Untersuchungen zur Analyse der O-Glykosylierung des IgA1-Moleküls im Krankheitsbild der IgA-Nephropathie
- Analysis of the O-glycosylation of the IgA1 molecule in IgA nephropathy
Keitel, Christine; Elling, Lothar (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2010)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2010
In this work, the binding characteristics of a Tn and a TF-Antibody and two lectins, HAA from Helix aspersa and Jacalin towards IgA1 O-glycans were characterized in detail. The aim was to evaluate existing techniques for the detection of undergalactosylated IgA O-glycans in IgA nephropathy and to develop and analyse new detection methods. A set of serum samples from IgAN patients and controls was analyzed with the antibodies and the HAA lectin. However, no significant difference in the glycosylation profile could be found. A new technique for the detection of undergalactosylated IgA O-glycans was developed, that employs the enzymatic labeling of undergalactosylated glycans. Therefore, an enzyme that was able to transfer galactose onto free GalNAc sites on IgA1 had to be cloned. For this purpose, three homologous core 1 beta1,3 galactosyltransferases, that transfer galactose onto GalNAc from human, drosophila melanogaster and E. coli were recombinantly expressed and characterized in detail. The human enzyme was only formed in inclusion bodies and showed almost no activity. The bacterial enzyme was also formed in inclusion bodies. The solubility and activity of this enzyme could be greatly enhanced by expressing it as fusion protein with a propeptide from a lipase of staphylococcus hyicus. However, it was not able to transfer galactose onto IgA1 O-glycans. The enzyme from drosophila melanogaster showed a high activity when expressed in Hi5 insect cells and was the only one able to transfer galactose onto IgA1 O-glycans. In the first approach, unlabeled galactose was transferred by the drosophila C1 GalT onto free GalNAc moieties on IgA1. The binding profile of the Tn- and TF-antibody and the HAA lectin to IgA O-glycans was analyzed before and after the transfer of galactose. This should establish a homogenously galactosylated glycan standard. The comparison of the galactosylation profile before and after the transfer should show a great difference in undergalactosylated samples and a little difference in normally galactosylated samples and thus identify IgAN patients. Unfortunately, this method, also was not able to show a significant difference in the IgA glycosylation of IgAN patients and controls. This was greatly due to the poor specificity of the antibodies and lectin for their sugars. Secondly, free GalNAc moieties on IgA1 from the set of serum samples from IgAN patients and controls were labeled by the drosophila C1GalT with radioactive galactose (3H galactose). By this technique, no significant difference in the glycosylation pattern of IgAN patients and controls could be demonstrated. It seemed that enzyme kinetics prevented a complete labeling of all free GalNAc moieties. The aim to develop and evaluate different methods for the analysis of IgA1 O-glycans was achieved. However, none of the analysed methods was able to discriminate between IgAN patients and controls significantly. No method that was suitable as a sero-diagnostic test for IgAN could be developed.