Abstract
Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.
Highlights
No available inhibitors target isoforms of the biologically and medically relevant enzyme that initiates mucintype O-glycosylation
T2 Sensor Based on ANGPTL3—To develop a sensor sensitive to the activity of T2 we used a sequence from angiopoietin-like 3 (ANGPTL3) as the linker in our previously described fluorescent glycosylation sensor (23)
In liver cells, which naturally express ANGPTL3 and lack T3, the sequence is a specific target of T2 (25) indicating that other family members expressed in these cells do not recognize it
Summary
No available inhibitors target isoforms of the biologically and medically relevant enzyme that initiates mucintype O-glycosylation. Results: Protein-based, fluorescent sensors were developed for T2 and T3 isoforms using modified GalNAc-transferase target sites and these were activated in HEK cells lacking their corresponding GalNAc-transferases. Humans express up to 20 isoforms of GalNAc-transferase ( T1–T20) that localize to the Golgi apparatus and initiate O-glycosylation Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Despite the biological and medical significance of the GalNAc-transferase family, GalNAc-transferase isoform-specific assays are lacking that monitor their activity in living cells and there are no known inhibitors To address these shortcomings, we initiated the development of cell-based fluorescence sensors whose fluorescence is a readout of GalNAc-transferase activity (23). With two or more isoform-specific sensors at hand, parallel screening can identify compounds selectively activating a single sensor thereby greatly reducing the likelihood of off-target effects
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