Extent of N-glycosylation of the metalloproteinase inhibitor and cytokine TIMP-1 determines pancreatic cancer cell proliferation and survival via CD63

Glycosylation emerges as a critical determinant of protein function in cancer, yet its impact on multifunctional secreted factors remains understudied. Here, we identified tissue inhibitor of metalloproteinases-1 (TIMP-1), a glycoprotein with glycosylation sites at N30 and N78 harboring both canonical antiproteolytic and noncanonical cytokine-like activity, as one of the most upregulated secreted glycoproteins circulating in the blood of pancreatic cancer (PC) patients. Whereas plasma from healthy donors contained similar amounts of double-glycosylated (TIMP-1^glyc1/1), single- glycosylated (N78 and not N30) (TIMP-1^glyc0/1), and nonglycosylated (TIMP-1^glyc0/0) TIMP-1, TIMP-1^glyc1/1 predominated in plasma from PC patients. scRNAseq and in vitro validation linked this shift to tumor progression-associated upregulation of the oligosaccharyltransferase complex in epithelial cells. In human PC cell lines, oligosaccharyltransferase complex activity was critical for the synthesis of TIMP-1^glyc1/1. Importantly, tumor cell survival and proliferation-promoting activity via CD63 were dependent on TIMP-1 glycosylation, which required N30-glycosylation. In contrast, glycosylation was not necessary for the antiproteolytic activity of TIMP-1 towards different matrix metalloproteinases (MMPs) (collagenases MMP-1, MMP-8; gelatinases MMP-2, MMP-9; stromelysin MMP-3; matrilysin MMP-7) but modulated the respective inhibitory efficacy. Analysis of a published glycoproteome data set, allowing assessment of individual glycosylation site occupancy in TIMP-1, revealed that N30 site occupation correlated with poor survival, while N78 site occupation showed no prognostic value, corroborating the impact of double glycosylation of TIMP-1, as observed in patients, on tumor promotion. The glycosylation-dependent modulation of the multifunctionality of tumor-secreted TIMP-1 thus provides a molecular basis for its long-debated cancer-promoting role. Finally, it exemplifies the impact of glycosylation macroheterogeneity on disease-relevant modulation of protein function.