Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering

Abstract

Metabolic oligosaccharide engineering (MOE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides where they can modulate a host of biological activities or be exploited as "tags" for bio-orthogonal and chemoselective ligation reactions. Undertaking a MOE experiment can be a daunting task based on the growing repertoire of analogs now available and the ever increasing number of metabolic pathways that can be targeted; therefore, a major emphasis of this article is to describe a general approach for analog design and selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell-surface glycans have been successfully remodeled by MOE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. Curr. Protoc. Chem. Biol. 2:171-194 © 2010 by John Wiley & Sons, Inc.

Keywords: ManNAc analogs; glycosylation; selectin‐based adhesion; sialic acid glycoengineering.

Figure 1

Overview of metabolic oligosaccharide engineering (MOE). MOE was pioneered for the sialic acid biosynthetic pathway, which is one of the glycosylation pathways of a cell that can accommodate non-natural analogs. Modifications in this pathway can be introduced as “R₁”-modified forms of (A) ManNAc, (B) Neu5Ac, or (C) CMP-Neu5Ac. These metabolic intermediates intercept the sialic acid pathway at various points, are utilized in place of the natural pathway components, and ultimately appear on the cell surface as chemically altered form of Neu5Ac, the most common form of sialic acid found in human cells. Examples of “R₁” modifications are shown in Figure 3.

Figure 2

Flow diagram depicting various steps involved in a typical MOE experiment. Procedures that are not described in this unit are denoted with a star; additional information on these steps is provided in the references cited in the main text.

Figure 3

Analog design features. (A) The “core” structures of ManNAc, GalNAc, fucose, and GlcNAc analogs used in MOE that are further derivatized with R₁ and R₂ groups. Categories of R₁ groups, which are carried through on to the cell surface and displayed as a modify glycan, include those with (B) enlarged hydrocarbon groups, (C) heteroatom (or other) containing functional groups, and (D) direct fluorophores. (E) Examples of R₂ groups include a proton (i.e., the -H found on hydroxyl groups) and 2- to 5-carbon short chain fatty acids that are ester-linked to the hydroxyl groups of the monosaccharide analog. Ac and Bu denote acetyl and n-butanoyl substitutions shown in Figure 4 and referenced throughout the main text.

Figure 4

Chemical structures of ManNAc analogs used in this unit.