Sialic acids are involved in many biological processes. In glycoproteins and glycolipids theyare essential for signalling and mediate molecular interactions as well as being targets formany pathogens such as influenza virus. The synthesis of sialylated glycoconjugates is ofgreat importance. The incorporation of sialic acid through chemical synthesis carries severaldifficulties, enzymatic strategies using glycosyltransferases are very attractive alternativestrategy, and have been used on a broad range of substrates forming glycosidic linkages withregio-and stereo-specificity.The work presented herein shows the study and application of two enzymes, Photobacteriumdamselae alpha2,6-sialyltransferase (Pd2,6ST) and Trypanosoma cruzi trans-sialidase (TcTS)which are used in the synthesis of sialyloligosaccharides. Both enzymes were expressed in E.coli and purified for biotransformations.In the first application new sialylated chromogenic compounds were generated through thisenzymatically by using TcTS and a Pd2,6ST. These compounds were used for the detectionof neuraminidase activity in a number of biological samples and led to the discovery ofneuraminidase activity from Bacillus pumilus and Arthrobacter aurescens, two differentbacteria in which the presence of neuraminidases had never been described. Secondly, TcTSwas used to study lipid glycosylations. Glycans in biological systems can be associated tocomplex lipidic microdomains and the presence of these microdomains can affect the activityof some enzymes. In case of Trypanosoma cruzi trans-sialidase, a decreased activity wasdetected when the acceptor substrate was part of the aggregated lipid rafts compared toactivity observed when the reaction was performed using fully dispersed substrate. Thirdly,the sialylation of glycoarrays using Pd2,6ST was studied. For the first time, sialylated glycanswith alpha2,6- glycosidic linkages were successfully incorporated into a gold glycoarrayplatform, which had been previously developed for the label-free detection of carbohydrateproteininteractions. Successful enzymatic incorporation of sialic acids onto the arrays wasconfirmed with commercial available lectins. Finally, by using the gold glycoarray platformcontaining both 2,3 and 2,6 linked sialic acids as well as other common glycans, thecarbohydrate-binding properties of the surface proteins of the bacterium Lactobacillus reuteri10was studied using MALDI-ToF MS techniques. For first time, strong interactions wereobserved between a mucus binding protein and Neu5Acalpha2,6-linked glycans, with muchweaker binding to 2,3-linked analogues. Such glycan structures have been identified inabundant manner in colon mucins and this study contributes to the understanding of complexinteractions between mucins and probiotic organisms as well as pathogenic bacteria. Thesestudies show that glycan arrays can contribute both to the understanding of probiotics as wellas to the identification of glycan binding proteins as targets for new drugs.
|Date of Award||31 Dec 2015|
- The University of Manchester
|Supervisor||Sabine Flitsch (Supervisor)|
- Sialic acid
- MALDI-ToF MS