In-depth specificity profiling of Pro-Pro endopeptidases (PPEPs) using combinatorial synthetic peptide libraries

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Proteases comprise the class of enzymes that catalyze the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility towards protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. We developed a new method which combines the high diversity of a combinatorial synthetic peptide library with the sensitivity and detection power of mass spectrometry to determine protease cleavage specificity. We applied this method to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e. Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) which has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

Authors 10

1. Bart Claushuis Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

2. Robert A. Cordfunke Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Leiden, The Netherlands

3. Arnoud H. de Ru Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

4. Annemarie M. Otte Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

5. Hans C. van Leeuwen

   Netherlands Organisation for Applied Scientific Research

   Affiliation as printed

   Department of CBRN Protection, Netherlands Organization for Applied Scientific Research TNO, Rijswijk, The Netherlands

6. Oleg I. Klychnikov

   Lomonosov Moscow State University

   Affiliation as printed

   Department of Biochemistry, Moscow State University, Moscow, Russian Federation

7. Peter A. van Veelen Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

8. Jeroen Corver Aachen Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands

9. Jan Wouter Drijfhout Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Leiden, The Netherlands

10. Paul J. Hensbergen corresponding Aachen Center for Proteomics and Metabolomics

    Leiden University Medical Center

    Affiliation as printed

    Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands