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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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Substrate specificity of AF secreted proteases and human serum.A. Cleavage of an IQFP library by AF 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 AF culture supernatant. B. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only AF culture supernatant, only human serum, or both. D. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by AF secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in Table 1.
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pone-0021001-g002: Substrate specificity of AF secreted proteases and human serum.A. Cleavage of an IQFP library by AF 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 AF culture supernatant. B. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only AF culture supernatant, only human serum, or both. D. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by AF secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in Table 1.

Mentions: Comparative protease activity profiling of AF H237 culture supernatant and complement preserved normal human serum revealed striking differences in their proteolytic signatures (Figure 2A–B). Of the 512 wells in the library, 212 exhibited greater than 2-fold fluorescence enhancement upon exposure to AF culture supernatant but were not detectably cleaved by human serum (Figure 2C). In addition, 93 library wells exhibited greater than 4-fold fluorescence enhancement upon exposure to AF culture supernatant. 92 of these 93 substrate motifs contained isoleucine, leucine, phenylalanine, or tyrosine at one or more variable positions; 46.2% of the sequences contained one of these residues at one position, 45.2% at two positions, and 7.5% at all three positions (Figure 2D). These four residues accounted for 44.1% of Xaa, 59.1% of Yaa, and 57.0% of Zaa (Table 1). Positively charged residues (Lys, Arg) accounted for less than 10% of the residues at each variable position, hydroxyl-containing residues (Ser, Thr) less than 15%, negatively charged residues (Asp, Glu) less than 5%, and proline less than 5%. Of the 93 wells exhibiting 4-fold fluorescence enhancement or greater, several motifs (two constant positions and one variable position) appeared five or more times (Table 2). Four of the five motifs contained Ile/Leu at the Yaa position. The Xaa and Zaa positions were more varied but hydrophobic residues still predominated at the fixed positions of all five motifs except one, which contained Ser/Thr at the Xaa position.


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)

Substrate specificity of AF secreted proteases and human serum.A. Cleavage of an IQFP library by AF 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 AF culture supernatant. B. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only AF culture supernatant, only human serum, or both. D. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by AF secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0021001-g002: Substrate specificity of AF secreted proteases and human serum.A. Cleavage of an IQFP library by AF 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 AF culture supernatant. B. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only AF culture supernatant, only human serum, or both. D. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by AF secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in Table 1.
Mentions: Comparative protease activity profiling of AF H237 culture supernatant and complement preserved normal human serum revealed striking differences in their proteolytic signatures (Figure 2A–B). Of the 512 wells in the library, 212 exhibited greater than 2-fold fluorescence enhancement upon exposure to AF culture supernatant but were not detectably cleaved by human serum (Figure 2C). In addition, 93 library wells exhibited greater than 4-fold fluorescence enhancement upon exposure to AF culture supernatant. 92 of these 93 substrate motifs contained isoleucine, leucine, phenylalanine, or tyrosine at one or more variable positions; 46.2% of the sequences contained one of these residues at one position, 45.2% at two positions, and 7.5% at all three positions (Figure 2D). These four residues accounted for 44.1% of Xaa, 59.1% of Yaa, and 57.0% of Zaa (Table 1). Positively charged residues (Lys, Arg) accounted for less than 10% of the residues at each variable position, hydroxyl-containing residues (Ser, Thr) less than 15%, negatively charged residues (Asp, Glu) less than 5%, and proline less than 5%. Of the 93 wells exhibiting 4-fold fluorescence enhancement or greater, several motifs (two constant positions and one variable position) appeared five or more times (Table 2). Four of the five motifs contained Ile/Leu at the Yaa position. The Xaa and Zaa positions were more varied but hydrophobic residues still predominated at the fixed positions of all five motifs except one, which contained Ser/Thr at the Xaa position.

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