Translational glycobiology: from bench to bedside

Abstract

The importance of sugars to protein function is real and is of significant clinical relevance. Technology advances enable large population studies to be carried out, shedding light on individual sugar variation and variations with time. Three-dimensional mass spectroscopy on solid pathological specimens is going to open up a whole new world of pathology visualisation. The door is now open to exploit carbohydrate recognition in new therapeutics by identifying novel biomarkers in cancer to aid diagnosis, and also providing therapeutic targets for treatment. Glycan age correlates with biological age. This means we can map the reversal of biological age with exercise and diet.

The relevance of glycobiology to medicine first became apparent in the early 1990s in relation to rheumatoid arthritis and immunoglobulin glycosylation. Since then, there has been an exponential increase in our knowledge of the importance of sugars to protein function, and hence their significant clinical relevance. One of the big quantum leaps has been with regard to technology. High-throughput glycan analysis is now so sophisticated that what once took a week can now be carried out in an hour. This has enabled experiments that can accommodate analyses of samples from thousands of patients.¹ Large population studies have identified significant variations of glycosylation between individuals, but also within an individual through time.² This, together with our increase in molecular genetic knowledge, enables us to link genetic associations with glycan analysis. As an example, if a gene is known to be associated with a particular disease (e.g. HLA-B27 and axial spondylitis), the direct association between the presence of this gene with a particular glycan can be established, hence enabling the unravelling of disease pathways. In addition, it is possible to link glycome data of body fluids, such as serum, to genomics, transcriptomics, proteomics, lipidomics and metabolomics data, as well as pathological pathways, to give a more complete overview of disease.

The first genome-wide association studies of the glycome have been published^3–5 and this provided the first insights into the complex genetic control of protein glycosylation. The epigenetic control of protein glycosylation is also being investigated^6,7 and it seems to account for part of the normal glycosylation variation that has been mapped from week to week. Several large clinical studies of the glycome have been initiated.^8–12 These studies identified significant potential of the glycome for biomarker development, but also for understanding physiological relevance of inter-individual differences in protein glycosylation.¹ Hence you will gain a better understanding of what are normal sugar changes associated with maintaining body function, and what the aberrations are in disease. Glycan age correlates with biologic age. It is going to be exciting to chart the reversal of an abnormal biological age with exercise and diet.

Our understanding of sugar biochemistry has been aided significantly by the development of interacting visual glycan databases. The research workers’ motto is ‘building bridges and knocking down walls’ and they certainly have done so, as the relationship between biochemistry, pathology and clinical medicine can now be appreciated. Three-dimensional computer-generated images of glycan structures that once caused regular computer crashes during lectures can now be visualised on the iPhone. The ease of glycan analysis has enabled the development of a number of novel biomarkers that enable clinicians to differentiate normal from abnormal, and active and inactive disease, e.g. inflammatory bowel disease and disease subtypes such as antiphospholipid syndrome and whether or not you are likely to suffer thrombosis. These advances are likely to help clinicians significantly, as even in the 2000s we have few tests of normality and at times have to revert to whole-body scanning to look for disease.

Histopathology is central to diagnosis and treatment and the advent of immunochemistry has made this a very powerful tool. The provision of three-dimensional mass spectroscopy on solid pathological specimens is going to open up a whole new world of pathology visualisation. This will enable the pathologist to be more exact with regard to the pathological mechanisms occurring in the organ tissue under investigation and this will lead to more targeted treatment.

Cellular glycosylation is the product of highly complex biosynthetic pathways controlled by both genes and influenced by the environment. Glycosylation in cells and in specific proteins is therefore highly sensitive to genetic mutations, changes in gene activation or silencing, or environmental factors such as infections, the microbiome, diet, alcohol consumption and smoking. In cancer, several studies have shown that glycosylation can intervene in key steps of the disease progression.^13,14 Glycosylation modulation of receptor function in cancer is an example, where alteration of branched and terminally sialylated glycans have been shown to have major impact on the activation of tyrosine kinase receptors, which leads to increased cancer cell aggressive phenotypes.^15–17 Additionally, specific Asn-glycosylation structures of adhesion molecules, such as E-cadherin, have been shown to induce altered cell adhesion and increased tumour cell invasion.^18,19 Chronic Helicobacter pylori infection initiates a glycosylation-mediated pathway leading to gastric cancer. This is achieved by changes in the gastric cell glycophenotype with increased expression of sialyl-Lewis x antigens due to changes in transcription of glycosyltransferase genes.^20,21

Attached oligosaccharides can protect glycoproteins against proteolysis and this has therapeutic consequences.²² Natural glycosylated interferon-beta is produced mainly by fibroblasts. Recombinant glycosylated interferon-beta used for the treatment of multiple sclerosis is less degraded by extracellular proteases than Escherichia coli-derived and non-glycosylated, recombinant interferon-beta. The consequence is that glycosylated interferon-beta is more stable as proteases have difficulty degrading the protein. In comparison, E. coli-derived non-glycosylated interferon-beta protease degradation is quicker and ‘remnant epitopes’ are produced. Glycosylated interferon-beta leads to less ‘remnant epitope’ activation of reactive T cells and to less neutralising antibody formation against this cytokine.²³ The glycobiology of innate immune molecules, including cytokines, chemokines, proteases and protease inhibitors, is also important in the pathology of common autoimmune diseases. It has been known for decades that large Asn-linked sugars regulate the specific activities of and interactions between cytokine ligands and receptors and the ensuing signal transduction events and biological effects.²⁴ The O-linked oligosaccharides in the gut are key to maintaining a normal microbiome and disruptions in this O-glycosylation pathway is associated with dysbiosis and gut inflammation, and in animal models these abnormal changes can lead to cellular transformation and colon cancer.²⁵ The two important post-translational modifications in autoimmune diseases, proteolysis and glycosylation, thus possess divergent functions. Whereas extracellular proteolysis leads to remnant epitopes and enhances autoimmune reactions, antigen glycosylation seems to have often protective effects.²⁶

The door is now open to exploit carbohydrate recognition in new therapeutics. Ser/Thr-O-linked oligosaccharides are the key ligands for selectins, which are involved in regulating leukocyte trafficking in inflammation and through the lymphatic system. Blocking antibodies to P-selectin have been tested and shown to block selectin function in humans. Crizanlizumab is such an antibody and is showing promise in blocking the vaso-occlusive crises in sickle cell disease.²⁷ Success in this area is likely to lead to its application to other inflammatory disorders, such as arthritis and Crohn’s disease. Therapeutic oral treatment with N-acetylglucosamine is now being tested in clinical trials for inflammatory bowel disease. Certain types of sugars that block angiogenesis may prevent cancer spread. Aging is central to everyone’s thoughts, and perhaps reversing glycan age will reverse biological age.

The field of glycotherapy and development of glycomimetics is indeed an exciting one that has enormous potential. Apart from heparin and its various glycan variants, there are currently a number of other successful commercial products that utilise either isolated, synthetic or glycan mimics (extensively discussed by Peter H Seeberger and Richard D Cummings in their chapter in Essentials of Glycobiology).²⁸ Examples include neuraminidase inhibitors such as zanamivir (Relenza) and oseltamivir (Tamiflu), which target sialic acid and are used to prevent influenza infections and imino sugar inhibitors, miglustat (Zavesca; a glucosylceramide synthase inhibitor) and imiglucerase (Cerezyme), for treating the lysosomal glycosphingolipidoses such as type I Gaucher disease.^28,29

Others include topiramate, a sulfamate-modified fructose diacetonide, which is used as an antiepilepsy drug, and voglibose, an α-glucosidase inhibitor, which is used for lowering blood glucose levels in diabetes. Other glycan-based drugs for the treatment of diabetes include miglitol and acarbose.²⁸

Glycans, coupled to carrier proteins, are also now being extensively utilised in the production of new and highly effective vaccines. These include conjugate vaccines against Haemophilus influenzae type b, against pneumococcal (Synflorix, GSK and Prevnar13, Pfizer), and against Neisseria meningitides.²⁸

The examples listed here demonstrate the fact that the therapeutic opportunities for glycans are immense, as are the innovations around molecule design and optimisation for this widely untapped drug class.

Declarations

Competing interests

None declared.

Funding

None declared.

Ethics approval

Ethical approval was not required as data were anonymised and published in aggregate form.

Guarantor

JA.

Contributorship

AA and JA were responsible for the design and formatting of this article. All authors contributed to data interpretation and revising and editing drafts produced by AA and JA. All authors had full access to and have approved the final version of this manuscript.

Acknowledgements

The authors would like to thank Prof Rita Gerardy-Schahn and the local organising committee for their help with this report (http://jenner-glyco.com/default.asp). Information used in this study are derived from the 12th Jenner Glycobiology and Medicine Symposium ‘Translational Glycobiology – From Bench to Bedside’, 6–9 May 2017, Sheraton Dubrovnik Riviera Hotel, Dubrovnik.

Provenance

Not commissioned; peer-reviewed by Raymond Dwek.