Prototypical Glycoconjugates On Cell Surfaces

Cell surface glycoconjugates can be grouped into four main classes: N-linked glycoproteins, O-linked glycoproteins, glycosyl phosphatidylinositol (GPI)-anchored proteins, and glycolipids. These are the major cell surface hosts of monosaccharides derived from intracellular metabolism. N-Linked glycosylation in eukaryotes is a cotranslational event that occurs in the endoplasmic reticulum (ER) [2,16]. It is found almost exclusively on proteins that contain the consensus sequence AsnXaaSer/Thr (where Xaa is any amino acid except Pro). Glycosylation of the asparagine residue in the appropriate sequon is mediated by a membrane-bound enzyme oligosaccharyl-

Figure 2 Metabolic substrate engineering as a strategy for modulating cell surface oligosaccharide structure. Modified metabolic substrates can intercept a biosynthetic pathway in two ways: the pathway might be inhibited, leading to truncated structures on the cell surface, or the modified substrate might be incorporated into oligosaccharides in place of the normal substrate.

Normal Monosaccharide Substrate

Modified Monosaccharide Substrate


Aminoglycoside Modified Enzyme

transferase. This enzyme transfers an oligosaccharide with the structure Glc3-Man9GlcNAc2 from a dolichol diphosphate precursor to the side chain of particular asparagine residues in the polypeptide chain (Fig. 3). During its journey through the secretory pathway, this tetradecasaccharide is first trimmed and then elaborated to form an N-linked glycan with a high-mannose, hybrid, or complex-type structure [16,17]. Examples of these structures are shown in Fig. 4 [25].

Unlike N-glycosylation, O-glycosylation begins with the transfer of a single monosaccharide residue, usually GalNAc, to a serine or threonine residue of the poly-peptide chain [12]. The GalNAc residue is then further elongated through stepwise enzymatic modifications by glycosyltransferases, giving rise to specific core structures. To date, eight core structures have been identified by NMR spectroscopy or mass spectrometry (Fig. 5) [26-30]. These glycans can then be further elaborated by the addition of a sialic acid or Fuc residue, sulfate, methyl, acetyl, or poly-V-acetyllac-tosamine units. A subset of O-glycoproteins are the proteoglycans, which present long glycosaminoglycan chains from a peptide-proximal xylose residue (Fig. 6).

GPI anchors are involved in cell signaling, protein targeting, and protein secretion [15,23]. The addition of a GPI anchor to a polypeptide is a posttranslational event that occurs in the endoplasmic reticulum (ER). The structures of GPI anchors are very complex. The core structure, consisting of ethanolamine, Man residues, GlcN, and phosphatidylinositol, is conserved from protozoan to mammalian organisms (Fig. 7). However, the peripheral structures vary through species and cell type. The conserved GPI core can be further modified by the addition to the core mannoses of ethanolamine phosphate residues, GalNAc residues, and Man residues. This entire structure is then bound to a lipid that is embedded in the membrane. Similar to N-glycosylation, GPI modification of proteins involves the transfer of a preassembled precursor, en bloc, to the C-terminus of a target protein by amide bond formation with the ethanolamine group.

Glycolipids present oligosaccharide epitopes immediately proximal to the plasma membrane [18-22]. Most glycolipids can be classified into three major groups: sphingolipids, gangliosides, and glycero- and isoprenol-glycolipids [31]. The biosynthesis of glycolipids proceeds by stepwise addition of monosaccharide units to a lipid carrier in the compartments of the secretory pathway. As in the case of glycoprotein biosynthesis, the transfer of nucleotide-activated monosaccharides is mediated by glycosyltransferases. An example of a ganglioside, GM3, is shown in Fig. 8 [32].

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