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Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
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Substrate O-glycosylation actively regulates extracellular proteolysis.
Elizabeta Madzharova1, Fabio Sabino1, Konstantinos Kalogeropoulos1
1Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Protein Science : a Publication of the Protein Society
|July 29, 2024
Summary
Truncated O-glycosylation, specifically the Tn antigen, enhances matrix metalloproteinase 9 (MMP9) activity and alters protein processing in breast cancer cells. This suggests a link between O-glycans and proteolysis, impacting cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Extracellular proteolysis regulates cellular functions and is implicated in diseases.
- Post-translational modifications (PTMs), including O-glycosylation and proteolysis, interact to control cellular homeostasis.
- Matrix metalloproteinases (MMPs) and N-acetylgalactosamine (GalNAc)-type O-glycosylation are key extracellular PTMs.
Purpose of the Study:
- To investigate how truncated O-glycans (Tn antigen) affect proteolytic processing, particularly MMP9 activity.
- To explore the impact of O-glycan truncation on the extracellular proteome and ectodomain shedding.
- To understand the interplay between O-GalNAc glycosylation and proteolysis in cancer.
Main Methods:
- Utilized MDA-MB-231 breast cancer cells with inactivated C1GALT1-specific chaperone 1 (COSMC) to induce Tn antigen.
- Employed terminal amine isotopic labeling of substrates (TAILS) for quantitative proteome and N-terminome analysis.
- Assessed MMP9-specific proteolytic processing and identified novel ectodomain shedding events.
Main Results:
- Truncated O-glycans (Tn antigen) led to enhanced MMP9-mediated proteolysis in the extracellular proteome.
- Significant alterations in the overall proteome were observed in cells with truncated O-glycans.
- Novel instances of ectodomain shedding regulated by O-glycan truncation were discovered.
Conclusions:
- Mature O-glycosylation is crucial for regulating proteolytic processing and maintaining proteome homeostasis.
- Truncated O-glycans can increase protein susceptibility to proteolytic degradation, particularly by MMP9.
- The interplay between proteolysis and O-GalNAc glycosylation may influence cancer phenotypes.
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