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- Large-scale approaches for glycobiology

mcith_gb-2005-6-11-236-1.jpg Figure 1  Systems and molecular complexity in glycobiology. (a) The glycosylation machinery consists of an intricate network of metabolic pathways that interconvert monosaccharides and produce high-energy sugar nucleotides (full details of the pathways are available in [9]). The hexosamine pathway [46] that converts glucosamine (1) to UDP-N-acetylglucosamine (UDP-GlcNAc) (2) is highlighted in blue. The versatility of the glycosylation machinery is epitomized by the conversion of UDP-GlcNAc into N-acetylmannosamine (ManNAc) (3), a sugar that is metabolically converted to CMP-sialic acid (CMP-Sia; 4) by the pathway highlighted in red. UDP-GlcNAc and CMP-Sia, together with seven other sugar nucleotides, are transported into the endoplasmic reticulum (ER) and Golgi apparatus (5), where they are used for the production of complex oligosaccharides (6) that comprise the glycosylation profile of the cell surface. This profile is made up of proteins (such as the prion protein and CD34, shown here) and glycolipids such as ganglioside GM3, a glycosphingolipid. Sialic acid (Sia) is a ubiquitous terminal modification. (b) The chemical structures of glucosamine, UDP-GlcNAc, UDP-ManNAc, and CMP-Sia. (c) As well as being used to build complex oligosaccharides, UDP-GlcNAc is a high-energy building block that provides the GlcNAc residue required for O-GlcNAc protein modification in the cytosol [13]. (d) Slight modifications to the chemical structure of CMP-Sia elicit profound changes in biological activity. The membrane glycosphingolipid ganglioside GM3 (center) is converted to pro-apoptotic gangliosides GD3 by addition of Sia (top), whereas deacetylation of GM3 yields de-N-acetyl GM3, which has a growth stimulatory effect.

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mcith_gb-2005-6-11-236-2.jpg Figure 2  Conventional low-throughput glycoconjugate characterization and steps that will improve throughput. Current strategies for oligosaccharide identification include multiple time-consuming steps including, but not limited to, (1) isolation of individual glycoconjugates, such as prions or CD34 (see Figure 1), from a cell or tissue; (2) the detachment and purification of each oligosaccharide from a particular glycoconjugate; and (3) a one-at-a-time structural characterization and identification. Each of these steps currently requires multiple procedures and method of analysis [21], as illustrated in the boxes for steps (1) and (3). Streamlined methods now under development, such as (4) the coupling of isolation by glycoblotting with identification by mass spectrometry (MS) [35], and automated interpretation of spectra [30], are also shown. These methods, along with array-based technologies (see Figure 3), offer hope for high-throughput glycan characterization in the near future.

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mcith_gb-2005-6-11-236-3.jpg Figure 3  Oligosaccharide and carbohydrate-binding protein arrays. (a) Oligosaccharide microarrays are used to detect and characterize carbohydrate-binding proteins. They are constructed by (1) spotting known oligosaccharides (either synthetic or naturally isolated) onto a solid surface such as a treated glass slide in a predetermined array. Whole cells can be bound to the array (2), but it is more common to first fractionate cells or tissues to isolate (3) putative carbohydrate-binding proteins. (b) Arrays of known carbohydrate-binding proteins (either lectins or monoclonal antibodies) are used to detect and characterize oligosaccharides. They are produced by printing spots of the proteins onto a suitable surface (1). Again, whole cells (2) can be bound to the array, but more usually (3) their cell-surface oligosaccharides will be isolated and used. Both types of array can be used for a variety of purposes.

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