Pauline M. Rudd

Chapter 3:Changes in Serum N-Glycosylation Profiles: Functional Significance and Potential for Diagnostics

Considering that glycans are ubiquitously present on all cell surfaces, the fact that several human diseases involve disturbed glycan processing pathways comes as no surprise to the emerging field of Glycobiology. Alterations in cellular signalling pathways (either caused by disease specific mutatio...

HPLC Strategies for Profiling and Sequencing Oligosaccharides

The sugars released from a pure glycoprotein often consist of a heterogeneous population containing both neutral and charged oligosaccharides. For example, the single N-glycosylation site in human erythrocyte CD59 is associated with more than 100 neutral and sialylated complex glycans, each representing a different glycoform (1). The existence of such extensive heterogeneity in biologically important glycoproteins requires refined approaches to the analysis of oligosaccharides. The adaptable technology which is described here represents a significant advance towards faster, more automated and more detailed strategies for the rapid profiling and analysis of sugars. Such technologies may be required for major studies, such as the human genome project, which defines DNA in normal and diseased states, and the proteome project, which sets out to analyse the total amount of protein in a living cell. It is worthy of note that genetic diseases are not caused by the genes themselves, but by the products for which the genes code and their post-translational modifications, which include glycosylation. In this chapter two strategies for rapid oligosaccharide analysis are described: Oligosaccharide profiling and detailed structural analysis.

Systems Glyciobiology: from Genome to Glycome

As more therapeutic options become available there is an increasing need for clinical markers that will provide more sensitive and specific early detection of disease. At the same time, improved technologies for monitoring disease progression and response to therapy are required. In many cases, single assays of existing biomarkers are neither sensitive nor specific enough for use as sole screening methods and in general a combination of markers is required. Many systemic diseases, particularly cancer, have been linked with many systems, from genomics to glycomics. Therefore we have developed an automated 96-well plate based strategy for identifying, quantifying and screening potential glycans released from proteins in body fluids as clinical markers. We have constructed a data base of the serum glycome of healthy controls to compare with that of clinical controls and of patients with various diseases including schizophrenia, rheumatoid arthritis, breast, ovarian, lung, stomach, prostate and pancreatic cancers and compared the specificity and sensitivity of the glycan markers with the current markers used in the clinics. Automated data analysis is subsequently fine tuned for each disease opening the way for undertaking large scale clinical trials that may prove useful for diagnosing disease and monitoring progression and therapy. Importantly, the technology has enabled links to be made from the serum glycome to individual glycoproteins, glycoprocessing pathways, signaling transduction pathways and to the genome itself, demonstrating the possibility of probing a whole system for disease associated changes and providing a deeper insight into pathogenesis.

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).