via cysteinyl (Cys)-thiolate bonds, how these MTs originated, and how similar mechanisms are present in other fungal species. Size exclusion chromatography revealed that a substantial fraction of Cd in A. crocodilinus sporocarps was sequestered in a 3.4 kDa complex containing Cys-rich peptides. Screening a sporocarp cDNA expression library in a Cd-sensitive Saccharomyces cerevisiae strain identified two MT transcripts, AcMT1 and AcMT2, encoding 49-amino acid (AA) AcMT1 with 10 Cys and 32-AA AcMT2 with 7 Cys. The presence of AcMT2 in the 3.4 kDa Cd-peptide complex isolated from sporocarp was confirmed by mass spectrometry. In mycelial isolates exposed to heavy metals, AcMT1 was more strongly upregulated, while AcMT2 was more expressed under normal conditions. Sequence comparisons revealed that AcMT2 is closer to the ancestral gene, whereas AcMT1 is a more recent duplicate. Combined bioinformatic and functional evidence supports AcMT2 as a constitutively expressed MT involved in Cd binding in the sporocarp, while AcMT1, though more inducible in mycelia and more protective in yeast, appears to serve a transient detoxification role. Moreover, the gene duplication and domain rearrangement mechanism underlying this MT diversification was also identified in other Agaricales and Boletales species.

Keywords: Agaricaceae; Gene duplication; Gene internal duplication; Heavy metals.

Fig. 1

Metal distribution and characterization of SEC fractions from Agaricus crocodilinus sporocarp. A Distribution of metals in cell-free extracts analyzed by size exclusion chromatography. B Electropherogram showing the protein content of the metal-containing fractions 29–32. The sporocarp extract was fractionated by SEC, and the concentrations of Cd (solid line) and Zn (dotted line) in the eluate were monitored by ICP-MS using the ¹¹¹Cd and ⁶⁶Zn isotopes. The elution maxima of molecular mass standards are indicated by arrows; the 3.4 kDa metal–peptide complex is marked with an asterisk. Panel B shows an electrophoreogram of fluorescence-labeled Cys-containing peptides present in the 3.4 kDa metal–peptide complex, alongside a 6.1 kDa rabbit MT1A standard used for size comparison

Fig. 2

Metal distribution analysis of cell-free extracts from Agaricus crocodilinus. Mycelia were grown in the presence of 5 µM Cd²⁺ (A) or 250 µM Zn²⁺ (B). Mycelial extracts were fractionated by size-exclusion chromatography (SEC), and the concentrations of Cd (solid line) or Zn (dotted line) in the eluates were monitored by inductively coupled plasma mass spectrometry (ICP-MS) using the ¹¹¹Cd and ⁶⁶Zn isotopes. Elution maxima of molecular mass standards are indicated by arrows; the 3.4 kDa metal–peptide complex is marked with an asterisk. A small Cd peak, likely mycophosphatin, was formed between 24 and 26 min

Fig. 3

Amino acid alignment of A. crocodilinus AcMTs with characterized fungal MT homologues. Cysteinyl residues are highlighted in grey and the conserved heptaCys motif is marked below the alignment. Duplicated fragment of AcMT1 is marked above the alignment. Aligned AcMT homologues (GenBank accession numbers): AsMT2 (AGO04615), PiMT1 (AAS19463), PaMT1 (AJO67962), ShMT1/2 (AUS94322/AUS94321), LbMT1/2a/2b (AHI43933/AHI43934/AHI43935), CcMT1/2 (QNN26299/QNN26300), HcMT1/2 (ACJ65191/ACJ65192), HmMT1/2/3 (AHA31878/AHA31879/AHL29949)

Fig. 4

Phylogenetic tree of fungal MTs with known function in metal detoxification. The tree was constructed using Neighbor-joining method, with 10,000 bootstraps, support values over 60 are displayed. Protein sequences for the alignment were obtained from GenBank and their identifiers and names are displayed. Branch lengths represent evolutionary distances measured in substitutions per site. Scale bar indicates 0.10 substitutions per site. The tree is rooted using Saccharomyces cerevisiae CUP1-1 metallothionein as an outgroup

Fig. 5

Internal duplications in AcMT1 and other Agaricomycete MTs. A and B show schematic representations of proposed internal duplication events in AcMT1 and in other long Agaricomycete MT homologues, respectively. Duplicate fragments are labeled G1 and G2, with their nucleotide (DNA or mRNA) identity indicated as % G1 to G2. Exons (ex) are shown as grey rectangles. In panel A, exon organization is compared between AcMT2 and the computationally derived ancAcMT1 (AcMT1 lacking G2). cDNA sequences of AcMT2 and ancAcMT1 are aligned below, with identical nucleotides marked by asterisks

Fig. 6

Metal tolerance in S. cerevisiae expressing AcMTs. Tolerance to Cd, Zn, and Cu was assessed in metal-sensitive S. cerevisiae mutants (ycf1Δ, zrc1Δcot1Δ, and cup1Δ) transformed with the p426GPD vector carrying either no insert or the AcMT1 or AcMT2 cDNA, as indicated. Shown are results from spot plate assays (left) on SD agar with or without the indicated metal supplements, and corresponding metal dose-dependent growth inhibition in liquid media (right), expressed as IC₅₀ₘₑₜₐₗ values. The IC₅₀ values represent means ± standard deviation from at least three independent biological replicates. Different letters in superscript denote statistically significant differences (one-way ANOVA with Tukey’s post hoc test, p < 0.05). For turbidity data, see Supplementary Figure S2

Fig. 7

Metal-induced expression of AcMT1 and AcMT2 in Agaricus crocodilinus mycelia. A AcMT gene expression was analyzed by qRT-PCR in A. crocodilinus mycelia exposed for 24 h to the indicated concentrations of Cd²⁺, Zn²⁺, or Cu²⁺. Relative mRNA levels of AcMT1 and AcMT2 are shown in comparison to untreated mycelium grown in PD medium (expression set to 1, dashed line). B Basal expression of AcMT1 and AcMT2 in the absence of metal treatment. Transcript levels were normalized to the expression of A. crocodilinus β-tubulin (GenBank accession PV055698). Values represent means ± standard deviation from three independent biological replicates. Different letters above the bars indicate statistically significant differences (one-way ANOVA followed by Tukey’s test, p < 0.05)