A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

Abstract

Polysialic acid (polySia) is a long homopolymer consisting of α2,8–linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF–7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF–7 cells. PolySia was found predominantly on soluble proteins in MCF–7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF–7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins – AGR2 and QSOX2 – were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV–depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF–7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.