Postproline Cleaving Enzymes also Show Specificity to Reduced Cysteine

Area of Science:

• Proteomics
• Enzymology
• Biochemistry

Background:

• Postproline cleaving enzymes (PPCEs) are crucial in proteomics for cleaving after proline residues, a common stop site for many proteases.
• Established PPCEs include Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, expanding the available proteolytic tools.

Purpose of the Study:

• To investigate the unexpected cleavage specificity of AnPEP towards reduced cysteine residues observed during online proteolysis experiments.
• To systematically analyze the factors influencing AnPEP's cleavage preference, particularly its interaction with cysteine.

Main Methods:

• Utilized Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to analyze AnPEP cleavage products.
• Investigated the effect of various cysteine modifications (disulfide bonds, oxidation, alkylation) on AnPEP activity.
• Compared the cleavage specificity of AnPEP with neprosin under similar experimental conditions.

Main Results:

• AnPEP demonstrated significant cleavage specificity towards reduced cysteine residues, in addition to proline and alanine.
• Cysteine modifications, especially alkylation, effectively blocked postcysteine cleavage, explaining why this activity was previously undetected.
• Neprosin exhibited similar postcysteine cleavage specificity to AnPEP.

Conclusions:

• The cleavage specificity of PPCEs should be redefined to include cysteine, extending it from post-Pro/Ala to post-Pro/Ala/Cys.
• These findings necessitate a re-evaluation of PPCE applications, particularly for analyzing cysteine-rich proteins and assessing protein cysteine status.
• The enzymatic mechanism of PPCEs requires further investigation in light of their newly identified cysteine-cleaving activity.