Polysialic acid is a cell surface polysaccharide with important functions in neural development and plasticity, and in immune evasion by a few strains of pathogenic bacteria. Recent studies have highlighted the potential for enzymatic polysialylation of therapeutic proteins1, cells in culture, and neural tissues in vivo2 using the polysialyltransferase from Neisseria meningitidis serogroup B (polySTNmB).
Polymerising glycosyltransferases have a tendency to synthesise mixtures of disperse polymer length. However, oligo- or polysaccharides of uniform length are required for evaluation and application as therapeutic reagents. Here we address this hurdle by engineering the mechanism of chain elongation used by the polySTNmB. Using recently developed methods for high throughput screening3 and detailed characterisation of polyST activity4 we conduct a neutral drift experiment. The polySTNmB was subjected to high mutagenesis rates followed by purifying selection to remove inactive variants to explore functional regions of the polySTNmB sequence space. Detailed analysis of over 50 drifted variants (7.3 ± 3.0 amino acid exchanges per sequence) revealed that some sequences had acquired new modes of chain elongation which correspond to either a processive or distributive mechanism of polymerisation. The study reveals sequence elements which control the mechanism of elongation and enable the synthesis of uniform polymeric products.
1. Lindhout, T. et al. Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proceedings of the National Academy of Sciences (2011). doi:10.1073/pnas.1019266108
2. Maarouf, A. E. et al. Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase. J. Biol. Chem. 287, 32770–32779 (2012).
3. Keys, T. G., Berger, M. & Gerardy-Schahn, R. A high-throughput screen for polysialyltransferase activity. Analytical Biochemistry 427, 60–68 (2012).
4. Keys, T. G. et al. A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases. Analytical Biochemistry 427, 107–115 (2012).
