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May 1, 2005

GLYCOSCIENCE & NUTRITION

TM

VOL. 6, NO. 3 VOL. 6, NO. 3

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.

The potential for glycobiology to improve the that inhibit morphogenesis and/or viral entry. practice of medicine has been well-recognized, Infection by human immunodeficiency virus which is why the Jenner Glycobiology and type-1 (HIV-1) is characterized by low levels of Medicine biennial symposiums, concerning this neutralizing antibodies. One broadly neutralizing association, have been taking place for the last human monoclonal antibody is 2G12.10 This has 1,2,3,4,5,6 14 years. The science of glycobiology three possible combining sites and recognizes has matured rapidly, and with it the far-reaching a cluster of oligomannose residues on the clinical implications are becoming understood. “immunologically silent” face. This recognition The next decade is going to see this final provides exciting challenges for immunogen frontier of science conquered. The impact this design. N- and O-linked glycosylation of understanding of glycobiology will have upon our enveloped glycoproteins permits Ebola virus practice of medicine is going to be exciting. The 7th binding to host cells.11,12,13 It is thought that an Jenner Glycobiology and Medicine Symposium, alternative pathway to the calnexin-calreticulin whose major sponsor was Mannatech, Inc., was folding and quality control pathway is being used designed to reflect these advances. All the major by the viral glycoproteins. clinical areas were involved, with contributions Immune mechanisms will be a major focus from pivotal players in science and medicine. for clinical intervention over the next decade. As with our previous meetings, junior This will involve modulation of both the innate scientists were involved, as we recognize that and adaptive immune defenses. at the end of the next decade they will be in the The innate immune system provides the driving seat. This introduction serves as a taster first line of defense to invading pathogens, and to whet your appetite. recognition of pathogens governs the induction From embryogenesis to pathogenesis, and type of pathogen-specific adaptive responses. glycosylation plays a pivotal role. Complex and Schistosomiasis is a major tropical parasitic hybrid N-glycans and O-fucose glycans are critical disease. Recently several antigen presenting in oocyte development and function.7 This area cell-associated lectins, such as the dendritic must surely be a fertile ground for glycosylation cell-specific DC-SIGN, L-SIGN on liver sinusoid research. endothelial cells, macrophage galactose-type The pathogenesis of viral infections involves lectin (MGL), galactin-3 and mannan binding sugars at every turn.8,9 Hepatitis C virus and lectin (MBL), have been shown to interact with bovine viral diarrhea egg glycoproteins of virus (BVDV) are opening Schistosoma mansoni.14 The science of glycobiology has themselves to scrutiny. These sugars occur on matured rapidly, and with it the The BVDV has proven very far-reaching clinical implications many parasitic helminths, useful in the evaluation of the and it is thought that they are becoming understood. antiviral activity of molecules may constitute common

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GLYCOSCIENCE & NUTRITION STAFF EDITORS Eileen Vennum, RAC, Editor-in-Chief Jane Ramberg, MS, Managing Editor Bill H. McAnalley, PhD, Executive Editor Stephen Boyd, MD, PhD, FRSM, Associate Editor Gary Carter, BS, Associate Editor Alexis Eberendu, PhD, Associate Editor John Hall, DDS, Associate Editor C. Micheal Koepke, RAC, Associate Editor Ronda Sisak, RD-LD, Associate Editor Dennis Sparkman, PhD, Associate Editor Charlene Wang, MS, MD, Associate Editor EDITORIAL STAFF Kay Chichilla Barbara Kinsey Mary Wood GRAPHIC ARTISTS Lynn An Jennfier Aponte

VOL. 6, NO.3 sugar profiles utilized gelatinase B-deficient for lectin-mediated Immune mechanisms will be a major mice are resistant to immune recognition. antibody-induced arthritis. focus for clinical and intervention For example, Lewis X Determination of T cell over the next decade. interacts with DC-SIGN reactivity against the on dendritic cells, and gelatinase B-cleaved it is thought that this interaction may play a fragments of collagen II indicates that there are role in triggering dendritic cells to mount a Th2 many glyco-epitopes present in collagen II, which response. reinforces the role of glycopeptide antigens in MBL is an oligomeric protein designed autoimmunity. to recognize pathogen-associated molecular It is, however, always exciting when clinical patterns. The biological importance of MBL was anecdotes translate into therapeutic and indicated when opsonin-deficient children with diagnostic possibilities. Glycobiology is at that recurrent infections were found to be genetically transition. deficient in MBL.15 Further interest in this molecule Once disease mechanisms have been was sparked by the observation of complement understood, the next step is to determine activation upon binding to carbohydrates. MBL whether this information can be used to devise reacts with sugar groups on microorganisms, therapeutic options. Predictably, therapeutic and recently two other plasma proteins of similar hypotheses are plentiful. For example, there is a structure and activities, H- and L-ficolin, have possibility that sugars may be used to block skin been described.16 Glycan structures that can act inflammation. as potential ligands for MBL have been identified There have been new developments in on all the immunoglobulins. In human serum treating glycosphingolipid storage diseases.22 only agalactosyl-IgG and polymeric and dimeric This may not be a common group of diseases, IgA have been shown to bind MBL and initiate but for those who have it, this opens the door to the lectin pathway of complement.17,18 This is improved quality of life. The glycosphingolipid thought to occur through GlcNAc-terminating lysosomal storage diseases result from defects glycan structures. in glycosphingolipid catabolism. They are Disease associations with sugar changes progressive disorders, the majority of which are plentiful when the adaptive immune system involve central nervous system pathology. is considered. This may involve fundamental A new approach to treatment is substrate processes, for example glycosylation related reduction therapy (SRT), using small molecule molecular mechanisms are thought to involve the inhibitors to reduce the rate of glycosphingolipid function of the T cell co-receptor CD8,19 which biosynthesis.22 One of these drugs, NB-DNJ, will have far-reaching implications if abnormal. has recently been approved for clinical use in Sugar associations with cancer have been type 1 Gaucher’s disease. There is also the recognized for some time. There continues to potential of combining SRT with drugs that target be new data generated concerning ovarian the downstream consequences of storage. cancer and arthritis, but research is expanding Rheumatoid arthritis (RA) is a common into new areas. Sugars have now been shown to disorder where the available diagnostic tests, be associated with the pathological mechanism e.g. rheumatoid factor and anticitrulinated associated with the glycosylphosphatidylinosit cyclic peptide, lack sensitivity. The diagnostic ol (GPI) anchorage of the prion protein, pigeon potential of IgG glycosylation has been fanciers’ lung20 and muscular dystrophy.21 previously discussed, and we await the results At least six different forms of muscular from prospective trials.5,23 Indeed, abnormal dystrophy are caused by genes that encode galactosylation of polyclonal IgG in antineutrophil glycosyltransferases,21 and when malfunctioning cytoplasm antibody-(ANCA) associated systemic results in a secondary deficiency in the vasculitis patients has now been reported24 and glycosylation of dystroglycan. the diagnostic potential of this technology for other Autoimmune arthritis has been associated autoimmune rheumatic diseases is significant. with the generation of remnant glyco-epitopes Experiments looking at the cause behind by gelatinase B/matrix metalloproteinase-9, these sugar changes indicate both quantitative which is an inflammatory mediator and effector. and qualitative changes in the RA serum Considerable amounts of gelatinase B are galactosyltransferase (GTase) isoform profile.25 released by neutrophils in the synovial cavity of This is likely to be due to a greater proportion patients with rheumatoid arthritis. This is thought of hypersialylated isoforms, which have the to be linked to the pathogenesis of arthritis as potential to adversely affect the catalytic activity

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antibody is therefore important to get to grips with. It has been of the enzyme. This provides a possible mechanism for postshown that the in vivo micro-environment can have a profound translational regulation of GTase activity in RA. It also provides influence on the glycosylation profile of IgG-Fc.29 This may reflect further evidence that RA glycosylation changes may be more the unique structural relationship between the oligosaccharide general than previously indicated and encompass proteins other and the protein. The “core” heptasaccharide is essential for than IgG. These observations can only strengthen the potential FcγRI, FcγRII, FcγRIII and C1 activation whilst outer arm sugar of sugars as RA disease biomarkers. residues can influence these and other functions, e.g. FcγRIII, The selectin family of adhesion molecules mediates the FcγRn, MBL, MR. Thus, fidelity of glycosylation is essential to initial attachment of leukocytes to venular endothelial cells the effector function profile of antibodies, and in the future the at sites of tissue injury and inflammation. For example, in oligosaccharide will be used to function as a structural rheostat staphylococcal arthritis, fucoidin, a sulfated polysaccharide from to generate specific glycoforms exhibiting optimal effector seaweed, binds to and blocks the function of L- and P-selectins, activities for a particular disease target. thereby inhibiting leukocyte rolling and adhesion to endothelial Cellular glycol-engineering for fully human glycosylation and surfaces.26 Treatment with fucoidin has been shown to reduce optimized sialylation of proteins is therefore going to be important the severity of septic arthritis within the first 3 days following if these molecules are going to be fully and specifically active. bacterial infection. It is suggested that the efficient treatment of Most pharmaceutical proteins are expressed in bacteria, yeast septic arthritis should encompass a combination of antibiotics or mammalian cells resulting in proteins lacking glycosylation or and immunomodulation. carrying glycans which largely differ from human carbohydrate The attachment and rolling of leukocytes on the vascular chains in various aspects, including sialylation. However, endothelium can also be mediated by CD44 through its binding relationships between the N-glycan structures and biological to the glycosaminoglycan hyaluronan.27,28 This interaction can activities of, for example, recombinant human erythropoietins be upregulated in the context of inflammation via the removal, produced using different culture conditions and purification or remodeling, of N-glycans on the CD44 molecule that both procedures are now better understood.30 It is nevertheless unblocks the hyaluronan-binding site and facilitates receptor apparent that novel glycoprotein expression technology will clustering. The production of cross-linked hyaluronan fibrils need to be developed to address this problem, and data are in the presence of the inflammation-associated protein TSG-6 now available to demonstrate how this can be done. First case could represent another way in which CD44 becomes activated studies using a novel glycol-engineered expression system show through a clustering mechanism. that the bioactivity of human recombinant glycoproteins can be Gastroenterologists want to know more about normal dramatically improved by generating a fully human glycosylation and abnormal bacteria that inhabit our bowels. O-acetyl sialic and optimizing the sialylation degree, as shown e.g. by a ~500acid expression in colorectal mucosa has been shown to be fold activity increase for rhGM-CSF or a potency increase of a regulated by enteric microflora; as demonstrated by the loss glycoprotein vaccine.31 of sialic acid and oligo-O acetylation after elimination of the The above introduction adds up to the fact that glycobiology fecal flow. There is therefore potential to use this observation is an extremely exciting science in which to be involved. to quantitate bacterial colonization and perhaps interfere with Additionally, if you are a clinician, it is even more gripping as you disease-associated pathology. will be at the forefront of important clinical developments. Biological therapies will be the new treatments of the next I hope these proceedings stimulate decade. The impact of glycosylation From embryogenesis to pathogenesis, you as much as they have me, and I look on the structure and function of natural forward to seeing you at Jenner 8! glycosylation plays a pivotal role and recombinant (therapeutic) IgG

REFERENCE LIST nd 1. Axford JS. 2 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

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