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Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

Watson DS, Feng X, Askew DS, Jambunathan K, Kodukula K, Galande AK - PLoS ONE (2011)

Bottom Line: A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints.However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia, United States of America.

ABSTRACT

Background: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and principal findings: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.

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Properties of AF secreted proteases.A. Effect of buffer pH on activity of AF secreted proteases. Cleavage of each of the IQFP consensus motifs was optimal at approximately pH 8. Cleavage of three probes (Thr-Ile-Gln, Lue-Phe-Tyr, Ala-Ile-Leu) showed a narrow activity peak at pH 8 and a loss of activity at pH 9, whereas cleavage of Thr-Ile-Phe was near maximal in a broad range of assay buffer pH values. B. Cleavage of 8 IQFPs by 4 independent preparations of AF H237 culture supernatant. Proteolytic activities did not differ significantly among preparations.
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pone-0021001-g004: Properties of AF secreted proteases.A. Effect of buffer pH on activity of AF secreted proteases. Cleavage of each of the IQFP consensus motifs was optimal at approximately pH 8. Cleavage of three probes (Thr-Ile-Gln, Lue-Phe-Tyr, Ala-Ile-Leu) showed a narrow activity peak at pH 8 and a loss of activity at pH 9, whereas cleavage of Thr-Ile-Phe was near maximal in a broad range of assay buffer pH values. B. Cleavage of 8 IQFPs by 4 independent preparations of AF H237 culture supernatant. Proteolytic activities did not differ significantly among preparations.

Mentions: From each of the first four IQFP motifs shown in Table 2, a representative well that was not cleaved by human serum was selected for further study. The IQFPs selected were: Ile/Leu–Phe/Tyr–Phe/Tyr, Ala/Val–Ile/Leu–Ile/Leu, Ser/Thr–Ile/Leu–Phe/Tyr, and Ser/Thr–Ile/Leu–Asn/Gln. The eight constituent sequences of each well were individually synthesized and confirmed by mass spectrometry. Fine amino acid substrate specificity was determined by quantifying endpoint fluorescence fold change following incubation of AF culture supernatant with each individual substrate (Figure 3). Three of the four motifs demonstrated a clear preference at one or more variable positions. The Ser/Thr–Ile/Leu–Asn/Gln motif required glutamine at the Zaa position, whereas the Ala/Val–Ile/Leu–Ile/Leu motif required leucine at the Zaa position. The Ile/Leu–Phe/Tyr–Phe/Tyr motif required phenylalanine at the Yaa position and also preferred tyrosine at the Zaa position. In contrast, the 8 constituent peptides of the Ser/Thr – Ile/Leu – Phe/Tyr motif were cleaved to a similar extent without dramatic preferences at any of the variable positions. From each motif, the two most efficiently cleaved IQFP sequences were selected for further characterization: Ser-Ile-Phe, Thr-Ile-Phe, Ser-Ile-Gln, Thr-Ile-Gln, Leu-Phe-Tyr, Leu-Phe-Phe, Ala-Ile-Leu, and Ala-Leu-Leu. Cleavage of each of these IQFPs was optimal at pH 8.0, although some exhibited broader pH maxima (Ser-Ile-Phe, Thr-Ile-Phe, Leu-Phe-Phe, Leu-Phe-Tyr) than others (Ser-Ile-Gln, Thr-Ile-Gln, Ala-Ile-Leu, Ala-Leu-Leu) (Figure 4A). Cleavage of these sequences was consistent across multiple independent culture preparations (Figure 4B).


Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

Watson DS, Feng X, Askew DS, Jambunathan K, Kodukula K, Galande AK - PLoS ONE (2011)

Properties of AF secreted proteases.A. Effect of buffer pH on activity of AF secreted proteases. Cleavage of each of the IQFP consensus motifs was optimal at approximately pH 8. Cleavage of three probes (Thr-Ile-Gln, Lue-Phe-Tyr, Ala-Ile-Leu) showed a narrow activity peak at pH 8 and a loss of activity at pH 9, whereas cleavage of Thr-Ile-Phe was near maximal in a broad range of assay buffer pH values. B. Cleavage of 8 IQFPs by 4 independent preparations of AF H237 culture supernatant. Proteolytic activities did not differ significantly among preparations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117871&req=5

pone-0021001-g004: Properties of AF secreted proteases.A. Effect of buffer pH on activity of AF secreted proteases. Cleavage of each of the IQFP consensus motifs was optimal at approximately pH 8. Cleavage of three probes (Thr-Ile-Gln, Lue-Phe-Tyr, Ala-Ile-Leu) showed a narrow activity peak at pH 8 and a loss of activity at pH 9, whereas cleavage of Thr-Ile-Phe was near maximal in a broad range of assay buffer pH values. B. Cleavage of 8 IQFPs by 4 independent preparations of AF H237 culture supernatant. Proteolytic activities did not differ significantly among preparations.
Mentions: From each of the first four IQFP motifs shown in Table 2, a representative well that was not cleaved by human serum was selected for further study. The IQFPs selected were: Ile/Leu–Phe/Tyr–Phe/Tyr, Ala/Val–Ile/Leu–Ile/Leu, Ser/Thr–Ile/Leu–Phe/Tyr, and Ser/Thr–Ile/Leu–Asn/Gln. The eight constituent sequences of each well were individually synthesized and confirmed by mass spectrometry. Fine amino acid substrate specificity was determined by quantifying endpoint fluorescence fold change following incubation of AF culture supernatant with each individual substrate (Figure 3). Three of the four motifs demonstrated a clear preference at one or more variable positions. The Ser/Thr–Ile/Leu–Asn/Gln motif required glutamine at the Zaa position, whereas the Ala/Val–Ile/Leu–Ile/Leu motif required leucine at the Zaa position. The Ile/Leu–Phe/Tyr–Phe/Tyr motif required phenylalanine at the Yaa position and also preferred tyrosine at the Zaa position. In contrast, the 8 constituent peptides of the Ser/Thr – Ile/Leu – Phe/Tyr motif were cleaved to a similar extent without dramatic preferences at any of the variable positions. From each motif, the two most efficiently cleaved IQFP sequences were selected for further characterization: Ser-Ile-Phe, Thr-Ile-Phe, Ser-Ile-Gln, Thr-Ile-Gln, Leu-Phe-Tyr, Leu-Phe-Phe, Ala-Ile-Leu, and Ala-Leu-Leu. Cleavage of each of these IQFPs was optimal at pH 8.0, although some exhibited broader pH maxima (Ser-Ile-Phe, Thr-Ile-Phe, Leu-Phe-Phe, Leu-Phe-Tyr) than others (Ser-Ile-Gln, Thr-Ile-Gln, Ala-Ile-Leu, Ala-Leu-Leu) (Figure 4A). Cleavage of these sequences was consistent across multiple independent culture preparations (Figure 4B).

Bottom Line: A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints.However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia, United States of America.

ABSTRACT

Background: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and principal findings: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.

Show MeSH
Related in: MedlinePlus