Characterization of Protein Glycosylation
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The majority of proteins are posttranslationally modified, the most significant change being glycosylation, i.e., the attachment of one or more oligosaccharide chains. Because of their long history, but also relative neglect until recently, the terminology for saccharides is diverse. Also a major problem in the glycosciences is that many different methods are necessary for oligosaccharide analysis, and this does not at first seem straightforward. I hope this chapter will demystify the structures and the analysis of glycoconjugates (glycoproteins, GPI-anchored proteins, glycolipids, and proteoglycans). The terminology is in fact easy to follow. It has simple beginnings: from glucose comes the generic term glycose, which is used in words such as glycosidic ring, glycoprotein, and so forth; from sucrose (a disaccharide of glucose and fructose) comes the word saccharide and, hence, oligosaccharide chain. In addition to glucose (Glc), there are seven other possible orientations of hydroxyl groups in hexoses of the formula C 6 H 12 O 6 (from whence comes the term carbohydrate) in the series allose (All), altrose (Alt), Glc, mannose (Man), gulose (Gul), idose (Ido), galactose (Gal), talose (Tal). However, in addition to hydroxyl groups on the ring carbons, there are also acetamido groups (Fig. 1 ), e.g., at C-2 in n -acetylglucosamine (GlcNAc) and n -acetylgalactosamine (GalNAc), and at C-5 in n -acetylneuraminic acid (NeuAc). There may also be present sulfate and phosphate esters. Other commonly occurring monosaccharides are the 6-deoxyhexose fucose (Fuc), the pentose xylose (Xyl), and the C-6 carboxyl uranic acids, glucuronic acid (GlcA), iduronic acid (IdoA), and galacturonic acid (GalA). The monosaccharides are linked together between the hydroxyl groups numbered around the glycosidic ring as shown in Fig. 1 and with α or β (anomeric) configuration, depending on the ring geometry ( 4 C 1 or 1 C 4 for hexopyranoside rings) and linkage above or below the plane of the ring (Fig. 1 ).
Fig. 1. (A) There are two alternative forms for portraying monosaccharides as shown here for β- D - N -acetylglucosamine (GlcNAc). Different monosaccharides vary by the number and orientation of then functional groups, i.e., OH, NHAc, and the like Compared to GlcNAc, GalNAc has the C-4 hydroxyl group above the plane of the ring. In addition to linkage to each other via one or more (giving branching) hydroxyl group, monosaccharides and oligosaccharides are also linked to protein and lipid. The main linkages are GalNAcα to the hydroxyl group of Ser or Thr ( O -linked, mucin type), Xylα to the hydroxyl group of Ser (proteoglycan type), GlcNAcβ to the acetamido nitrogen of Asn ( N -linked) or to the hydroxyl group of Ser ( see Chapter 2 ), and Glcβ to ceramide (glycolipids). (B) Sialic acids are a family of monosaccharides where R = CH 3 -CO-( N -acetylneuraminic acid) or CH 2 OH-CO-( n -glycolylneuraminic acid); the hydroxyl groups can be substituted with various acyl substituents, and those at C-8 and C-9 by additional sialic acid residues.
