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that inhibit morphogenesis and/or viral entry. Infection by human immunodeficiency virus type-1 (HIV-1) is characterized by low levels of neutralizing antibodies. One broadly neutralizing human monoclonal antibody is 2G12.10 This has three possible combining sites and recognizes a cluster of oligomannose residues on the “immunologically silent” face. This recognition provides exciting challenges for immunogen design. N- and O-linked glycosylation of enveloped glycoproteins permits Ebola virus binding to host cells.11,12,13 It is thought that an alternative pathway to the calnexin-calreticulin folding and quality control pathway is being used by the viral glycoproteins.

Immune mechanisms will be a major focus for clinical intervention over the next decade. This will involve modulation of both the innate and adaptive immune defenses.

The innate immune system provides the first line of defense to invading pathogens, and recognition of pathogens governs the induction and type of pathogen-specific adaptive responses. Schistosomiasis is a major tropical parasitic disease. Recently several antigen presenting cell-associated lectins, such as the dendritic cell-specific DC-SIGN, L-SIGN on liver sinusoid endothelial cells, macrophage galactose-type lectin (MGL), galactin-3 and mannan binding lectin (MBL), have been shown to interact with

egg glycoproteins of Schistosoma mansoni.14 These sugars occur on many parasitic helminths, and it is thought that they may constitute common

The potential for glycobiology to improve the practice of medicine has been well-recognized, which is why the Jenner Glycobiology and Medicine biennial symposiums, concerning this association, have been taking place for the last 14 years.1,2,3,4,5,6 The science of glycobiology has matured rapidly, and with it the far-reaching clinical implications are becoming understood. The next decade is going to see this final frontier of science conquered. The impact this understanding of glycobiology will have upon our practice of medicine is going to be exciting. The 7th Jenner Glycobiology and Medicine Symposium, whose major sponsor was Mannatech, Inc., was designed to reflect these advances. All the major clinical areas were involved, with contributions from pivotal players in science and medicine.

As with our previous meetings, junior scientists were involved, as we recognize that at the end of the next decade they will be in the driving seat. This introduction serves as a taster to whet your appetite.

From embryogenesis to pathogenesis, glycosylation plays a pivotal role. Complex and hybrid N-glycans and O-fucose glycans are critical in oocyte development and function.7 This area must surely be a fertile ground for glycosylation research.

The pathogenesis of viral infections involves sugars at every turn.8,9 Hepatitis C virus and bovine viral diarrhea virus (BVDV) are opening themselves to scrutiny. The BVDV has proven very useful in the evaluation of the antiviral activity of molecules

GLYCOBIOLOGY AND MEDICINE: THE FINAL FRONTIER

By John S. Axford, DSc, MD, FRCP

A review of the 7TH Jenner Glycobiology and Medicine Symposium, London, England, from the 5th to the 8th of September, 2004.

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The science of glycobiology has matured rapidly, and with it the

far-reaching clinical implications are becoming understood.

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STAFF

EDITORSEileen Vennum, RAC, Editor-in-ChiefJane Ramberg, MS, Managing EditorBill H. McAnalley, PhD, Executive EditorStephen Boyd, MD, PhD, FRSM, Associate EditorGary Carter, BS, Associate EditorAlexis Eberendu, PhD, Associate EditorJohn Hall, DDS, Associate EditorC. Micheal Koepke, RAC, Associate EditorRonda Sisak, RD-LD, Associate EditorDennis Sparkman, PhD, Associate EditorCharlene Wang, MS, MD, Associate Editor

EDITORIAL STAFFKay ChichillaBarbara KinseyMary Wood

GRAPHIC ARTISTSLynn AnJennfier Aponte

sugar profiles utilized for lectin-mediated immune recognition. For example, Lewis X interacts with DC-SIGN on dendritic cells, and it is thought that this interaction may play a role in triggering dendritic cells to mount a Th2 response.

MBL is an oligomeric protein designed to recognize pathogen-associated molecular patterns. The biological importance of MBL was indicated when opsonin-deficient children with recurrent infections were found to be genetically deficient in MBL.15 Further interest in this molecule was sparked by the observation of complement activation upon binding to carbohydrates. MBL reacts with sugar groups on microorganisms, and recently two other plasma proteins of similar structure and activities, H- and L-ficolin, have been described.16 Glycan structures that can act as potential ligands for MBL have been identified on all the immunoglobulins. In human serum only agalactosyl-IgG and polymeric and dimeric IgA have been shown to bind MBL and initiate the lectin pathway of complement.17,18 This is thought to occur through GlcNAc-terminating glycan structures.

Disease associations with sugar changes are plentiful when the adaptive immune system is considered. This may involve fundamental processes, for example glycosylation related molecular mechanisms are thought to involve the function of the T cell co-receptor CD8,19 which will have far-reaching implications if abnormal.

Sugar associations with cancer have been recognized for some time. There continues to be new data generated concerning ovarian cancer and arthritis, but research is expanding into new areas. Sugars have now been shown to be associated with the pathological mechanism associated with the glycosylphosphatidylinositol (GPI) anchorage of the prion protein, pigeon fanciers’ lung20 and muscular dystrophy.21

At least six different forms of muscular dystrophy are caused by genes that encode glycosyltransferases,21 and when malfunctioning results in a secondary deficiency in the glycosylation of dystroglycan.

Autoimmune arthritis has been associated with the generation of remnant glyco-epitopes by gelatinase B/matrix metalloproteinase-9, which is an inflammatory mediator and effector. Considerable amounts of gelatinase B are released by neutrophils in the synovial cavity of patients with rheumatoid arthritis. This is thought to be linked to the pathogenesis of arthritis as

gelatinase B-deficient mice are resistant to antibody-induced arthritis. Determination of T cell reactivity against the gelatinase B-cleaved

fragments of collagen II indicates that there are many glyco-epitopes present in collagen II, which reinforces the role of glycopeptide antigens in autoimmunity.

It is, however, always exciting when clinical anecdotes translate into therapeutic and diagnostic possibilities. Glycobiology is at that transition.

Once disease mechanisms have been understood, the next step is to determine whether this information can be used to devise therapeutic options. Predictably, therapeutic hypotheses are plentiful. For example, there is a possibility that sugars may be used to block skin inflammation.

There have been new developments in treating glycosphingolipid storage diseases.22 This may not be a common group of diseases, but for those who have it, this opens the door to improved quality of life. The glycosphingolipid lysosomal storage diseases result from defects in glycosphingolipid catabolism. They are progressive disorders, the majority of which involve central nervous system pathology. A new approach to treatment is substrate reduction therapy (SRT), using small molecule inhibitors to reduce the rate of glycosphingolipid biosynthesis.22 One of these drugs, NB-DNJ, has recently been approved for clinical use in type 1 Gaucher’s disease. There is also the potential of combining SRT with drugs that target the downstream consequences of storage.

Rheumatoid arthritis (RA) is a common disorder where the available diagnostic tests, e.g. rheumatoid factor and anticitrulinated cyclic peptide, lack sensitivity. The diagnostic potential of IgG glycosylation has been previously discussed, and we await the results from prospective trials.5,23 Indeed, abnormal galactosylation of polyclonal IgG in antineutrophil cytoplasm antibody-(ANCA) associated systemic vasculitis patients has now been reported24 and the diagnostic potential of this technology for other autoimmune rheumatic diseases is significant. Experiments looking at the cause behind these sugar changes indicate both quantitative and qualitative changes in the RA serum galactosyltransferase (GTase) isoform profile.25 This is likely to be due to a greater proportion of hypersialylated isoforms, which have the potential to adversely affect the catalytic activity

Immune mechanisms will be a major focus for clinical and intervention

over the next decade.

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of the enzyme. This provides a possible mechanism for post-translational regulation of GTase activity in RA. It also provides further evidence that RA glycosylation changes may be more general than previously indicated and encompass proteins other than IgG. These observations can only strengthen the potential of sugars as RA disease biomarkers.

The selectin family of adhesion molecules mediates the initial attachment of leukocytes to venular endothelial cells at sites of tissue injury and inflammation. For example, in staphylococcal arthritis, fucoidin, a sulfated polysaccharide from seaweed, binds to and blocks the function of L- and P-selectins, thereby inhibiting leukocyte rolling and adhesion to endothelial surfaces.26 Treatment with fucoidin has been shown to reduce the severity of septic arthritis within the first 3 days following bacterial infection. It is suggested that the efficient treatment of septic arthritis should encompass a combination of antibiotics and immunomodulation.

The attachment and rolling of leukocytes on the vascular endothelium can also be mediated by CD44 through its binding to the glycosaminoglycan hyaluronan.27,28 This interaction can be upregulated in the context of inflammation via the removal, or remodeling, of N-glycans on the CD44 molecule that both unblocks the hyaluronan-binding site and facilitates receptor clustering. The production of cross-linked hyaluronan fibrils in the presence of the inflammation-associated protein TSG-6 could represent another way in which CD44 becomes activated through a clustering mechanism.

Gastroenterologists want to know more about normal and abnormal bacteria that inhabit our bowels. O-acetyl sialic acid expression in colorectal mucosa has been shown to be regulated by enteric microflora; as demonstrated by the loss of sialic acid and oligo-O acetylation after elimination of the fecal flow. There is therefore potential to use this observation to quantitate bacterial colonization and perhaps interfere with disease-associated pathology.

Biological therapies will be the new treatments of the next decade. The impact of glycosylation on the structure and function of natural and recombinant (therapeutic) IgG

antibody is therefore important to get to grips with. It has been shown that the in vivo micro-environment can have a profound influence on the glycosylation profile of IgG-Fc.29 This may reflect the unique structural relationship between the oligosaccharide and the protein. The “core” heptasaccharide is essential for FcγRI, FcγRII, FcγRIII and C1 activation whilst outer arm sugar residues can influence these and other functions, e.g. FcγRIII, FcγRn, MBL, MR. Thus, fidelity of glycosylation is essential to the effector function profile of antibodies, and in the future the oligosaccharide will be used to function as a structural rheostat to generate specific glycoforms exhibiting optimal effector activities for a particular disease target.

Cellular glycol-engineering for fully human glycosylation and optimized sialylation of proteins is therefore going to be important if these molecules are going to be fully and specifically active. Most pharmaceutical proteins are expressed in bacteria, yeast or mammalian cells resulting in proteins lacking glycosylation or carrying glycans which largely differ from human carbohydrate chains in various aspects, including sialylation. However, relationships between the N-glycan structures and biological activities of, for example, recombinant human erythropoietins produced using different culture conditions and purification procedures are now better understood.30 It is nevertheless apparent that novel glycoprotein expression technology will need to be developed to address this problem, and data are now available to demonstrate how this can be done. First case studies using a novel glycol-engineered expression system show that the bioactivity of human recombinant glycoproteins can be dramatically improved by generating a fully human glycosylation and optimizing the sialylation degree, as shown e.g. by a ~500-fold activity increase for rhGM-CSF or a potency increase of a glycoprotein vaccine.31

The above introduction adds up to the fact that glycobiology is an extremely exciting science in which to be involved. Additionally, if you are a clinician, it is even more gripping as you will be at the forefront of important clinical developments.

I hope these proceedings stimulate you as much as they have me, and I look forward to seeing you at Jenner 8!

REFERENCE LIST1. Axford JS. 2nd Jenner International Glycoimmunology Meeting. Immunol Today. 1993;14(3):104-106.2. Axford JS. 3rd Jenner International Glycoimmunology Meeting. Immunol Today. 1995;16(5):213-215.3. Axford JS. 4th Jenner International Glycoimmunology Meeting. Immunol Today. 1997;18(11):511-513.4. Axford J, Kieda C, van Dijk W. Meeting report. 5th Jenner Glycobiology and Medicine. Glycobiology. 2001;11(2):5G-7G.5. Alavi A, Axford J. Glycobiology of the rheumatic diseases: an update. Adv Exp Med Biol. 2003;535:271-280.6. Axford J, Kieda C, van Dijk W. 6th Jenner Glycobiology and Medicine. CPD Bulletin.Immunology and Allergy. 2004;3(3):85-88.7. Shi S, Williams SA, Seppo A, et al. Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycans develop and implant. Mol.Cell Biol. 2004;24(22):9920-9929.8. Branza-Nichita N, Lazar C, Dwek RA, et al. Role of N-glycan trimming in the folding and secretion of the pestivirus protein E(rns). Biochem Biophys Res Commun. 2004;319(2):655-662.

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9. Dwek RA, Butters TD, Platt FM, et al. Targeting glycosylation as a therapeutic approach. Nat Rev Drug Discov. 2002;1(1):65-75.10. Calarese DA, Scanlan C, Zwick MB, et al. Antibody domain exchange in an immunological solution to carbohydrate cluster recognition. Science. 2003;300:2065-2071.11. Alvarez CP, Lasala F, Carrillo J, et al. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol. 2002;76(13):6841-6844.12. Lin G, Simmons G, Pohlmann S, et al. Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. J Virol. 2003;77(2):1337-1346.13. Takada A, Fujioka K, Tsuiji M, et al. Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry. J Virol. 2004;78(6):2943-2947.14. Klabunde J, Berger J, Jensenius JC, et al. Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms. Exp Parasitol. 2000;95(4):231-239.15. Super M, Thiel S, Lu J, et al. Association of low levels of mannan-binding protein with a common defect of opsonisation. Lancet. 1989;2(8674):1236-1239.16. Holmskov U, Thiel S, Jensenius JC. Collections and ficolins: humoral lectins of the innate immune defense. Annu Rev Immunol. 2003;21:547-578.17. Malhotra R, Wormald MR, Rudd PM, et al. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat Med. 1995;1(3):237-243.18. Roos A, Bouwman LH, Van Gijlswijk-Janssen DJ, et al. Human IgA activates the complement system via the mannan-binding lectin pathway. J Immunol. 2001;167(5):2861-2868.19. Merry AH, Gilbert RJ, Shore DA, et al. O-glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8. J Biol Chem. 2003;278(29):27119-27128.20. Baldwin CI, Todd A, Bourke SJ, et al. Pigeon fanciers’ lung: identification of disease-associated carbohydrate epitopes on pigeon intestinal mucin. Clin Exp Immunol. 1999;117(2):230-236.21. Martin-Rendon E, Blake DJ. Protein glycosylation in disease: new insights into the congenital muscular dystrophies. Trends Pharmacol Sci. 2003;24(4):178-183.22. Platt F , Butters T. Inhibition of substrate synthesis: A phamacological approach for glycosphingolipid storage disease therapy. Chapter 15: 382-408. In: Lysosomal Disorders of the Brain. Oxford Univ Press, 2004.23. Axford JS, Cunnane G, Fitzgerald O, et al. Rheumatic disease differentiation using immunoglobulin G sugar printing by high density electrophoresis. J Rheumatol. 2003;30(12):2540-2546.24. Holland M, Takada K, Okumoto T, et al. Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis. Clin Exp Immunol. 2002;129(1):183-190.25. Alavi A, Axford JS, Pool AJ. Serum galactosyltransferase isoform changes in rheumatoid arthritis. J Rheumatol. 2004;31(8):1513-1520.26. Verdrengh M, Erlandsson-Harris H, Tarkowski A. Role of selectins in experimental Staphylococcus aureus-induced arthritis. Eur J Immunol. 2000;30(6):1606-1613.27. Teriete P, Banerji S, Noble M, et al. Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44. Mol.Cell. 2004;13(4):483-496.28. Lesley J, Gal I, Mahoney DJ, et al. TSG-6 modulates the interaction between hyaluronan and cell surface CD44. J Biol Chem. 2004;279(24):25745-25754.29. Watt GM, Lund J, Levens M, et al. Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function. Chem Biol. 2003;10(9):807-814.30. Yuen CT, Storring PL, Tiplady RJ, et al. Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Br J Haematol. 2003;121(3):511-526.3� activity, fully human glycosylation and optimised sialyation (GlycoExpress): Biotechnological features. Bioprocessing J. 2005;in press