Direct Identification of Proteolytic Cleavages on Living Cells Using a Glycan-Tethered Peptide Ligase

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...

Full description

Saved in:
Bibliographic Details
Published in:ACS central science 2022-10, Vol.8 (10), p.1447-1456
Main Authors: Schaefer, Kaitlin, Lui, Irene, Byrnes, James R., Kang, Emily, Zhou, Jie, Weeks, Amy M., Wells, James A.
Format: Article
Language:English
Subjects:
Cell activation
Cell interactions
Cell migration
Cell surface
Cells
Cells (biology)
Cleavage
Enzymes
ErbB-2 protein
Flow cytometry
Genetic engineering
Glycan
Immune system
Immunotherapy
Labeling
Membrane proteins
Microscopy
Nucleophiles
Oxidation
Peptides
Phenotypes
Polysaccharides
Proteins
Proteolysis
Proteomics
Reagents
Receptor mechanisms
Signal transduction
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:2374-7943
2374-7951
DOI:10.1021/acscentsci.2c00899