Glycosylation and its role in clearance:
Once glycoproteins have been synthesized, perhaps a more general purpose for the N- and O- linked glycans is to control the persistence of proteins in circulation. Indeed, glycans serve as sorting tags for the intracellular and extracellular trafficking of glycoproteins as a result of their interactions with sugar-binding receptors.
Oligosaccharides terminating with sulphated N-acetylgalactosamine (SO4-GalNAc) play a central role in the expression of biological activity by the pituitary hormone lutropin (LH). The glycosyltransferase that adds GalNAc to the oligosaccharides on LH is protein specific. A highly specific GalNAc-4-sulfotransferase then adds sulphate to the terminal GalNac. As a result LH, but not other glycoproteins synthesized in the gonadotroph, bears this terminal SO4-GalNAc. Although binding and activation of the LH receptor is not dependent on the terminal sugars present on LH, these sugars determine the hormone circulatory half-life. Indeed, the hormones containing the terminal SO4-GalNAc are rapidly cleared by a receptor on the endothelial cells of the liver. Rapid removal from the blood results in a pulsatile rise and fall of circulating LH levels. It can be hypothesized that attachment of this sugar structure is a general tag that signals clearance, so that a brief spike of these hormones is delivered to their target cells.
The rapid removal of desialylated glycoproteins from serum via the hepatic aialoglycoprotein receptor is another saccharide-based turnover system. Since almost all serum glycoproteins are secreted with sialic acid-terminated N-linked oligosaccharides, action of a neuraminidase, which cleaves it off, is first required before the glycoproteins can be cleared by this receptor. The rate of removal from the serum may be greater for desialylated glycoproteins with tri- and tetra-antennary oligosaccharides. Thus the turnover rate of serum glycoproteins mediated by this single recptor could be tailored to individual glycoproteins by attachment of appropriate oligosaccharides.
The serum half-life of a glycoprotein is therefore likely to be a complex function of its interaction with a variety of oligosaccharide receptors.
Prevention of non-specific interactions and protease protection:
Oligsaccharides are particularly well adapted for a role in preventing non-specific interactions between glycoproteins. In the first place, the sugars are large and are able to provide a “fence” around an individual protein molecule that cannot readily be entered by other proteins attached to the cell membrane. Aggregation between proteins can hence be prevented. Moreover, glycans tend to be highly hydrated, and thus shield various portions of protein surfaces from the aqueous solvent . Secondly, sugar-protein interactions that involve single monosaccharide residues have low binding constants. Therefore, non-specific interactions of a sugar with a protein are likely to be short-lived. An example of a case where such non-specific interactions are prevented by the presence of O- and N- linked glycans shall be provided later on when considering the roles of glycosylation in the immune system.
As we saw earlier, glycan structure during glycoprotein synthesis can often provide a signal for ER associated degradation. However, by virtue of their size, oligosaccharides can sometimes may also have the reverse effect in that their shielding of large regions of protein surface may provide protease protection. This is the case for example with the microbial tetanus toxin, whose N-glycosylation blocks the action of an asparagines-specific cysteine endopeptidase. In contrast, microbial organisms which do not contain N-linked sugars can be fully processed to antigenic peptides. This indicates that that glycans can also protect proteins from degradation.
Effects on protein dynamics and stabilisation:
N- and O-linked glycans also affect the dynamics and stability of a glycoprotein. This was exemplified in a comparative study between unglycosylated ribonuclease A (RNase A) and its glycosylated counterpart ribonuclease B (RNase B). Both these enzymes catalyze the hydrolysis of 3’-5’ phosphodiester linkages of ribonucleic acids. RNase B consists of five glycoforms (Man5-9GlcNAc2) and contains a single N-glycosylation site at Asn 34. Crystallographic and NMR studies show that the gross global structure of the enzyme is unaffected by glycosylation. However local differences were observed in 1D 1H-NMR spectra since the rates of exchanges of backbone amide protons for deuterium in both RNase A and B were found to be different for a large number of residues. In general, the presence of oligosaccharide was found to protect residues close to the site of glycosylation (Asn 34) from hydrogen exchange as well as a large number of residues throughout RNase B which are remote from Asn 34. This happens because the presence of oligosaccharide results in reduced dynamic fluctuations in the glycosylated enzyme relative to its unglycosylated counterpart. This in turn may confer stability to the RNase B protein structure. Supporting evidence came from a study of the folding and unfolding of RNase which showed that unfolding by guanidium chloride was decreased for RNase B relative to RNase A. Furthermore, CD analysis of the thermal denaturation of glycosylated and unglycosylated ribonuclease enzymes has shown that the carbohydrate moiety has a small stabilizing effect on the protein. Thus the presence of oligosaccharide results in increased stability of the protein in partially folded states.
Glycosylation and T cell recognition of antigen presenting cells (APCs):
The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. Multiple stages and events are involved in the T cell recognition of APCs, and as we will see, N- and O-linked glycans play an important role in most of these processes.
The specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the APC. Their respective cell adhesion glycoproteins such as CD2 (located on the surface of killer T cells) and CD48 in mice (or CD58 in humans), help to form a cell junction providing a molecular spacer between opposing cells. Interaction between CD2 and its ligands occurs through homologous binding surfaces located in the amino-terminal domains distal to the membrane surface. Oligosaccharides located on the membrane proximal domains of CD2 and CD48 seem likely to restrict the orientations of binding faces of the cell adhesion molecules CD2 and CD48. This defined orientation increases affinity of CD2 and CD48 for each other, contributing to the alignment of opposing cell surfaces.
In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T cell receptors (TCRs) requires mobility on the membrane surface. The size and location of glycans prevent non-specific protein-protein interactions such as aggregation of TCRs as well as limiting the geometry of the interactions of the proteins in central clusters. In general, prevention of certain interactions with molecules on the same surface results in greater translational freedom, and hence faster diffusion. Oligosaccharides hence play a role in the transport of pMHC into the centre of the junction.
In the third stage, the sugars play a role in stabilising the conformation of the complexes in the synapse and protecting them from the action of proteases during signal transduction, a process which may take several hours.
N- and O-linked glycans hence serve a variety of purposes and have a significant effect on the functions of the glycoproteins to which they are attached. The essential role for carbohydrate in a biological function of a glycoprotein does not necessarily imply a direct role in mediating that function. Indeed, many N- and O-linked oligosaccharides help stabilise protein structures and serve to shield various portions of protein surfaces from the aqueous solvent and from proteases. In other cases, these sugars serve as targets and hence influence intracellular and intercellular trafficking.
However, once the general question “What is the function of glycosylation?” has been answered satisfactorily, specific questions of the form “What is the function of this particular glycan on this particular glycoconjugate?” have to be tackled, and it is they who will probably form the basis for a great deal of future work.
