Selektive Markierung von Glykokonjugaten mit modifizierten Donorzuckern als Substrate von Glykosyltransferasen und Glykosidasen
type: CreativeWork
description: The main goal of this Ph. D. thesis has been the selective labelling of disease-related glycoconjugate structures with glycosyltransferases. In this context the main focus has been on the agalacto structures of the N-glycans of IgG. These structures are associated with rheumatoid arthritis as described in the literature. The modified nucleotide sugar UDP-6-biotinyl-Gal should be used for the selective labelling experiments. Several constructs of human beta4Gal-T1, available in our research group for the selective labelling, should be characterized. With one of the mentioned constructs a mutagenesis should be performed to make a labelling with UDP-6-biotinyl-GalNAc possible. This nucleotide sugar could not yet be transferred successfully. Further on it should also be shown if the technique of selective transfer can be applied to the enzyme class of glycosidases. The main goal of this Ph.D. thesis could be fulfilled. First the synthesis and most important the isolation of UDP-6-biotinyl-Gal have been optimized. Instead of a two step procedure for purification UDP-6-biotinyl-Gal can now be obtained in a one step isolation process and with greater purity. The mutagenesis of human beta4Gal-T1 has also been successful. By bringing in a point mutation we have been able to obtain an enzyme which has a UDP-GalNAc transfer activity. Together with three constructs of human beta4Gal-T1, the free, luminal and His6-TagPropeptid-beta4Gal-T1, the mutant His6-TagPropeptid-beta4Gal-T1(Y284L) has also been characterized. The substrate spectrum, the pH optimum as well as the rest activity with several metal ions has been examined. Further all constructs have been characterized kinetically. Rather surprisingly we could find constructs that according to their specifity can transfer UDP-6-biotinyl-Gal to the monosaccharide acceptor GlcNAc beta1-Bn or to the glycoprotein rat IgG. A transfer of UDP-6-biotinyl-GalNAc with His6-TagPropeptid-beta4Gal-T1(Y284L) could not be demonstrated. Nevert…
abstract: The main goal of this Ph. D. thesis has been the selective labelling of disease-related glycoconjugate structures with glycosyltransferases. In this context the main focus has been on the agalacto structures of the N-glycans of IgG. These structures are associated with rheumatoid arthritis as described in the literature. The modified nucleotide sugar UDP-6-biotinyl-Gal should be used for the selective labelling experiments. Several constructs of human beta4Gal-T1, available in our research group for the selective labelling, should be characterized. With one of the mentioned constructs a mutagenesis should be performed to make a labelling with UDP-6-biotinyl-GalNAc possible. This nucleotide sugar could not yet be transferred successfully. Further on it should also be shown if the technique of selective transfer can be applied to the enzyme class of glycosidases. The main goal of this Ph.D. thesis could be fulfilled. First the synthesis and most important the isolation of UDP-6-biotinyl-Gal have been optimized. Instead of a two step procedure for purification UDP-6-biotinyl-Gal can now be obtained in a one step isolation process and with greater purity. The mutagenesis of human beta4Gal-T1 has also been successful. By bringing in a point mutation we have been able to obtain an enzyme which has a UDP-GalNAc transfer activity. Together with three constructs of human beta4Gal-T1, the free, luminal and His6-TagPropeptid-beta4Gal-T1, the mutant His6-TagPropeptid-beta4Gal-T1(Y284L) has also been characterized. The substrate spectrum, the pH optimum as well as the rest activity with several metal ions has been examined. Further all constructs have been characterized kinetically. Rather surprisingly we could find constructs that according to their specifity can transfer UDP-6-biotinyl-Gal to the monosaccharide acceptor GlcNAc beta1-Bn or to the glycoprotein rat IgG. A transfer of UDP-6-biotinyl-GalNAc with His6-TagPropeptid-beta4Gal-T1(Y284L) could not be demonstrated. Nevert…
author: Namdjou, Darius-Jean
language: ger
keywords: info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Glykokonjugate, Enzymatische Oxidation, Autoaggressionskrankheit, Markierung, Transferasen
date published: 2006
date modified: 2026-07-30T19:43:02Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University