An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

Abstract

BackgroundThe ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation.ResultsA majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified.ConclusionsWe were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…In this study, we observed that 13 out of 22 peptidases with increased abundance after GT exposure were also expressed in A. niger grown under carbon starvation [31,32]. These proteases include An18g01320, An01g01750, PepF (An07g08030), An01g00370, An03g01010, An06g00190, PepA (An14g04710), An02g04690, An08g04640, An09g02830, An12g05960, An03g01660, PepC (An07g03880).…”

“…The proteases with the highest levels of abundance after GT exposure were classified as members of the A1 pepsin family proteases and S53 sedolisin family proteases, based on MEROPS database (merops.sanger.ac.uk) ( Table 4). Interestingly, these two families have been observed to be the most abundant under carbon starvation conditions, in A. niger [31,32].…”

Chitinases CtcB and CfcI modify the cell wall in sporulating aerial mycelium of Aspergillus niger

“…Gene annotations were manually curated using literature reports, BLAST searches (Altschul et al, 1990) and orthologue clusters from the Aspergillus Genome Database (Arnaud et al, 2010). The presence of secretion signal peptides was predicted following Braaksma et al (2010). The prediction of glycosylphosphatidylinositol (GPI)-anchor sequences was assessed by analysing amino acid sequences obtained from the Aspergillus Genome Database using the fungal BIG-PI prediction server (Eisenhaber et al, 2004) as well as by manual inspection for the C-terminal consensus sequence (de Groot et al, 2003) when automated prediction and literature on (orthologues of) the protein were not in line.…”

Genetic and Genomic Manipulations in Aspergillus niger

“…Investigation into the proteomes of A. niger has focused on the exo-proteomes, or the collection of extracellular proteins, under different growth conditions (Adav et al 2010 ;Braaksma et al 2010 ;Lu et al 2010 ;Tsang et al 2009 ). The microsomal proteome following xylose induction has also been examined by shot-gun proteomics (Ferreira de Oliveira et al 2010 ).…”