Metabolic Delivery of Ketone Groups to Sialic Acid Residues

Abstract

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.It has been known for several decades that the cell surface is richly decorated with a dense covering of complex oligosaccharides. Even before many of the specific biological functions of these carbohydrates were elucidated, it was apparent that remodeling the molecular landscape of the cell surface transformed the behavior of cells (1, 2). The discovery of their participation in cell-cell recognition events has brought cell surface glycoconjugates to the forefront of biological research in recent years. Numerous cell surface oligosaccharides have been sequenced and their interactions with receptors on opposing cells are understood in some molecular detail (3-7). Nevertheless, the interactions of cell surface oligosaccharides with protein receptors in solution and on opposing cells remain difficult to study at the molecular level in comparison to protein-protein interactions on cell surfaces.The lag in carbohydrate research can be attributed, in part, to the difficulty in controlling the presentation of well defined carbohydrate epitopes on cell surfaces (for a perspective, see Ref. 8). While cell surface oligosaccharide structures can be conservatively altered by the introduction, overexpression, or deletion of genes encoding specific glycosyltransferases (9 -17), the complexities of oligosaccharide biosynthesis impose some limitations on the use of genetic methods for modulating the structures of cell surface oligosaccharides. As a consequence, alternative methods for decorating cells with chemically defined oligosaccharides are the subject of much recent attention (18).Owing to adv...

Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides

“…3 A-D, respectively. Previously, we reported the incorporation of both natural and͞or 13 C-isotopically labeled sialic acid under identical conditions as described here. The MALDI-MS spectra of those experiments, of which Fig.…”

“…N- (Table 1) was added to the growth media, and its incorporation into LOS was determined by MS. 13 C-Sialic acid ( 13 C-NeuAc) was used as a control. The incorporation of unnatural sialic acids into LOS was first determined by MS analysis (MALDI-TOF) of isolated O-LOS from H. ducreyi grown on solid media impregnated with the various synthetic analogs.…”

“…When fed to cells, unnatural variants of N-acetylmannosamine (ManNAc) are transformed by the sequential action of five enzymes (ManNAc-6-kinase, NeuAc-9-phosphate synthase, NeuAc-9-phosphate phosphatase, CMP-NeuAc synthetase, and a sialyltransferase) to the corresponding unnatural sialosides resident on the cell surface. A variety of mannosamine derivatives successfully navigate this biosynthetic process, including analogs with extended N-acyl groups (11) and analogs modified at the N-acyl position with reactive electrophiles (12)(13)(14). Cells bearing modified sialic acids have shown altered profiles of virus binding (15) and altered responses to adhesion molecules and growth factors (16,17), effects that implicate sialic acid as a determinant of each process.…”

“…In mammalian cells, alterations in the structure of sialic acid can be achieved by metabolic conversion of unnatural substrates (11)(12)(13)(14). When fed to cells, unnatural variants of N-acetylmannosamine (ManNAc) are transformed by the sequential action of five enzymes (ManNAc-6-kinase, NeuAc-9-phosphate synthase, NeuAc-9-phosphate phosphatase, CMP-NeuAc synthetase, and a sialyltransferase) to the corresponding unnatural sialosides resident on the cell surface.…”

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

“…Thereafter, the activated immune system will recognize and eradicate cancer cells that are marked by the artificial antigen. The modification of cell surface glycans via giving cells an unnatural monosaccharide precursor is broadly known as cell glycoengineering, pioneered by 27 Since the glycoengineering of cell surface N-acetyl sialic acid (Neu5Ac) with unnatural derivatives of N-acetyl-D-mannosamine (ManNAc) as the bioengineering precursors has been proven easy and effective, 23, 27-34 our study of the new strategy has been focused on sialylated TACAs.…”