Carbohydrate characterization

- Large-scale approaches for glycobiology

Structures of sugars have long fascinated chemists and biologists, beginning with Emil Fischer's landmark efforts to decipher the isoforms of hexoses more than a century ago [11]. Since then, even with modern techniques, biologists have been outpaced by the difficulty of obtaining a glycosylation profile - the specific complement of glycoconjugates present - of even a single cell. To illustrate that there is no simple task in carbohydrate analysis, Figure 1 shows a few biologically significant glycoconjugates. Even the addition of a single N-acetylglucosamine moiety to a protein to give the O-GlcNAc modification, which regulates numerous biochemical pathways by acting in a yin-yang manner with phosphorylation [12] (Figure 1c), is complicated by its occurrence on hundreds of different cytosolic and nuclear proteins, and on multiple sites within a single protein. The various biological activities of glycosphingolipids, relatively simple sugar-bearing biomolecules exemplified by the ganglioside GM3 (Figure 1d), demonstrate that very subtle changes to sialic acid (N-acetylneuraminic acid or Sia), an unusual nine-carbon sugar found in more than 50 different chemically distinct forms [13], can regulate apoptosis, senescence, and proliferation, thereby highlighting the need for careful analysis of fine structural details.

Moving to larger glycoconjugates, prions are glycosylated proteins that possess only two sites where oligosaccharides attach (Figure 1a). Even so, any one of several dozen different sugar chains can reside at either site; consequently, prions exist as hundreds of distinct entities. The discovery of the influence of carbohydrates on prion infectivity and on the development of spongiform encephalopathies [14,15] underlines the importance of fully defining structural heterogeneity of this kind. As a final example, the heavily glycosylated cell-surface glycoprotein CD34 (Figure 1a), found on hematopoietic cells and epithelial cells, serves as a developmental marker for hematopoietic cells, mediates leukocyte homing, and contributes to cancer metastasis. It bears 20 or more separate oligosaccharide chains [16], implying that, if ten different oligosaccharide structures randomly occur at each site (a conservative estimate), 10²⁰ different forms of CD34 can exist and each of the approximately 10⁴ to 10⁵ copies of this protein found in a typical cell has a reasonable probability of being unique.