Anthony H. Merry

Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds

Symbolic diagrams are commonly used to depict N‐ and O‐linked glycans but there is no general consensus as to how individual constituent monosaccharides or linkages are shown. This article proposes a system that avoids ambiguities inherent in most other systems and is appropriate for both hand drawing and computer applications. Constituent monosaccharides are depicted by shapes modified to show OAc, deoxy, etc. Linkage is indicated by the bond angle and anomericity by solid (β) or dashed (α) lines.

A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.

The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N‐linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N‐glycans from the protein in a gel by optimised peptide‐N‐glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2‐amino benzamide. The labelled glycan pools (containing 50–100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix‐assisted laser desorption/ionization‐mass spectrometry, which requires 10–20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.

Symbol nomenclature for representing glycan structures: Extension to cover different carbohydrate types.

This Viewpoint article addresses comments made on our original article describing a symbolic system for the depiction of N‐ and O‐linked carbohydrate structures and proposes a method for extending the symbol set to include monosaccharides commonly found in carbohydrates present in bacteria and plants. As before, basic monosaccharides are shown by shape with one or more additions such as solid fill or additions of lines, crosses or dots to represent functional groups. The use of colour to differentiate constituent monosaccharides is avoided, thus enabling the system to be used in a variety of formats. Linkage and anomericity are shown by the angle and type of line connecting the symbols. In this extended version, new symbols are proposed for additional hexoses and it is proposed that pyranose and furanose forms of the monosaccharides could be shown by solid or broken outlines to the symbols. Conventions for depicting the presence of multiple functional groups such as deoxy‐(NH2)2 are also discussed. It is hoped that these proposals will stimulate discussion so that a consensus can be reached as to how the glycobiology community can best convey complex information in a simple manner.

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12–15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N‐linked and O‐linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone‐forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O‐glycans of both control groups and the N‐glycans of the black control samples were significantly more sialylated than those of the white stone‐formers. This demonstrates a possible association between low‐percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal–cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.

ROLES FOR GLYCOSYLATION IN RECEPTOR- LIGAND INTERACTIONS IN THE IMMUNE SYSTEM

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDG). In these diseases, where sugars may be entirely absent or incorrectly processed, many proteins are affected [c1] such that their biological functions are compromised. This disorder gives rise to a wide range of severe clinical conditions, including some that involve the immune system. Most of the key molecules involved in the innate and adaptive immune response are glycoproteins and the glycans play important roles in recognition events that are critical for triggering the biological functions of the proteins. Such roles include quality control to ensure correct protein folding, the loading of major histocompatibility class I (MHCI) peptide antigens, the geometry of the T-cell synapse and the interaction of MHCI with CD8. In the humoral immune response, multiply presented IgA glycans bind pathogenic bacteria and specific glycoforms of IgG can, when clustered, activate the complement pathway through engaging the mannose binding lectin. The recognition of an unusual cluster of mannose residues on the HIV gp120 envelope glycoprotein by a novel domain swapped antibody, isolated from a patients serum, represents a new paradigm for antigen recognition. The broadly neutralising 2G12 IgG antibody has four closely spaced potential combining sites and may suggest a novel means to target clustered glycan arrays on pathogens.

Glycoproteomics: High-Throughput Sequencing of Oligosaccharide Modifications to Proteins

Genomics establishes the relationship between biological processes and gene activity. Proteomics (James 1997), which relates biological activity to the proteins expressed by genes, is fundamental to our understanding of biology. It is the proteins, rather than the genes that encode them, which engage in biological events (Wilkins et al. 1995). Furthermore, most proteins contain post-translational modifications which are the products of enzyme reactions. Since the enzymes are coded for by different genes, the complete structure of an individual protein cannot be determined by reference to either a single gene or the protein sequence alone. One of the most common ways that a protein is modified is by the process of glycosylation, in which oligosaccharides are attached to specific sites encoded in the primary sequence of the protein (Dwek 1996).