Methods to investigate glycogen structure in Glycogen Storage Diseases (GSDs)

Fancellu, Gaia (2022) Methods to investigate glycogen structure in Glycogen Storage Diseases (GSDs). Masters thesis, University of East Anglia.

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Abstract

Glycogen is the primary source of energy in mammals. It acts as a metabolic buffer, storing or releasing glucose from liver and skeletal muscles in response to an excess or demand of energy. Inherited metabolic disorders that affect glycogen metabolism, such as Glycogen Storage Diseases (GSDs), are related to the appearance of aberrant glycogen structures, which impact on the rate and extent of glucose availability. To date, details on the structure and metabolism of glycogen are still not sufficient to provide an accurate insight into GSDs. The aim of this project is to develop easy-to-use methodologies to analyse and better define the structure of glycogen from healthy and GSDs sources.

To achieve the goal of this project, two methodologies were developed. The first one consisted in the enzymatic debranching of glycogen to determine the chain length distribution and the degree of branching. The method was established on commercially available glycogen (from oyster) and validated on glycogen extracted from HepG2 cells and HepG2 cells bearing GSD type Ia (glucose 6-phosphatase deficiency). The second method aimed at elucidating the position of the branching points by a stepwise enzymatic digestion of the branches and the external chains of the standard glycogen. The results obtained from this second method were then integrated with a theoretical model (called DP15) to have a better understanding on the branches arrangement. The development of both methodologies was supported by analytical techniques, such as thin-layer chromatography (TLC), high-performance anion exchange chromatography coupled with pulsed amperometric detector (HPAEC-PAD), and bicinchoninic acid (BCA) assay to assess the hydrolysed products qualitatively and quantitatively.

The application of the first methodology showed that the structure of the glycogen standard contained 10% of branching points, and the chains were predominantly longer than DP6 in agreement with literature. The same structural analysis was applied on glycogen from mammalian HepG2 cells and mouse liver after the development of a novel extraction protocol. Preliminary studies showed that HepG2 glycogen had 20% of branching points, equivalent to twice the amount observed in oyster glycogen, and was mainly made of chains longer than DP7. The comparison of the chain length distribution between HepG2 cells and GSDIa HepG2 cells showed that this genetic mutation does not impact the length of the branches and the activity of the glycogen branching enzyme. The same methodology was also applied to glycogen from mouse liver, and the results showed a group of predominant chains between DP4 and DP8. The lack of a peak in the chain length distribution of mouse liver glycogen was attributed to the activity of glucosidases present in liver tissues.

The results obtained from the second methodology showed that the glycogen β- and phosphorylase limit dextrins have chain length distributions (CLDs) shifted towards a low DP, namely DP2, DP3 and DP4, when compared to oyster glycogen. The combination of the CLDs collected experimentally into the DP15 model led to select branched structures bearing branching points at the 3rd or 4th glucose residues from the reducing end of the main chain, suggesting that glycogen branching enzyme potentially branches every three or four glucose units.

To conclude, the methodologies presented in this thesis were demonstrated to be feasible and applicable on commercially available standards or samples isolated from mammalian cells for the investigation of the structure of glycogen. Further applications on samples from GSDs sources could elucidate the impact of these genetic mutations on glycogen structure.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Chemistry
Depositing User:	Chris White
Date Deposited:	15 Feb 2023 09:38
Last Modified:	15 Feb 2023 09:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/91135
DOI:

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