Using active site mutagenesis to change the substrate selectivity of transsialidase from T.cruzi

Abstract

Glycoconjugates such as glycoproteins and glycolipids form the glycocalyx at the surface of cell membranes. These glycoconjugates have been shown to play key roles in many biological processes, such as cell-cell adhesion, protein folding, protein interactions and virus recognition processes. The biosynthesis of glycoconjugates is more complicated than that of proteins because they are not encoded directly in the genome, but biosynthesis is under control of a set of 'glycoenzymes' such as glycosyltransferases, glycosidases and transglycosidases. This project has investigated a transsialidase (TcTS) from Trypanosoma cruzi which transfers sialic acids from sialoglycoconjugates to terminal galactosides forming alpha2,3-sialyllactosides. This enzyme has been extensively used in glyco-engineering and synthesis of sialosides, but its application has been limited by narrow substrate range and selectivity. The aim of the project was to change the substrate specificity to transfer of alpha2,6-sialosides by mutagenesis.A number of mutant libraries R35X, R53X, S57X, D59X, Y119X, R245X, W312X, R314X, Y342X, E362X, and Y364X around active site positions were generated and several screening protocols developed.Of these, the application of liquid chromatography combined with mass spectrometry (LCMS) was studied. Unfortunately, no transfer activity of alpha2,6-sialosides was observed in any of the mutant generated. However, the hydrolysis of alpha2,6-sialosides could be measured in several of these samples. To visualise the mutant colonies which can hydrolysealpha2,6-sialosides, a high-throughput detection method using sialic acid linked to X-Gal as a substrate was developed. Upon hydrolysis of this sialoside by any transsialidase present, X-Gal was generated resulting in a blue coloured colony. These results should provide a good foundation for future quests for transsialidase with novel activities.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor)