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Detection of Protease Activity by Fluorescent Peptide Zymography
Published on: January 20, 2019
Direct Identification of Proteolytic Cleavages on Living Cells Using a Glycan-Tethered Peptide Ligase
Kaitlin Schaefer1, Irene Lui1, James R Byrnes1
1Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, United States.
Abstract:
Proteolytic cleavage of cell surface proteins triggers critical processes including cell-cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2, induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies.
Insights
A new proteomic method, cell surface N-terminomics, identifies precise protein cleavage sites on living cells. This approach reveals how cancer-driving genes remodel cell surfaces, offering targets for immunotherapy.
Area of Science:
- Proteomics
- Molecular Biology
- Cancer Research
Background:
- Proteolytic cleavage of cell surface proteins is crucial for cell signaling and interactions.
- Dysregulated extracellular proteases are implicated in cancer development and progression.
- Existing methods lack the ability to broadly identify precise cleavage sites on living cells.
Purpose of the Study:
- To develop and validate a novel proteomic approach for identifying extracellular protein cleavage sites on living cells.
- To characterize the landscape of extracellular neo-N-termini across various cell types.
- To investigate the impact of oncogenes on extracellular proteolytic remodeling.
Main Methods:
- Development of cell surface N-terminomics using engineered peptide ligase (stabiligase) for tagging neo-N-termini.
- Functionalization of stabiligase with an N-terminal nucleophile for glycan attachment.
- Application of biotinylated peptide ester for proteomic analysis of tagged neo-N-termini.
Main Results:
- Identification and characterization of 1532 extracellular neo-N-termini across diverse cell types, including primary immune cells.
- Discovery of numerous novel cleavage sites not previously identified by proteomic studies.
- Demonstration that oncogenes KRAS(G12V) and HER2 induce significant extracellular proteolytic remodeling.
Conclusions:
- Cell surface N-terminomics is a versatile platform for broadly identifying extracellular neo-N-termini.
- This method reveals proteolyzed neoepitopes that can be targeted for immunotherapies.
- Understanding oncogene-induced proteolytic remodeling provides insights into cancer cell phenotypes.
