γ-Glutamyl transpeptidase (GgtA) of Aspergillus nidulans is not necessary for bulk degradation of glutathione

Abstract

Aspergillus nidulans exhibited high γ-glutamyl transpeptidase (γGT) activity in both carbon-starved and carbon-limited cultures. Glucose repressed, but casein peptone increased γGT production. Null mutation of creA did not influence γGT formation, but the functional meaB was necessary for the γGT induction. Deletion of the AN10444 gene (ggtA) completely eliminated the γGT activity, and the mRNA levels of ggtA showed strong correlation with the observed γGT activities. While ggtA does not contain a canonical signal sequence, the γGT activity was detectable both in the fermentation broth and in the hyphae. Deletion of the ggtA gene did not prevent the depletion of glutathione observed in carbon-starved and carbon-limited cultures. Addition of casein peptone to carbon-starved cultures lowered the formation of reactive species (RS). Deletion of ggtA could hinder this decrease and resulted in elevated RS formation. This effect of γGT on redox homeostasis may explain the reduced cleistothecia formation of ΔggtA strains in surface cultures.

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups

“…All GSTFs under analysis from bacteria, protists, and ascomycete fungi are predicted to associate with these enzymes, except ascomycete fungal GSTFs of clade I, the mycorrhizal group of clade II, and the group with an intron phase of 22 of clade V, which lack one or two of the enzymes, (Supplementary Table 4). These enzymes are involved in glutathione modification and cycling, cellular redox regulation, and secondary metabolism in both fungi and plants, [54][55][56] suggesting that nonplant GSTFs generally play an important role following redox balance shift during oxidative stresses and secondary metabolism. Except for these highly conserved partners for almost all nonplant GSTFs, there are some taxonomy-or even clade-specific partners.…”

Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans

“…The presence of the following proteins in the fermentation broth was demonstrated (Table S4): AbnC (AN8007, putative extracellular endo-1,5-α-L-arabinosidase); EglB (AN3418, cellulase); BglA and BglL (AN4102 and AN2828; putative β-glucosidases); ChiB (AN4871, chitinase); EglC (AN7950, putative GPI-anchored glucan endo-1,3-β-D-glucosidase); PepJ (AN7962, protease); CatB (AN9339, catalase); and SodA (AN0241, Cu/Zn-SOD). The presence of AbnC, CatB, EglB, PepJ, SodA, and the AN8445 peptidase in the fermentation broth of lactose-containing cultures has already been demonstrated [39]. van Munster et al [24] found that upregulation of CAZyme genes by carbon stress is accompanied by the secretion of the corresponding proteins in A. niger even in the case of carbon starvation.…”

“…Intracellular glutathione reductase, nitrate reductase, and β-galactosidase activities were determined according to Pinto et al [35], Bruinenberg et al [36], and Nagy et al [37], respectively, from the cell-free extracts. Extracellular chitinase [34], N-acetyl-glucosaminidase [38], βglucosidase [30], cellulase [30], γ-glutamyl transpeptidase (γGT) [39], and protease [40] activities were determined from the fermentation broth. In the case of cellulase determination, which was based on measuring the increase in the reducing sugar content in the carboxymethylcellulose substrate solution, fermentation broth was dialyzed against 0.1 mol L −1 K-phosphate buffer (pH 6.5) prior to measurement to remove reducing sugars.…”

“…Selected spots were cut from gels manually, and, after trypsin digestion, peptides were analyzed on a 4000 QTRAP (AB Sciex, Framingham, MA, USA) mass spectrometer coupled to an Easy nLC II nanoHPLC (Bruker, Billerica, MA, USA). The obtained LC-MS/MS data were used for protein identification based on the ProteinPilot 4.5 (ABSciex) search engine, and the SwissProt database and minimum two peptides with 99% confidence were required for identification of the protein [39].…”

“…Accordingly, the genes of the proteins detected here were upregulated by the appropriate carbon stress (Table S2) with the following exceptions: We could not detect the upregulation of eglB in carbon-starved cultures or the upregulation of sodA and AN8445 in lactose-containing cultures (Table S2). However, we could detect the presence of the encoded proteins in the fermentation broth (Table S4, [39]). In the case of SodA and EglC (detected during carbon starvation), as well as of CatB (detected both in carbon-starved cultures and on lactose) (Table S4, [39]), the corresponding genes even showed downregulation by carbon stress (Table S2).…”