8 DOI 10.1002/pmic.200700917 Proteomics 2008, 8, 8–20 Frontiers in glycomics: Bioinformatics and biomarkers in disease An NIH White Paper prepared from discussions by the focus groups at a workshop on the NIH campus, REPORT Bethesda MD (September 11–13, 2006) Nicolle H. Packer1*, Claus-Wilhelm von der Lieth2* {, Kiyoko F. Aoki-Kinoshita3, Carlito B. Lebrilla4, James C. Paulson5, Rahul Raman6, Pauline Rudd7, Ram Sasisekharan6, Naoyuki Taniguchi8 and William S. York9 1 Macquarie University, Sydney, Australia 2 German Cancer Research Center, Heidelberg, Germany 3 Soka University, Tokyo, Japan 4 University of California Davis, CA, USA 5 Scripps Research Institute, La Jolla, CA, USA 6 MIT, Cambridge, MA, USA 7 University College Dublin, Dublin, Ireland 8 Osaka University, Osaka, Japan 9 Complex Carbohydrate Research Center, Athens, GA, USA Key issues relating to glycomics research were discussed after the workshop entitled “Frontiers in Received: September 26, 2007 Glycomics: Bioinformatics and Biomarkers in Disease” by two focus groups nominated by the Accepted: November 1, 2007 organizers. The groups focused on two themes: (i) glycomics as the new frontier for the discovery of biomarkers of disease and (ii) requirements for the development of informatics for glycomics and glycobiology. The mandate of the focus groups was to build consensus on these issues and develop a summary of findings and recommendations for presentation to the NIH and the greater scientific community. A list of scientific priorities was developed, presented, and dis- cussed at the workshops. Additional suggestions were solicited from workshop participants and collected using the workshop mailing list. The results are summarized in this White Paper, authored by the co-chairs of the focus groups. Keywords: Bioinformatics / Biomarkers / Glycobiology / Glycomics Correspondence: Professor Nicolle H. Packer, Department of 1 Mandate Chemistry and Biomolecular Sciences, CORE of Biomolecular Frontiers, Rm 307, Building E8C, Macquarie University, Sydney, To describe and identify the state and the potential of glycans New South Wales, NSW 2109, Australia as biomarkers for diseases and to recommend the tools that E-mail: [email protected] need to be supported so as to develop and enhance bio- Fax: 161-2-9850-8313 marker discoveries that employ the glycome. Abbreviations: ADCC, antibody-dependent cell-mediated cyto- toxicity; AFP, alpha-fetoprotein; FSH, follicle-stimulating hor- mone; GAGs, glycosaminoglycans; N-glycans, N-linked oligosac- * Both authors contributed equally to this work. charides; O-glycans, O-linked oligosaccharides { Deceased; see note added in proof. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2008, 8, 8–20 9 2 The need for glycomics in biomarker The surface of all cells are elaborated by the addition of discovery sugars to the membrane macromolecules—the ability of the structure of these sugars to be fine-tuned affects the com- Glycans include short carbohydrate chains (i.e., oligo- munication between cells, serves as docking pads for bacteria saccharides) and larger molecules containing many carbo- or viruses, and provides clinically useful markers for dis- hydrate residues (e.g., glycosaminoglycans) that are bound to eases. proteins or lipids but may also be free. Glycomics—the study of the biological role of carbohydrates—is opening up new research fronts, and pharmaceutical and biotechnology 2.1 Glycans are involved in a host of disease-related companies are probing the glycome for targets for novel functions drugs or new therapies for infectious disease, cancer, and metabolic disorders (Fig. 1). The importance of carbohydrates in general metabolic Two years ago, in the Massachusetts Institute of Tech- processes and the malfunctions resulting in disease are nology’s Technology Review, Dr. Terry van der Werff nomi- evidenced by the extremely diverse physical and mental nated glycomics as one of ten emerging technologies that deficiencies evident in the 16 or so congenital disorders of will change the world. glycosylation that have been classified (CDG Syndrome). “The glycome was regarded as a much bigger challenge There are also several congenital muscle dystrophies than the genome or proteome—the language of sugars was designated as alpha-dysglycanopathy due to the mutation just too complex. But all that has changed dramatically in the of glycosyltransferases involved in O-mannosylation of past decade. New technologies are facilitating exploration and alpha-dystroglycan. These genetic defects are often a a new understanding of the glycome, and chemists now have mutation in a single glycosyltransferase gene causing the tools to assemble large, complex carbohydrate molecules widespread phenotypic effects usually devastating to the from simple monosaccharide components. It’s now obvious patient. that carbohydrates play significant roles in healthy biology as The primarily extracellular location of the glycans means well as disease. Glycomics should be ranked equally with the that they are intrinsic to cell–cell interactions such as patho- genome and proteome, and developed as rapidly as possible” genic infection, fertility, immunity, and cancer. Glycans thus (Prof. Mark Von Itzstein 2006, Institute of Glycomics, Aus- provide an alternative, and to date an under-exploited, mo- tralia, and one ofthe discoverersofthe sugar enzyme inhibitor lecular class with which to look for disease biomarkers, drug ™ of influenza infection (Relenza )). targets, and therapeutics. Figure 1. The critical role of gly- comics in systems biology. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 10 N. H. Packer et al. Proteomics 2008, 8, 8–20 2.1.1 Glycans are potential biomarkers for cancer At the workshop there was much discussion of this most important question of carbohydrates as cancer biomarkers, Most tumor antigens are glycoproteins or glycolipids and with the obvious potential to collaborate and learn from the their monoclonal antibodies were generated against peptide established Early Detection Research Network (EDRN; portions of the glycoprotein, or sugar portions of glycolipid, http://edrn.nci.nih.gov/) whose mission is to discover, one or two decades ago. Although N-glycans are not known develop, and validate biomarkers for early detection of can- to be immunogenic, a combined protein–sugar epitope on a cer. The principles that EDRN operates by in validating di- glycoprotein can form a very specific immunogen. Well- agnostic biomarkers can serve as a good model system for known clinical cancer diagnostic tests use such existing gly- carbohydrate biomarkers of cancer and other diseases. coprotein cancer markers (CA125, CA19-9, CEA, CA15-3, MUC1), which are on the whole not specific to a particular 2.1.2 Glycans are potential targets for drugs cancer and for which the exact epitope has largely not been defined. Alpha-fetoprotein (AFP) and prostate specific anti- Since glycans are located at the cell surfaces of both the gen (PSA) are glycoproteins which are tissue specific and microorganism and the mammalian host cell, they present thus are being used for monitoring primary hepatoma and the perfect targets for the development of new antibiotic prostate cancer, respectively. Unfortunately only the poly- drugs to prevent or treat the infective process of bacteria, peptide component of most of these cancer biomarkers is viruses, and fungi. being exploited in clinical tests and needs to be re-evaluated, as these proteins may be elevated in patients with benign or inflammatory diseases. It is highly likely that glycoform var- 2.1.2.1 The influenza story iants of these cancer-specific markers will provide greater di- agnostic performance in terms of sensitivity and specificity. One of the most topical stories today is the potential threat of The biomarker for early cancer diagnosis will then need to be an influenza pandemic spread of infection in humans by a evaluated by longitudinal studies with statistically relevant highly pathogenic avian virus. Influenza is a highly con- numbers of human samples. tagious, acute, viral infection of the respiratory tract. The causative agents of the disease are immunologically diverse, 2.1.1.1 The alpha-fetoprotein story single-strand RNA viruses. Type A viruses are the most pre- valent and are associated with most serious health risks and Quite recently the FDA has approved AFP-L3, the core fuco- epidemics. Glycosylation of both the host cell receptor and sylated form of AFP as a tumor marker for primary hepato- the two main viral membrane proteins is intrinsically cellular carcinoma. The performance of AFP-L3, with a sen- involved in many aspects of the pathogenicity of this organ- sitivity of about 50% in detection of early-stage hepatocellular ism. carcinoma, is significantly better than that of total AFP. Fur- The action of a major viral envelope protein, neur- thermore, the fraction of AFP contributed by AFP-L3 has aminidase, is to cleave the sialic acid from the membrane shown considerable promise as a predictor of progression of glycolipid so the new virus particles can be released from cirrhosis to cancer within the next year. These findings host cells. An additional glycosylation site within the neur- highlight the improvement in diagnostic efficacy of a serum aminidase (NA) protein globular head has been reported to glycoprotein when a change in glycosylation is examined, as contribute to the high virulence of the H5N1 virus. In addi- opposed