Substrate O‐glycosylation actively regulates extracellular proteolysis

Abstract

Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post‐translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)‐mediated cleavages and N‐acetylgalactosamine (GalNAc)‐type of O‐glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O‐glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1‐specific chaperone 1 (COSMC) on the general and MMP9‐specific proteolytic processing in MDA‐MB‐231 breast cancer cells. Quantitative assessment of the proteome and N‐terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA‐MB‐231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O‐glycans. These results highlight the critical role of mature O‐glycosylation in fine‐tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O‐GalNAc glycosylation, possibly affecting cancer phenotypes.