Development of BODIPY labelled sialic acids as sialyltransferase substrates for direct detection of terminal galactose on N- and O-linked glycans.

Glycans on proteins and cell surfaces are useful biomarkers for determining functional interactions with glycan binding proteins, potential disease states, or indeed level of differentiation. The ability to rapidly and sensitively detect or tag specific glycans on proteins provides a diagnostic tool with wide application in chemical glycobiology. The monosaccharide N-acetylneuraminic acid (sialic acid) is a key player in these interactions and the manipulation and control of sialylation levels has been an important research focus, particularly in the development of therapeutic proteins. Using sialyltransferases to tag specific glycans provides a rapid means of determining what types of glycans are present. We have synthesized two variants of sialic acid carrying the fluorophore BODIPY (4,4 -Difluoro-4-boro-3a,4a-diaza-s-indacene) and examined its use with several different sialyltransferases on a variety of protein substrates and cell surface glycans. Our data show that there are significant differences between various enzymes ability to transfer the labelled sialic acids, and that the type of N-glycan and target protein strongly influences this activity.

Assay Methods for the Glycosyltransferases Involved in Synthesis of Bacterial Polysaccharides.

Glycans play many important roles in bacterial biology and the complexity of the glycan structures requires biochemical assays in place to help characterize the biosynthetic pathways. Our focus has been on the use of enzymes from pathogens which make molecular mimics of host glycans. We have been examining glycosyltransferases that make strategic linkages in biologically active glycans which can be also exploited for potential therapeutic glycoconjugate synthesis. This chapter will provide details on assays for a variety of bacterial glycosyltransferases that we and others have used for the characterization of pathogen glycoconjugate biosynthetic pathways, and for the in vitro synthesis of human-like glycans produced by bacterial pathogens. The methods presented here should enable other assays to be developed for new pathway characterization.