4.3 Glycosylation sequences and protein glycosylation
Polysachharide units on proteins may be simple or branched and are almost completely confined to those proteins destined for the cell surface or secretion. The sites and types of glycosylation are determined by the primary structure of the protein and by the availability of enzymes to carry out glycosylation (glycosyltransferases).
N-linked polysaccharides are attached to the –NH2 groups of asparagine and O-linked polysaccharides are attached to the –OH groups of serine and threonine. N-linked glycosylation occurs in the rough ER as the protein is synthesised.
Glycosylation of proteins is important for two reasons: it alters the properties of proteins, changing their stability, solubility and physical bulk, and carbohydrate units may act as recognition signals that are central to aspects of protein targeting and cellular recognition.
N-glycosylation begins in the ER lumen and carbohydrates are further processed after transport of the protein to the Golgi apparatus. A specific sequence NXT/S is required, in which N is asparagine and T/S is either threonine or serine and X may be any amino acid except proline or aspartic acid. Not all NXT/S sequences in protein molecules are glycosylated because in some cases they are masked by protein folding. The process of linking the carbohydrate unit through the amide nitrogen atom of asparagine occurs in the ER, whereas the process of linking the carbohydrate unit through the hydroxyl group of serine or threonine occurs in the Golgi. N-glycosylation is the most conserved form of protein glycosylation in eukaryotes, but the modifications of N-linked oligosaccharides in plants and invertebrates often differ greatly from those in vertebrates.
The initial step in N-glycosylation is the transfer of a pre-assembled branched oligosaccharide from a special lipid molecule dolichol to the target asparagine residue, in a single enzymatic step (Figure 28). Dolichol acts as an anchor upon which the carbohydrate unit is assembled by successive addition of monosaccharide units. Assembly of the carbohydrate starts on the cytosolic side of the ER and is completed in the lumen of the ER.
After the addition of the oligosaccharide, and provided the protein has folded correctly, the terminal glucose units are trimmed back by an ER glucosidase, and an ER mannosidase (Figure 29), before being despatched to the Golgi network.
The protein will then bind to chaperone molecules, such as calnexin and calreticulin, and will be retained in the ER until properly folded and released for transport to the Golgi. If it is incorrectly folded it will be released for degradation.
Once in the Golgi network the carbohydrate is further modified by enzymes that add or remove monosaccharide residues (Figure 30). Some carbohydrate residues on glycoproteins are heavily sulfated. For example, the molecules perlecan and agrin, which are components of the extracellular matrix carry heparan sulfate groups, which are polymeric carbohydrates that are highly negatively charged under physiological conditions. The enzymes that promote the sulfation of the carbohydrates are located in the Golgi network, and this is the final stage in the maturation of such proteins.
The implications of glycosylation for the structure and function of a glycoprotein are so far-reaching that the regulation of protein glycosylation and its effect on the subsequent structure and function of proteins are extremely important in cell biology. For example, recent research into prion diseases has highlighted the impact of glycosylation on the structure of the prion protein, PrP, and its propensity to misfold if incorrectly glycosylated. Misfolded PrP can recruit further PrP molecules that are highly resistant to degradation, which leads to PrP deposition in the nervous system and death of nerve cells.