MGAT5 drives melanoma aggressiveness via TIMP1 glycosylation-dependent CD63-β1-integrin signaling.

MGAT5-mediated N-glycosylation plays a central role in tumor progression and metastasis by affecting proteins involved in tumor cell behavior. One relevant target is Tissue inhibitor of metalloproteases 1 (TIMP1), which, beyond its classical role as a metalloproteinase inhibitor, promotes oncogenic signaling through interactions with CD63 and β1-integrins. Emerging evidence suggests that aberrant TIMP1 N-glycosylation may regulate these diverse functions. Here, we investigated the role of MGAT5 in melanoma cell behavior and how MGAT5-mediated aberrant N-glycosylation influences TIMP1 function, including its activity as a signaling molecule and as an inhibitor of metalloproteinases. We performed Mgat5 knockout using CRISPR/Cas9 in metastatic melanoma cells to understand the role of MGAT5 in cell proliferation, migration, invasion, and tumor growth. Through site-directed mutagenesis, we generated melanoma cells expressing aglycosylated TIMP1 and evaluated the effect of N-glycosylation on TIMP1 function, including its role as a signaling molecule, by co-immunoprecipitation and anoikis resistance assays, and as an inhibitor of metalloproteinases, by zymography. We demonstrated that Mgat5 knockout reduced metastatic melanoma cell proliferation, migration, invasion, and tumor growth, TIMP1 is a target of MGAT5 in metastatic melanoma cells, and aberrant glycosylation of TIMP1 favors its interaction with CD63/β1-integrins and cell survival while decreasing its ability to inhibit metalloproteases (MMPs). Therefore, MGAT5 plays a pro-tumor role in metastatic melanoma cells and the aberrant glycoform of TIMP1 directs its function as a signaling molecule, orchestrating the pleiotropic functions of TIMP1.