Date of Award

Winter 12-2016

Embargo Period

1-16-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Adam D. Linstedt

Abstract

Humans express at least twenty isoforms of GalNAc transferase (termed GalNAc-T1 to T20) that localize to the Golgi apparatus and initiate mucin-type O-glycosylation. Regulation of this family of enzymes affects the function of a vast array of cellular proteins transiting the secretory pathway. This has an impact on many disease states including cancer, which frequently involves profoundly altered cell surface glycan profiles. As a consequence, druglike modulators of GalNAc-Ts hold promise as entirely new therapeutics for major diseases such as osteoporosis, dyslipidemia, heart disease, and cancer. Additionally, specific modulators will be instrumental in elucidating the basic biology of this complex family of 20 isozymes carrying out a single reaction yet showing developmental and tissue specific expression and partially overlapping substrate specificities. However, there are currently no known inhibitors or activators of these initiating enzymes. To address this shortcoming, we developed a set of cell-based fluorescent sensors to be used for high-throughput screening to identify isoform specific modulators of O-glycosylation. The sensors traffic in the secretory pathway becoming activated if an added inhibitor blocks glycosylation. Simultaneous screening of compounds with two or more isoform specific sensors was used to minimize offtarget effects allowing identification of candidates that directly target a particular GalNAc-T. Indeed, one of the lead compounds identified, named T3Inh-1, specifically inhibited purified GalNAc-T3 in vitro. T3Inh-1 also blocked glycosylation of FGF23, a specific substrate of GalNAc-T3, in cultured cells and in mice. Further, T3Inh-1 opposed the effect of upregulated GalNAc-T3 in driving metastatic-like cell invasion without affecting cell proliferation. Our hope is that this inhibitor, as well as others from our ongoing screening, can be used for new discoveries and therapeutics involving mucin-type O-glycosylation.

Available for download on Tuesday, January 16, 2018

Share

COinS