MGAT3 and MGAT5 overexpression alters the protein cargo of extracellular vesicles released by metastatic melanoma cells

Abstract

Extracellular vesicles (EVs) are potential non-invasive diagnostic, prognostic and therapeutic tools. Additionally, they are important contributors to tumorigenesis. Glycosylation has been found to modulate the composition of the EV proteome. Increased amounts of β1,6-branched N-glycans, synthesized by N-acetylglucosaminyltransferase V (GnT-V), are most commonly observed in melanoma and are associated with decreased cell adhesion and increased metastasis. The opposite effect is caused by the addition of bisecting GlcNAc by N-acetylglucosaminyltransferase III (GnT-III). To date, the impact of these enzymes on EV cargo in melanoma remains unexplored. Flow cytometry was used to study the surface glycosylation of genetic variants of WM266-4 melanoma cells with induced overexpression of GnT-III or GnT-V encoding genes (MGAT3 or MGAT5) and EVs released by these cells. LC-MS/MS proteomics was applied to analyze the effect of altered glycosylation on the proteome of released EVs, followed by detailed bioinformatic analysis. Flow cytometry analysis revealed dynamic changes in the surface glycosylation of EVs derived from melanoma cells overexpressing MGAT3 or MGAT5. Induced overexpression of MGAT3 or MGAT5 also caused significant changes in the proteome of EVs. The proteomic analysis identified a total of 1770 microvesicular and 704 exosomal proteins that play different roles in melanoma progression, including those with established diagnostic/prognostic potential and those closely associated with melanoma onset. Proteomic profiling of EVs derived from cells overexpressing MGAT3 and MGAT5 revealed functional changes in EV protein content driven by glycosylation modifications. The study presented a potential multifaced application of melanoma-derived EVs for diagnostic and prognostic purposes.

Keywords: Biomarkers; Extracellular vesicles; Glycosylation; Melanoma; Proteomics.