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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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Strategy for protease activity profiling of AF secreted proteases.A. Structure and description of a typical IQFP probe from the library (variable sequence shown: Ala-Ile-Leu). B. Graphical depiction of IQFP cleavage by AF culture supernatant. N- and C-terminal substrate fragments were readily detected by MALDI-TOF mass spectrometry following ZipTip® C18 sample preparation. C. As an example, spectra of the sequence Ala-Ile-Leu before and after proteolytic cleavage are shown, with calculated fragments identified. A summary of all MS data is presented in Table 3.
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pone-0021001-g001: Strategy for protease activity profiling of AF secreted proteases.A. Structure and description of a typical IQFP probe from the library (variable sequence shown: Ala-Ile-Leu). B. Graphical depiction of IQFP cleavage by AF culture supernatant. N- and C-terminal substrate fragments were readily detected by MALDI-TOF mass spectrometry following ZipTip® C18 sample preparation. C. As an example, spectra of the sequence Ala-Ile-Leu before and after proteolytic cleavage are shown, with calculated fragments identified. A summary of all MS data is presented in Table 3.

Mentions: We have recently reported a robust method for profiling protease substrate specificities of complex biological mixtures using a concise combinatorial IQFP library [38]. In this library, 15 amino acids that are commonly found in protease substrates are represented at each of three variable positions, flanked by appropriate spacer sequences and fluorophore or quencher moieties (Figure 1). The resulting 3375 IQFP sequences are combined into pools of up to 8 similar sequences and the resulting 512 peptide pools are arrayed in six 96 well microplates. Library “hits” are then deconvoluted by synthesizing the constituent IQFPs of a particular well to obtain fine substrate specificity information. In our previous study, we used this library to define the quantitative proteolytic fingerprints of two body fluids, bronchoalveolar lavage and serum, that have clinical relevance for diagnosis of AF infection.


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)

Strategy for protease activity profiling of AF secreted proteases.A. Structure and description of a typical IQFP probe from the library (variable sequence shown: Ala-Ile-Leu). B. Graphical depiction of IQFP cleavage by AF culture supernatant. N- and C-terminal substrate fragments were readily detected by MALDI-TOF mass spectrometry following ZipTip® C18 sample preparation. C. As an example, spectra of the sequence Ala-Ile-Leu before and after proteolytic cleavage are shown, with calculated fragments identified. A summary of all MS data is presented in Table 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117871&req=5

pone-0021001-g001: Strategy for protease activity profiling of AF secreted proteases.A. Structure and description of a typical IQFP probe from the library (variable sequence shown: Ala-Ile-Leu). B. Graphical depiction of IQFP cleavage by AF culture supernatant. N- and C-terminal substrate fragments were readily detected by MALDI-TOF mass spectrometry following ZipTip® C18 sample preparation. C. As an example, spectra of the sequence Ala-Ile-Leu before and after proteolytic cleavage are shown, with calculated fragments identified. A summary of all MS data is presented in Table 3.
Mentions: We have recently reported a robust method for profiling protease substrate specificities of complex biological mixtures using a concise combinatorial IQFP library [38]. In this library, 15 amino acids that are commonly found in protease substrates are represented at each of three variable positions, flanked by appropriate spacer sequences and fluorophore or quencher moieties (Figure 1). The resulting 3375 IQFP sequences are combined into pools of up to 8 similar sequences and the resulting 512 peptide pools are arrayed in six 96 well microplates. Library “hits” are then deconvoluted by synthesizing the constituent IQFPs of a particular well to obtain fine substrate specificity information. In our previous study, we used this library to define the quantitative proteolytic fingerprints of two body fluids, bronchoalveolar lavage and serum, that have clinical relevance for diagnosis of AF infection.

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