3rd Jenner International Glycoimmunology Meeting

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Tzantzoglou, 5. ct ~1. I 1Y%I r1ll1.S X, 655-660 10 Eamularo, G., De S~nione. C.. Tzantzoglou. S. and Trrnchierr, \;. (1994) lmmutx~l. Today I S, 49.5-4Y6 11 Dalakas, M.C., Leon-Xlonzon, M.E., Bernardim, 1. and Gahl. W.i\. (1994) Ann. Ne~ol. 3 5, 482-48’ 12 Semino-Mora. C., Leon-~lonzon. M.E. and Dalakas, M.C. I 19941 Lab. Inzest. 71, 102-l I7 13 De Simone, C., Tzantzoglou. 5..

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1-12 14 Famularo, G., De Simone, C., Xrrrgoni hlartelh, E. and Jirillo. E.J. ~~:ndr~toxi~zRes. (in press) 15 Eoresta, I’., Ruggiero, V., D’Urso, C. et dl. (19941 l~7tefzszfv Caw Med. 20, SlS6 16 Kuratsune, H., \iamaguti. K., Takahashi, Cl. ct J/. I 19941 Cl%. lP7f&f. Dis. 1x, 562-567 1.5,

3rd Jenner International GlycoimmunologyMeeting John S. Axford Glycoimmunology has swiftly hecome an area of inttwse research interest. A recent meeting’” dis-

In 1993, Immunology Today reported on the 2nd Jenner International Glycoimmunology Meetwhich focused upon how ing’,

oligosaccharides could be relevant both to normal and diseaseassociated biological mechanisms and how interference with the oligosaccharide-protein interaction may have therapeutic implications. The past two years have seen an explosion of activity in these two areas. On the functionality front, there has been frantic interest by pharmaceutical companies to understand, and thus to manipulate, the of protein glycomechanisms sylation in order to alter the pharmacokinetic and dynamic profiles of their products. Therapeutically, intensive research has focused on the development of carbohydrate compounds, such as the simple oligosaccharide structure known as sialyl-Lewis x (sLex), which has been shown to have dramatic, protective effects in a P-selectindependent model of lung injury. This area of research is particularly exciting, since these synthetic compounds and their analogues have enormous potential and can be used in a wide variety of therapeutic applications.

cussed current technchgical aduances in glycoprotein analysts, and focused on our mcreasing knowledge in the areas of glycosyltransferase enzyme actitlity, the function of oligosaccharides and their association with disease. The topics covered ‘it the 3rd Jenner International Glycoimmunology Meeting reflected this expansion of research interest. The meeting comprised nine sessions that covered: techniques employed in glycoprotein analysis; the glycosyltransferase enzymes that dictate protein glycosylation; the relationship between oligosaccharides and their biological function and disease; and how oligosaccharides may be utilized as novel therapeutic agents. Glycoprotein analysis Significant advances have been made in our ability to analyse carbohydrates. Fluorophore-assisted carbohydrate electrophoresis is developing into a potentially useful tool for basic research and clinical ‘fThe 3rd Jenner International Gl~coimmunology Meeting was held in II Ciocco, Tuscany, Italy. on 1l-14 Ocroher 1994.

diagnosis for measuring carbohydrate fluctuations in human body fluids (J. Klock, Novato, CA). The utility of this approach was demonstrated in the context of the analysis of IgG-associated oligosaccharide changes, which may be of specific use as a diagnostic tool in earlv arthritis and the differentiation of arthritic conditions (K. Martin, London). Further leaps are being made in automated technology, particularly in automated hydrazinolysis glycan-release, which is a fluorescent-labelling system used for carbohydrate detection (R. Dennis, Oxford), and in the use of molecular modelling simulations to construct a visual image of the molecule in question, thereby helping to understand its interactions (L. Hounsell, London). Y.C. Lee (Baltimore) described studies that focused on the chemometrics of oligosaccharide analysis. Conventional analytical techniques in conjunction with known standards have previously enabled the determination of sugar structures. However, if standards are not available, correlation can still be made between the mobility of the oligosaccharides during chromatography or electrophoresis and their structural features. This

has enabled the determination of previously unknown oligosaccharide structures and has even led to the discovery of new ones. The glycosyltransferases h model of B-1,4galactosyltransferase (B-1,4-GTase) transcriptional regulation has been developed (J. Shaper, Baltimore). The distal region of the gene encoding B-1,4GTase functions as a housekeeping promoter, while the proximal region functions as a cell-specific promoter in tissues such as the mammary gland. It appears that the 3.9 kb start site is generally used but the 4.1 kb start site is preferentially used in mid-to-late pregnant and lactating mammary glands. This is coincident with the cellular requirement for increased B-1,4-GTase enzymatic levels required for lactose biosynthesis. B-1,4-GTase is also unusual among the glycosyltransferases in that it is found in two subcellular compartments (the trans-Golgi complex and the cell surface), where it performs two distinct functions (B. Shur, Texas). Little is known concerning how it is targeted to these different sites, but it is possible that a sequence unique to one GTase isoform is responsible for targeting to the plasma membrane, where it can function as a cell adhesion molecule. Glycoproteins exhibit N,N’-di(LacdiNAc) acetyllactosadiamine units and N-acetyllactosamine-type N-linked glycan (LacNAc) units on N- and O-linked oligosaccharide chains (D. van den Eijnden, Amsterdam). The factor that determines whether either of these type of chains prevail on protein products are the relative proportions of B-1,4-N-acetylgalactosamine transferase (B-1,4-GalNAcTase) and B-1, 4-GTase existing in the cell. The level of fucosyltransferase (FucT) activity and of mRNA for FucT III-VI drops sharply when a promyelocytic cell line is induced to differentiate along the granulocytic pathway, whilst the expression of sLe”, which is fucosylated, is increased. Perhaps FucT VII is the gene responsible for fucosylation leading to the synthesis of sLe’ (W. Watkins, London)? It has been suggested that the glycosyltransferase enzymes are

pivotal to disease-associated glycosplation variations. Indeed, it was previouslv reported that the activit! of B-1,4-GTase is reduced in lvmphocytes from rheumatoid arthritis (KA) patients, although it is now known that this is not due to significant genetic changes or to the presence of inhibitors (J. Axford, London). Isoenzyme variations of B- I ,4GTase have been observed in the serum of RA patients, and this ma\ in some way explain how a dowriregulation of 1gG galactosvlation is achieved. Perhaps these ‘&forms are glycoforms? In the same manner, Tn svndrome is characterized biochemically by repression of B1,3-GTase activity, causing a certain percentage of all haematopoietic cell lineages to express the Tn- epitope, which is cY-N-acetylgalactosamine (a-GalNAc) (F.. Berger, Switzerland). Oligosaccharides and biological function It is generally accepted that mucin-like glycoproteins function as lubricants and anti-pathogens. Two sialomucins, GlvCAM-1 and CD34, are involved in’inflammation and haematopoiesis (L. Lasky, San Francisco). It appears that both these mucins can function as ligands for I.-selectin. LJsing gene-knockout mice, it was found that GlyCAM-I functions as a regulator of lymphocyte trafficking during inflammation and may dampen the influx of leukocytes. The wide range of oligosaccharide binding exhibited by selectins is a result of variation in clustering of the calcium-dependent carbohydrate recognition sites (K. Childs, London). Certain growth factors and cytokines exert their effects by binding to the cell-surface polysaccharide heparan sulphate (J. Gallagher, Manchester). Fibroblast growth factor binds vi‘? a specific sequence termed oligo-H, which in some way modifies the structure of basic fibro blast growth factor, enabling it to be recognized by signal-transducing membrane receptors. I? Rudd (Oxford) has generated a ‘sugar library’ based on the IgAl molecule. This library has been used to demonstrate that the oligosaccharides present in the hinge region of IgA are the determinants recognized by the receptor for secretor!

1gA on T cells in patients with autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE), and IgA nephropathy. Glycosylation of CD8 may be important to the signal transduction and adhesion between T cells and antigen-presenting cells. The (:I)8 glycoprotein has 7-8 O-linked glycans closely spaced in the stalk region between the single immunoglobulin (Ig)-like domain and the transmembrane segment. This is crucial since it keeps the first ectodomain of CD8 in an extended form and prevents proteolytic degradation. The fact that the expression of glycosyltransferases is enhanced following T-cell activation may indicate that the function of activated T cells may vary according to different processing of the O-linked oligosaccharides (F. Serafini-Cessi, Bologna). The importance of IgG glyco$ylation has been much debated, and the debate continued at this meeting. It is now certain that the oligosaccharide-protein relationship in the IgG Fc region can be crucial to the expression of Ig effector function (R. Jefferis, Birmingham), since replacing an amino acid that IS in contact with a core sugar can result in loss of IgG biological ‘tctivity. The IgG glycosylation debate was advanced by the demonstration that, whatever role IgG sugars play in the etiopathogenesis of RA, measurement of agalactose forms of IgG Fc provides the clinician with useful prognostic information (D. Isenberg, London). Recent studies of patients with RA have shown that a discriminant functional analysis utilizing IgG galactosylation, grip strength, age and sex could predict the course of the disease in 94% of patients; IgG galactosylation was ranked as the most powerful parameter for this prediction. Surely it is now time to carry out a prospective clinical trial? Especially as the possibility exists that agalactosylated 1gG may be pathogenic (F. Hay, London). A significant proportion of rheumatoid factors (RFs) derived from rheumatoid synovial cells were shown to have greater binding to IgG lacking galactose than to those with normal galactosylation. This may imply a greater tendency

of agalactosylated IgG to form immune complexes. Molecular modelling experiments of IgG Fc regions from healthy individuals revealed the partial masking of key epitopes for RF binding by oligosaccharides that were absent in RA patients. Therefore, it appears that the absence of galactose may expose RFreactive epitopes, which may be important to the inflammatory process. Oligosaccharides and disease Although antibodies against type II collagen have been demonstrated in diseases such as RA, their role in disease pathology is not clear. It may be that not only is the antigenic specificity of the antibody pathogenically important, but also its glycosylation status, as enzymatic removal of galactose from these antibodies prior to immunizing rats resulted in the development of arthritis (T. Rademacher, London). Antigenicity involving carbohydrates has also been demonstrated in the glycosaminoglycans, where sulphate esters combine with the structure of the carbohydrate backbone to present topographies that the immune system recognizes Using monoclonal as antigenic. antibodies generated against each of the different categories of sulphated glycosaminoglycans (V. Hascall, Cleveland), precise analysis of these epitopes is possible. Molecular events leading to variation in glycosylation patterns have predominantly focused upon synthetic (i.e. transferase) changes. However, we should remember that peroxy and hydroxy radicals can result in postsynthetic sugar degradation, and that inflammation itself could also affect IgG glycosylation (J. Lunec, Leicester). Interestingly, a reciprocal galactose reduction has been demonstrated at the Fc region of the IgAl and IgA2 isotypes in Sjiigren’s syndrome, together with increased sialylation of the IgAl and IgA2 isotypes (M. Dueymes, Brest). This was thought to be limited to Olinked sugars located in the hinge region (M. Tomana, Alabama). In animal experiments, injection of this agalactosylated, human, monomeric IgA into mice resulted in increased uptake by the kidney, and

this ma>’ be explained b\; the presence of a GalNAc-specific receptor on mesangial cells. Importantly, the glycoforms of other glycoproteins are now bemg studied (J. van Dijk, Amsterdam). Indeed, cw,-acid glycoprotein (A(;!‘) has been shown to contain five N-linked glycans that differ in the type of branching, as well as in rhe degree of fucosylation and tialvlation. Interestingly, in R.A. there ‘is a persistent increase in heavily fucosylated AGP glycoforms that express the sLe’ structure. Just what role inflammation-induced expression of sLe’ on AGP plays has still to be elucidated. Haptoglobin is another glycoprocein that may be useful in unravelling pathological processes occurring in diseases such as Crohn’s disease and active hepatitis, and ma)- also provide a potential marker for clinical investigations (G. Turner, Newcastle). The ‘Yin-Yang’ of serineithreonine phosphorylation refers to the reciprocal relationship that exists between protein glycosylation and phosphorylation (G. Hart, Alabama). For example, RNA polymerase 1 may be glycosylated with O-linked N-acetylglucosamine or phosphorylated at its C-terminal domain. In relation to disease, a microtubuleassociated protein named Tau forms hyperphosphorylated filaments in Alzheimer’s disease, whereas they would normally be expected to be glycosylated by O-linked N-acetylglucosamine (0-GlcNAc). Oligosaccharides and therapy Currently, two of the most difficult problems in glycobiology are how to enhance the binding affinity of carbohydrate to protein ligands

and how to recognize new ligands. Perhaps the answer to both will be provided by the production of biologically active oligosaccharide libraries (0. Hindsgaul, Alberta). This is in the process of being achieved in a mammoth strategy of randomly combining the nine most common mammalian sugars into all their possible trisaccharide combinations, which has proved surprisingly effective. Therapeutically, such a library may be used to design oligosaccharides that target certain body organs (K. Rice, Columbus, OH). The liver seems to be the organ of most potential for trials of novel drug delivery systems. Similarl!; conjugated forms of s1.e’ may provide potential antagonists for inhibition of the binding of cell adhesion molecules. G. Jacob (St Louis) was confident of the results of experiments conducred to demonstrate that such antagonism can be achieved, but was somewhat reticent concerning its therapeutic potential. Concluding remarks In summary, this multidisciplinar! meeting proved to be extremely useful in establishing the state-of-the-art in each research area. This is enabling the expanding field of oligosaccharide research to be viewed as a whole, if from different angles, and the potential for collaboration to be realized. John S. Axford 1s Jt the Academc Rheurnatolog?~ Group, Dept of Celluh md Molecular Sciences, St George’s Medical School, London. UK SK71 7 OR?:.

Reference 1 Axford, J.S.(199.3)/mmzfnr~/.Tdq 14, 104-106

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