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Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device

Title:

Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device

Leclerc, Laura (2018) Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device. Masters thesis, Concordia University.

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Abstract

Cell-surface carbohydrates—or glycans—influence growth, differentiation, and immune response mechanisms. Alterations to the glycome can be markers for diseases including diabetes, neurodegenerative disorders, and cancer. Fucosyltransferases catalyze the addition of a fucose sugar residue to specific cell-surface glycans, which are involved in intercellular cell rolling/adhesion interactions such as white blood cells homing to inflammation sites and sperm-egg binding in fertilization. Fucosylated glycans are also implicated in inflammatory disease and cancers. In viral and microbial infections, fucosyltransferases can play a role in the adhesion and colonization of the host organism, as in the case of Helicobacter pylori α(1,3)-fucosyltransferase (FucT).
To better our understanding of glycome alterations and improve medical diagnostics and treatments, screens for glycosyltransferase activity and inhibition are needed. Efficient screens for specific glycosylations tend toward costly materials, instrumentation, and specialized skillsets- here, we present a novel inhibition assay for FucT using the fluorogenically labeled disaccharide, MU-β-LacNAc. The assay shows good potential for high throughput (Z’=0.78 in 384-well plate), though such an application is not shown here. It was also implemented on a digital microfluidic (DMF) platform, where inhibition curves of FucT by GDP, a product of the glycosyltransferase reaction that exhibits an inhibitory feed-back loop, were generated on-device. Results of the assay on DMF (IC50 = 0.093 mM ± 0.037) were shown to be comparable to results in a 384-well plate (IC50 = 0.114 mM ± 0.086), achieving a 87.5% reduction in reaction volume and setting the groundwork for future fully automated screens for potential inhibitors of glycosyltransferases.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (Masters)
Authors:Leclerc, Laura
Institution:Concordia University
Degree Name:M.A. Sc.
Program:Biology
Date:1 September 2018
Thesis Supervisor(s):Kwan, David and Leclerc, Laura
Keywords:fucosyltransferase,α(1,3)-fucosyltransferase,FucT,inhibition,microfluidics,dmf,digital microfluidics,MU-β-LacNAc,LewisX,LeX,Lewis-X,glycosylation assay
ID Code:984453
Deposited By: LAURA LECLERC
Deposited On:16 Nov 2018 17:05
Last Modified:20 Dec 2018 01:00

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