IQFP motifs cleaved by <i>AF</i> secreted proteases.

<p>Motifs appearing at least five times among the 93 hits are shown. From each of the four motifs exhibiting the highest mean fold change fluorescence enhancement, a representative well not cleaved by human serum was selected for deconvolution and further analysis.</p

Inhibition of <i>AF</i> secreted protease activity by a serine protease inhibitor.

<p><i>A</i>. AEBSF (8 mM) completely inhibited cleavage of all four IQFP motifs. No inhibition was observed with a metalloprotease inhibitor (EDTA; 20 mM), an aspartic protease inhibitor (Pepstatin A, 20 µM), or a cysteine protease inhibitor (E-64; 20 µM). <i>B</i>. The effect of AEBSF was dose-dependent. For each IQFP, data are normalized to a positive control sample lacking inhibitor. For both panels, data represent fluorescence fold change after 1 h incubation. * p≤0.05 for all IQFPs as compared to each of the three inhibitors and the positive control.</p

Comparative activity of secreted proteases of <i>Aspergillus</i> strains and species.

<p><i>A</i>. Cleavage of individual IQFPs by <i>Aspergillus</i> culture supernatants. The brightness of a given square corresponds to the extent of cleavage of the IQFP probe in that well, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021001#pone-0021001-g002" target="_blank">Figure 2</a>. Fluorescence measurements were normalized by the biomass of each culture according to dry weight to facilitate absolute comparisons across strains. Data represent the mean of two independent experiments. <i>A. fumigatus</i> exhibited the greatest extent of proteolytic activity, followed by <i>A. nidulans</i>, then <i>A. flavus</i>. IQFP probes used for the experiments in panels <i>B</i> and <i>C</i> are highlighted in red. <i>B</i>. Cleavage of selected IQFPs by three <i>Aspergillus</i> species. Values represent the mean of three strains per species. <i>C</i>. Effect of heating at 50°C for 30 min on the proteolytic activity of <i>A. fumigatus</i> H237 and <i>A. nidulans</i> A4 culture supernatants. <i>A. nidulans</i> supernatant retained >80% of activity under these conditions, whereas <i>A. fumigatus</i> supernatant retained only 30–40%. * p≤0.01 as compared to <i>A. fumigatus</i>. For panels A and B, data represent fluorescence fold change after 2 h incubation; for panel C, data represent fluorescence fold change after 1 h incubation. Error bars represent standard deviations.</p

Thermostability and thermophilicity of <i>AF</i> secreted proteases.

<p><i>AF</i> culture supernatants were heated before (<i>A,B</i>) or during (<i>C</i>) IQFP cleavage assays. <i>A</i>. <i>AF</i> culture supernatant was heated for 30 min at the indicated temperatures before addition of IQFPs. Proteolysis of each sequence sharply declined after heating at 50°C and was undetectable after heating at 55°C. <i>B</i>. <i>AF</i> culture supernatant was heated at 56°C for the indicated times before addition of IQFPs. Proteolysis of each sequence decreased markedly after heating for 15 min and was undetectable after heating for 30 min. <i>C</i>. <i>AF</i> culture supernatant and IQFPs were pre-heated for 15 min, combined, and incubated at the indicated temperature. Fluorescence was promptly measured after 1 hr. All IQFPs exhibited slightly increased cleavage at 45°C as compared to room temperature except LFY, which increased significantly. For all panels, data represent fluorescence fold change after 1 hr incubation. * p≤0.01 compared to room temperature.</p

Assignment of IQFP cleavage sites by mass spectrometry.

<p>Calculated and found masses of parent, N-fragment, and C-fragment peptides are shown. All N-terminal fragments were found with Na⁺ adducts. Two sequences per IQFP motif were analyzed.</p

Deconvolution of <i>AF</i> protease substrate consensus motifs.

<p>For each IQFP motif with five or more wells demonstrating proteolytic cleavage by <i>AF</i> secreted proteases (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021001#pone-0021001-t002" target="_blank">Table 2</a>), a representative well was selected for deconvolution and further study. Individual IQFP sequences were synthesized and fine substrate specificity was determined. Although cleavage of the Ser/Thr–Ile/Leu–Phe/Tyr motif was promiscuous (<i>A</i>), others exhibited clear amino acid preferences, particularly at the <i>Zaa</i> position (<i>B, D</i>). Data represent fluorescence fold change after 2 h incubation.</p

Substrate specificity of <i>AF</i> secreted proteases and human serum.

<p><i>A</i>. Cleavage of an IQFP library by <i>AF</i> H237 culture supernatant. Each square corresponds to a single well of a 96 well microplate. The brightness of a given square corresponds to its fluorescence intensity, quantified as fluorescence fold change, which in turn indicates the extent of cleavage of the IQFP probes in that well. Of the 512 wells in the library, 93 exhibited greater than 4-fold fluorescence enhancement upon incubation with <i>AF</i> culture supernatant. <i>B</i>. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only <i>AF</i> culture supernatant, only human serum, or both. <i>D</i>. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by <i>AF</i> secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021001#pone-0021001-t001" target="_blank">Table 1</a>.</p

Amino acid preferences of IQFP sequences cleaved by <i>AF</i> secreted proteases.

<p>Left panel - for each possible residue, abundance at each of the three variable positions is reported. Right panel - the number of occurrences per IQFP sequence for each residue (once, twice, or three times) is shown as a percentage of all hits.</p