Leigh Revers

The chemoenzymatic synthesis of the core trisaccharide of N-linked oligosaccharides using a recombinant β-mannosyltransferase

The chemical synthesis of the beta-mannosyl linkage of N-glycans has presented a great challenge to synthetic carbohydrate chemists. We have therefore investigated the application of beta-mannosyltransferases to the preparative synthesis N-linked core oligosaccharides. In this paper we report the chemoenzymatic synthesis of beta-D-mannopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose on a preparative scale using a phytanyl-linked acceptor in the presence of a recombinant beta-(1-->4)-mannosyltransferase.

The chemoenzymatic synthesis of neoglycolipids and lipid-linked oligosaccharides using glycosyltransferases

The application of glycosyltransferases to the chemoenzymatic synthesis of neoglycosphingolipids and lipid-linked oligosaccharides allows the regio- and stereoselective formation of glycosidic bonds. In our laboratory galactosyl-, sialyl-, and fucosyltransferases have been used to assemble oligosaccharide headgroups directly on a sphingosine derivative without the need for any protection group strategies, including the Lewisx antigen. In complementary studies on N-linked oligosaccharide biosynthesis, chemically phosphorylated dolichol analogues have been tested as substrates for Dol-P-Man synthetase. Also, the substrate recognition of the core beta-1,4-mannosyltransferase from yeast has been investigated using a range of chitobiose derivatives as potential substrates.

And What About O-Linked Sugars?

To many glycobiologists, O-linked oligosaccharides are proverbial ‘Cinderellas,’ uninvited to the glycosylation ball. This tendency to side-line O-linked sugars is most likely to have arisen because these structures are less easily classified than their N-linked ‘ugly sisters:’ indeed, their presence is often difficult to predict, there being no readily-identifiable peptide consensus sequence as is the case for N-linkages. Moreover, while eukaryotic glycoproteins can only be elaborated with three types of N-linked oligosaccharide (complex, hybrid or oligomannose), containing the signature core Man(β1–4)GlcNAc(β1–4)GlcNAc(β1-N)Asn, this situation is not paralleled in the world of O-linkages. In fact, the number of structurally-diverse classes of O-linked oligosaccharides continues to grow and it is likely that many unexpected linkages await discovery. As we have already seen in Chapter 2, and as we discuss below, these O-linked oligosaccharides can have important structural and functional effects on their protein partners, and for this reason alone, they should not be discounted.

Expect The Unexpected: Unfamiliar Glycosylations

There is an understandable tendency in glycobiology to concentrate on mammalian glycosylation, particularly human glycosylation, or the glycosylation of laboratory or domestic animals, since this subject is considered the most relevant to medically-oriented research. However, the human body is challenged daily by glycoproteins in foods and pollens, as well as by microbial organisms whose cells are themselves possessed of their own glycocalyx which consists of a liberal covering of potentially immunogenic carbohydrate structures. Moreover, the implementation of glycotechnology has brought with it possible glycohazards in the shape of novel antigens that invoke an undesirable immune response. The prospect of human xenotransplantation using organs harvested from pigs (see Chapter 7), and the clinical use of recombinant glycoproteins that have been produced in cells derived from other mammals or phyla (see Chapter 9) will inevitably lead to humans being exposed to foreign, and hence potentially immunogenic forms of glycoproteins. Consequently, the majority of this chapter is concerned with a description of some of the alternative, medically-relevant forms of protein glycosylation that can give rise to these problems.

Branching Out: Constructing The Antennae Of N -Linked Sugars

The study of glycobiology in the 1970s and 1980s was dominated by the determination of oligosaccharide structures and the search for the enzyme sources that could make them. The wide variety of oligosaccharide structures that were subsequently discovered inevitably led to the classic questions of why this diversity exists and how it has come about. More recently, scientists have examined the function of protein glycosylation and the molecular biology of the enzymes that are involved in this complex process. Indeed the current surge of interest in glycobiology is founded on the widespread belief that oligosaccharides, on account of their diversity, must have a string of roles to play in modulating and altering interactions between proteins, lipids, cells or indeed, entire organisms. As a result, it has become clear that the multiplicity of mammalian oligosaccharides arises from a complex set of processing and elongation events, to which the nascent oligosaccharides are subjected following their initial transfer to protein.

Adding The Finishing Touches: Terminal Elaborations

The remarkable diversity of eukaryotic N- and O-linked oligosaccharide structures is not only a consequence of the branching phenomena we described in Chapters 5 and 6, but can also result from the modification of the non-reducing termini of these branches: that is, the ends of the ‘antennae.’ In fact, it is the terminal groups of these antennae that are the ‘business end’ of major oligosaccharide-ligand interactions, such as those which occur in some intercellular adhesion events, and also between numerous pathogens and their target host cells. Exemplary are the sialic acids which are often part of the ligands for microbial invaders, and also fucosylated structures which are important in determining intraspecies variation and the migrational behaviour of leukocytes.

Sugars and Proteins: Getting It Together

The discovery of major roles for carbohydrates in biology is far from a recent development. Indeed, our knowledge of the existence of the simple sugars found in natural foodstuffs, such as honey, and their use in the fermenting process is as old as civilised man himself. Moreover, beer- and wine-making is by far the earliest practical exploitation of any biological transformation. However, following the advent of modern science and the triumphant elucidation of the pathways of carbohydrate metabolism during the first half of the twentieth century (and of the defects responsible for many of the associated human disorders, such as galactosaemia, shortly afterwards), sugars were prematurely consigned to the margins of scientific research. By the spring of 1953, amid a flurry of activity, Francis Crick and James Watson had unveiled their proposed structure for DNA,1 heralding an unprecedented sea-change in scientific thinking and providing scientists with a new focus. Unfortunately, in contrast to the ‘life molecule,’ carbohydrates continued to hold little appeal for the vast majority of scientists: after all, their structures and functions were at the time considered well-defined, and their biology, at best, unexciting. As Nathan Sharon, a pioneer in the field of glycoproteins, notably remarked in 1993, carbohydrates were long regarded as ‘second-class citizens’ of the cell.2 This is not to say that researchers failed to appreciate the fundamental importance of carbohydrates in living systems: organisms as diverse as bacteria and humans had been found to metabolise dietary sugars (be they simple sugars such as glucose, sucrose and lactose, or much larger carbohydrates like starch and cellulose) to provide energy, or, in the higher species, to appropriate them for the production of polysaccharides, a class of high molecular-weight carbohydrate polymers.

Core Issues: Building The Groundwork for N-Linked Sugars

Proteins destined for export out of the eukaryotic cell, retention within the secretory-endocytotic pathways (including the Golgi apparatus and lysosomes), or incorporation into the plasma membrane, are synthesised by ribosomes that bind to the cytoplasmic face of the endoplasmic reticulum (ER), a continuous system of membraneous tubules extending throughout the cell. As shown in Figure 1, the ribosomes are targeted to this location by the N-terminal signal sequence of the nascent protein: here they associate with the signal recognition particle (SRP), an association which leads to translation being temporarily halted. The SRP dissociates from the complex, following binding to a receptor or ‘docking protein’ which is integral to the ER membrane, and this results in the translational block being lifted; thereafter translocation of the polypeptide chain across the membrane and into the interior space, or ‘lumen’, of the ER can proceed. Once the growing peptide chain is exposed to the ER lumen, various co-translational processes can take place such as cleavage of signal sequences, protein folding and, of course, N-linked glycosylation.

Complementing The Cell: Glycoform Synthesis In Vitro

One of the many challenges in the field of glycobiology is the isolation of the pure glycoforms of a glycoprotein since the study of these glycoforms, or their synthetic analogues, is likely to provide a unique insight into the very nature of the glycoprotein itself. In particular, since glycoforms contain a well-defined oligosaccharide side chain at each glycosylation site, they are invaluable tools in understanding the relationship between oligosaccharide sequence and glycoprotein function: one of the holy grails of biology.

Icing On The Cake: Summary And Future Directions

It has been said that glycobiology, the science that encompasses the study of protein glycosylation, is a ‘new frontier in biology.’ In the previous nine chapters we have attempted to support this assertion by discussing the what, why and how of protein glycosylation, thereby illustrating that it is not only a ubiquitous modification, but that it also has an important influence on the structure and function of glycoproteins.