Glycoproteins - Hughes, R. 1985

Biosynthesis
Localization of the glycosylation process within the cell

Up to this point, we have considered The properties of specific Glycosyltransferases studied in vitro. However, glycoprotein Biosynthesis is a highly ordered intracellular process. Consequently, we must now examine the temporal appearance of newly synthesized Glycoproteins during their assembly, as well as the Intracellular Localization of glycosylation reactions. Glycoproteins are integral components of most, and perhaps all, intracellular membrane systems; furthermore, the majority—though by no means all—secreted Proteins are glycoproteins. Nevertheless, CARBOHYDRATES are presumably not an obligatory signal for secretion, since certain proteins, such as procollagen, continue to be secreted from Cells in which glycosylation is blocked. This block may result either from genetic Mutations in the metabolic pathways responsible for glycosylation or from the action of various protein glycosylation inhibitors. The most widely used inhibitor of this type is tunicamycin, which prevents the synthesis of dolichol diphosphate-N-acetylglucosamine at the initial stage of N-glycan assembly. However, recent studies, discussed in Chapter 4, indeed indicate that preventing protein glycosylation frequently leads to the mislocalization of glycoproteins within The Cell or results in the secretion of glycoproteins that would normally remain intracellular. The orderly sequence of events in glycosylation ensures the proper trafficking of glycoproteins from the site of Polypeptide chain synthesis to their final terminal stage of synthesis. Therefore, it is essential to understand where within the cell protein glycosylation takes place.

There is little doubt that the polypeptide moiety of glycoproteins is synthesized by Ribosomes bound to the membrane system of The Endoplasmic reticulum (the rough endoplasmic reticulum). If radioactive Monosaccharides are administered to cells actively synthesizing glycoproteins, autoradiography of cell sections or subcellular fractionation can readily demonstrate that the attachment of N-acetylglucosamine and mannose to the polypeptide occurs in the rough endoplasmic reticulum. Pioneering work in this field was conducted by Claude Leblond from McGill University. Using Structure/131.html">Similar Methods, it was shown that fucose, galactose, and N-acetylmannosamine—a direct and specific marker for sialic acids—are added predominantly in smooth membranes, particularly those of the Golgi apparatus. These observations are supported by direct assays of specific glycosyltransferases in subcellular fractions isolated from homogenized cells. For instance, the Golgi membranes contain the transferases responsible for assembling trisaccharides containing sialic acid, galactose, and N-acetylglucosamine in N-glycans, as well as for fucosylation. Recently, The Use of fluorescent specific Antibodies against galactosyltransferase in whole-cell preparations revealed a dense 'cap,' which evidently represents a portion of the Golgi complex [45]. In contrast, dolichol-stimulated glycosyltransferases are localized in the rough endoplasmic reticulum. Taking all these data into account, glycoprotein biosynthesis is schematized in Fig. 3.14.

According to the model proposed by Blobel and Dobberstein [46], nascent Polypeptides possess a signal peptide sequence that directs the attachment of ribosomes to the endoplasmic reticulum membranes. This association is stabilized by specific endoplasmic reticulum Membrane Proteins that exhibit affinity for particular Regions of the ribosomes. Translation of mRNA continues with the vectorial discharge of the growing polypeptide into the lumen of the rough endoplasmic reticulum cisterna. At an early stage, the signal peptide is cleaved by specific proteolysis, after which folding of the polypeptide chain begins and a glycosylation-accessible site on the polypeptide is exposed. Glycosylation proceeds via dolichol intermediates and involves The transfer of the oligosaccharide (glc)₃(mаn) ₉(glcNAc)₂ to exposed asparagine residues of the peptide chain. The oligosaccharide-transferring transferase is tightly membrane-bound [47]. Soluble Glycoproteins are completely released into the cisternal lumen upon completion of mRNA Translation, whereas nascent membrane glycoproteins remain integrated within the endoplasmic reticulum membranes. They are anchored via a hydrophobic peptide segment, much like Glycophorin.

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The next stage in glycoprotein biosynthesis involves The conversion of the rough endoplasmic reticulum into ribosome-free smooth membranes through the dissociation of the endoplasmic reticulum or the lateral migration of glycosylated proteins to regions of the endoplasmic reticulum deficient in membrane ribosome-binding proteins. The transport of newly synthesized glycoproteins to the Golgi apparatus is apparently mediated by vesicles coated on the cytoplasmic side with the protein clathrin [48]. While these coated vesicles were previously thought to be involved in the endocytic uptake of extracellular material, their role in Intracellular Transport is becoming increasingly evident. The membranes of the Golgi apparatus, in turn, give rise to various vesicular compartments, including Lysosomes and secretory vesicles. Further elaboration of N-glycans occurs at this stage, since the glycosyltransferases responsible for the subsequent assembly and termination of N-glycans are localized in the Golgi membranes. Processing glycosidases (glucosidases, mannosidases) co-fractionate with the smooth membrane fraction. Other events also take place, such as the phosphorylation of N-glycans, which appears to be crucial for the proper routing of glycoproteins into lysosomes (Section 4.3.2). Vesicles budding from the Golgi apparatus are again coated with clathrin, a requirement for their participation in glycoprotein exocytosis. They subsequently fuse with The Plasma Membrane, and the biosynthetic products are either secreted or remain as integral Components of the surface membrane. Notably, the topology of fusion accounts for the experimentally observed Asymmetry of the plasma membrane. Thus, the glycosylated peptide regions that reside within the endoplasmic reticulum cisternae end up on the outer surface of the cell following fusion.

This simplified scheme of highly complex processes cannot, of course, elucidate specialized problems such as The biosynthesis of band 3 glycoprotein. One possibility is that the signal peptide of certain newly formed polypeptides remains permanently embedded in the endoplasmic reticulum membrane. The elongating polypeptide may be threaded back and forth across the endoplasmic reticulum membrane multiple times during elongation, ultimately becoming glycosylated at its C-terminal domains.

The formation of O-glycans appears to be a relatively late event in biosynthesis [49]. The attachment of monosaccharides immediately adjacent to the polypeptide and of sialic acids, as well as the conversion of N-acetylneuraminic acid via hydroxylation and Acetylation reactions, are all processes associated with the membranes of the smooth endoplasmic reticulum or the Golgi apparatus. The distinct subcellular localization of core N-glycan assembly and N-glycan Termination reactions, on the one hand, and O-glycan assembly, on the other, helps explain the existence of different assembly mechanisms. Such a close association of the nascent polypeptide with endoplasmic reticulum membranes evidently entails the obligatory participation of lipid-linked intermediates, whereas later biosynthetic events involving mature glycoproteins proceed via simpler pathways.