Selektive Markierung von Glykokonjugaten mit modifizierten Donorzuckern als Substrate von Glykosyltransferasen und Glykosidasen
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. Nevertheless the mutant can be of great value for the selective coupling approach. In this approach an only slightly modified UDP-GalNAc is transferred to a glycoconjugate and then chemoselectively coupled to a molecule bearing a reporter group, e.g. biotin. With the constructs free and luminal beta4Gal-T1 a transfer assay on agalacto structures has been performed successfully. The conditions of transfer and detection have been optimized with the model protein rat IgG. A sandwich ELSA has been established as the detection method. Further the quantitative differences in the agalactosylation of the IgG N-glycans of several species have been shown successfully. By the proof of success of selective transfer and the ability to discriminate between different levels of agalactosyltaion the main goal of this work has been met. In addition a strategy for the quantification of the biotinylation level of different IgGs has been established. It could be shown that the agalactosylated N-glycans of human IgG can nearly be biotinylated quantitatively. The simple detection method by the sandwich ELSA in microtiter plate format and the high level of biotinylation rise high expectations in the direction of an early diagnosis of rheumatoid arthritis. With pNP-6-biotinyl-Gal(NAc) potential substrates of the selective transfer with glycosidases have been synthesized and isolated on a 100 mg-scale. Hydrolysis or transfer could not be demonstrated. However the oxidation products pNP-6-oxo-Gal(NAc) could also be isolated. It could be shown for both sugars that they are substrates of glycosidases. A transglycosylation of pNP-6-oxo-GalNAc with GlcNAc and subsequent chemical oxidation at the aldehyde moiety has led to one of the best so far known high affinity ligands for natural killer cell receptors.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Glykokonjugate, Enzymatische Oxidation, Autoaggressionskrankheit, Markierung, Transferasen
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