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 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.

Automated summary

Proteolytic cleavage of cell surface proteins plays a crucial role in processes such as cell-cell interactions, receptor activation, and shedding of signaling proteins. This study introduces a proteomic approach called cell surface N-terminomics, which allows for the identification of precise cleavage sites on living cells. By utilizing a peptide ligase called stabiligase, extracellular neo-N-termini can be efficiently tagged for proteomic analysis. The study demonstrates the versatility of this approach by identifying and characterizing numerous extracellular neo-N-termini in different cell types, including primary immune cells. The findings also reveal the involvement of single oncogenes, KRAS(G12V) and HER2, in the proteolytic remodeling of proteins associated with cancerous cell growth, invasion, and migration. Cell surface N-terminomics provides a generalizable platform for targeting proteolyzed epitopes using immunotherapies.