All ETDs from UAB

Advisory Committee Chair

Susan L Bellis

Advisory Committee Members

Anita Hjelmeland

Douglas Hurst

Alexa Mattheyses

Ralph Sanderson

Document Type

Dissertation

Date of Award

2023

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

During the process of malignant transformation, cells have striking changes in their cell surface glycosylation. One of these changes is an enrichment for α2,6 sialic acid which is added by the sialyltransferase, ST6GAL1. ST6GAL1 is upregulated in numerous malignancies and acts by adding an α2,6 sialic acid onto receptors bound for the plasma membrane. This sialic acid, in turn, modulates the activity of the receptor by regulating conformation, clustering, and cell surface retention. The work presented in this dissertation focuses on how ST6GAL1-mediated sialylation of a specific cell surface receptor, epidermal growth factor receptor (EGFR), regulates many of its properties. We first identify that ST6GAL1-mediated sialylation of the receptor promotes its activation in several cell lines with modulated ST6GAL1 expression. Then, using a panel of ovarian cancer lines with ST6GAL1 knock-down or overexpression, we reveal that α2,6 sialylation of EGFR promotes the activation of specific downstream signaling pathways such as AKT and NFκB. To understand how sialylation of EGFR impacts downstream signaling, we assessed a variety of different properties of EGFR. By performing dimerization assays, we find that α2,6 sialylation of EGFR enhances its dimerization capabilities. Further, using total internal reflection fluorescent microscopy, we uncover iv that ST6GAL1-mediated sialylation of EGFR promotes higher-order clustering of the receptor. We next sought to determine if α2,6 sialylation of EGFR could modulate its localization. By performing recycling assays and immunofluorescent microscopy, we found that sialylation of EGFR by ST6GAL1 enhances receptor recycling to the cell surface. In line with this, we also assessed receptor degradation using degradation assays and immunofluorescent microscopy. Interestingly, we showed that α2,6 sialylation of EGFR decreased co-localization to the lysosomes, thus preventing receptor degradation. In the second part of this dissertation, we review how glycosylation of EGFR modulates its activity. From our review of the literature, we find that glycosylation of the receptor regulates EGFR activity at three different levels: the protein level, the cellular level, and the organismal level to modulate tumorigenic properties. Taken together, the studies presented in this dissertation highlight glycosylation as a major regulator of EGFR activity in malignant cells.

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