Activity profiling of peptidases in Angiostrongylus costaricensis first-stage larvae and adult worms

Abstract

Background: Angiostrongylus costaricensis is a relatively uncharacterized nematode that causes abdominal angiostrongyliasis in Latin America, a human parasitic disease. Currently, no effective pharmacological treatment for angiostrongyliasis exists. Peptidases are known to be druggable targets for a variety of diseases and are essential for several biological processes in parasites. Therefore, this study aimed to systematically characterize the peptidase activity of A. costaricensis in different developmental stages of this parasitic nematode.

Methodology/principal findings: A library of diverse tetradecapeptides was incubated with cellular lysates from adult worms and from first-stage larvae (L1) and cleaved peptide products were identified by mass spectrometry. Lysates were also treated with class specific peptidase inhibitors to determine which enzyme class was responsible for the proteolytic activity. Peptidase activity from the four major mechanistic classes (aspartic, metallo, serine and cysteine) were detected in adult worm lysate, whereas aspartic, metallo and serine-peptidases were found in the larval lysates. In addition, the substrate specificity profile was found to vary at different pH values.

Conclusions/significance: The proteolytic activities in adult worm and L1 lysates were characterized using a highly diversified library of peptide substrates and the activity was validated using a selection of fluorescent substrates. Taken together, peptidase signatures for different developmental stages of this parasite has improved our understanding of the disease pathogenesis and may be useful as potential drug targets or vaccine candidates.

Fig 1. Characterization of the proteolytic activity of adult worm lysates at pH 3.0 using MSP-MS and an IQ-FRET substrate.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold obtained with female lysates in the presence or absence of pepstatin (aspartic peptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 15 minutes hydrolysis at pH 3.0 using female lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) Adult worm lysates (female and male) (150 μg/mL) were incubated with internally quenched GKPILFFRL substrate (20 μM). Enzyme activity was expressed as picomoles of AMC per second. E) Female lysates were assayed with GKPILFFRL substrate in the presence of 10 μM E-64 and 1 μM pepstatin. The results are reported as mean ± standard deviation of three biological replicates.

Fig 2. Characterization of the proteolytic activity of adult worm lysates at pH 5.0 using MSP-MS and AMC substrates.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold obtained with female lysates in the presence or absence of E-64 (cysteine peptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 60 minutes hydrolysis at pH 5.0 using female lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) Adult worm lysates (female and male) (150 μg/mL) were incubated with Boc-Ala-Gly-Pro-Arg-AMC (100 μM). Enzyme activity was expressed as picomoles of AMC per second. E) Female lysates were assayed with the same fluorescent substrate in the presence of 10 μM E-64. The results are reported as mean ± standard deviation of three biological replicates.

Fig 3. Characterization of the proteolytic activity of adult worm lysates at pH 8.0 using MSP-MS and AMC substrates.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold obtained with female lysates in the presence of AEBSF (serine peptidase inhibitor) or 1,10 phenanthroline (metallopeptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 240 minutes hydrolysis at pH 8.0 using female lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) Adult worm lysates (female and male) (150 μg/mL) were incubated with Suc-Gly-Pro-Leu-Gly-Pro-AMC or H-Tyr-AMC (100 μM). Enzyme activity was expressed as picomoles of AMC per second. E) Female lysates were assayed with Suc-Gly-Pro-Leu-Gly-Pro-AMC in the presence of 1mM AEBSF or 5 mM 1,10 phenanthroline. F) Female lysates (150 μg/mL) were assayed upon H-Tyr-AMC in the presence of 1 mM AEBSF, 5 mM 1,10 phenanthroline or a mixture of these two inhibitors. The results are reported as mean ± standard deviation of three biological replicates.

Fig 4. Characterization of the proteolytic activity of L1 lysates at pH 3.0 using MSP-MS and an IQ-FRET substrate.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold obtained with L1 lysates in the presence or absence of pepstatin (aspartic peptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 240 minutes hydrolysis at pH 3.0 using L1 larvae lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) L1 lysates (125 μg/mL) were assayed with GKPILFFRL (20 μM) in the absence or presence of 1 μM pepstatin. The results are reported as mean ± standard deviation of three biological replicates.

Fig 5. Characterization of the proteolytic activity of L1 lysates at pH 8.0 using MSP-MS and AMC substrates.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold in the presence or absence of AEBSF (serine peptidase inhibitor) or 1,10 phenanthroline (metallopeptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 15 minutes hydrolysis at pH 8.0 using L1 larvae lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) L1 lysates (125 μg/mL) were incubated with Suc-Leu-Leu-Val-Tyr-AMC (100 μM) in the presence of 1 mM AEBSF or 5 mM 1,10 phenanthroline. E) L1 lysates (125 μg/mL) were assayed upon Boc-Ala-Gly-Pro-Arg-AMC (100 μM) in the presence of 1 mM AEBSF or 5 mM 1,10 phenanthroline. The results are reported as mean ± standard deviation of three biological replicates.