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Clinical Science (1997) 93,287-293 (Printed in Great Britain) 287 Editorial Review Glycoprotein changes in tumours: a renaissance in clinical applications Elizabeth F. HOUNSELL, Mia YOUNG' and Michael J. DAVIES2 MRC Glycoprotein StructurelFunction Group, Department of Biochemistry and Molecular Biology, University College London, Gower Street, London W C l E 6BT. U.K. 1. Oligosaccharides linked to protein (glycoprotein) or lipid (glycolipid) are the major components at the outer surface of mammalian cells. Studies using antibodies and lectins have shown in the past that the oligosaccharides they recognize exhibit tumourassociated changes, i.e. they are carbohydrate tumour-associated antigens. 2. The oligosaccharides have been further characterized in recent years by structural analysis using high-resolution chromatographic techniques, MS and NMR. NMR gives an oligosaccharide fingerprint that is characteristic of monosaccharide type and linkage and which can be correlated with magnetic resonance spectroscopic data on fine-needle tissue aspirates. 3. Also of relevance is the new understanding of the molecular biology of MUC genes, which code for mucin protein backbones, and of the glycosyltransferase genes, which determine oligosaccharide structure and immunological recognition. 4. For these reasons, we believe that tumour-associated oligosaccharide changes should be revisited in the context of what we now know about structure and expression. This review synopsizes the past data using the detection of carbohydrate tumourassociated antigens by binding of lectins and antibodies, and puts it into the context of NMR fingerprints or signatures. INTRODUCTION Oligosaccharides linked to protein (glycoprotein) or lipid (glycolipid) are the major components at the outer surface of mammalian cells. They are often characteristic of cell type and provide a protective layer against chemical and enzymic attack. It has become clear that they also function as highly specific recognition molecules which play an import- ant role in the physiological properties of the cell. Many structural changes during malignant transformation into tumours have been described, as discussed herein, but the functional implications of the changes are not completely understood. Alterations in the structure of cell-surface glycoconjugates are considered to be relevant to the abnormal properties of cancer cells, such as uncontrolled cell growth, altered cell adhesion, avoidance of immunological destruction, invasiveness and metastatic spread [l]. In medicine, a large amount of attention has been given to these structural changes for possible utilization in diagnosis (to distinguish between benign and malignant lumps) and in prognosis, which is dependent on the maturity of the tumour and its metastatic potential. In the absence of metastasis most cancers would be cured by surgery, chemotherapy and radiation therapy [2]. The desire is to measure the effects of medical treatment and identify recurrence as soon as possible. In addition, new therapeutic regimes are required. The oligosaccharide sequences that have been implicated in altered cell adhesion and metastasis are chain-terminating monosaccharides which are present in the correct spatial arrangement for recognition. Particularly important in this regard are the monosaccharides fucose (Fuc) and sialic acid (SA). They are held in distinct conformational space specific for each carbohydrate-binding protein by different linkage to backbones of galactose (Gal) and N-acetylglucosamine (GlcNAc). Shown in Fig. 1, for example is the sequence called sialyl Lea (SLea). The constituent monosaccharides are ring structures (most easily seen for GlcNAc on the right-hand side of Fig. 1) on which are appended different functional groups such as the CH3 of Fuc (shown top, middle of Fig. 1) and the NH-CO-CH3 (also abbreviated to NHAc) for GlcNAc and SA. The CH3, CH2, CH and NH (the unshaded circles in Key words: cancer, carbohydrate tumour antigens, magnetic resonance spectroscopy, monoclonal antibody, mucin, NMR spectroxopy. Abbreviations: AP. adenomatous polyps; Fuc, fucose; GlcNAc, N-acetylglucosamine; HP. hyperplastic polyps; HPA, Helix pomotio agglutinin; mAb, monoclonal antibody; MRS, magnetic resonance spectroscopy; PSA, prostate-specific antigen; SA, sialic acid; SLea,sialyl Lea;SLe", sialyl Le"; ST, sialyl T; ST,sialyl T"; TAA, tumour-associated antigen. Correspondence: Dr E.F. Hounsell. 'Present address: Skyehusapoleket I Fredribtad, Postbokj 1026, I06 I Fredrikstad,Norway. 'Present address: Lonza Biologics PIC,228 Bath Road, Slough, Berkshire SL14DY, U.K. E. F. Hounsell et al. 288 Fig. 1) give signals detected and characterized by NMR spectroscopy. The position of each of the signals in the NMR spectrum is dependent on the chemical environment of the atom within the molecule [3]. The CH3 of Fuc in SLe" can be seen to be surrounded by oxygen atoms, for example (shaded in Fig. 1, found within the ring and forming the linkages between the monosaccharides and unlinked hydroxyl groups). Each different type of linkage of Fuc to the backbone Gal and GlcNAc (Fig. 2) gives a distinct signature in the NMR spectrum [4]. The amount of each sequence present in tumours and cell lines can now be explored by magnetic resonance spectroscopy (MRS) using easy to obtain twodimensional MRS images from fine-needle aspirates of tumours or cancer cell lines. Studies so far have shown different patterns of fucosylation dependent on the stage of cancer [5, 61. This is an exciting new development in tumour diagnosis which we discuss here in the context of our knowledge about tumourassociated oligosaccharide changes. Carbohydrate antigen structures Historically, the first cellular carbohydrate (i.e. oligosaccharide) antigens that were characterized were the so called blood group antigens on human erythrocytes [7], based on the presence of three w Fig. I. A minimum energy conformation of the oligosaccharide sequence sialyl Leain the orientation shown Fucal-3, GlcNAc ,GalP1-4' SAa2-3 SA is in this case N-acetylneuraminic acid (NeuSAc). The N-acetyl groups (NH-CO-CH3) of NeuSAc and GlcNAc are indicated together with the CHI of Fuc. The hydroxyl groups, 0 - 0 , and oxygens, 0 , are available for hydrogen binding to the protein-combining site of lectins or antibodies. CHI groups and clustered ring C-Hs will stack onto aromatic side-chains of amino acids. Fucal-2Calpl-3GlcNAc Type 1 H Fucal-2Galpl-4GlcNAc ~ y p 2e n Fucal-2Gal~l-3GalNAc Typc3H Fucal-3[Gnlpl-4~ClcNAc Le' Fucal-4[Gal~l-3~ClcNAc Lea Also Le' with Fucal-2 on the Gal is L d and similarly Le' with Fucal-2 on the Gal is Leb. Gal may also have Galal-3 or CnlNAcal-3 attached which are blood group B or A. respectively when the Fucal-2 is present eg ALd. GalNAcal-3, Galol-4, Fucal-2' FlcNAc Fucal-3 ALd Repeating Le' can occur with or without sialylation cg. SAaZ-3Calol~ CjlcNAcpl-3Gnlpl-4 Fucal-3 (~ICNAC Fucal-f SLe'Le' Fig. 2. Different types of fucose-containing sequence structurally different fucosylated antigens, H, A, or B (Fig. 2). The H structure, Fucal-2Gal/31-, may be considered as a precursor giving rise to A or B by the action of specific glycosyltransferases which add, respectively, GalNAcal-3 or Galcrl-3 to the Gal in the H structure. Later it was discovered that these and related oligosaccharide sequences are found on cells in most tissues [8-lo]. They may be presented on glycolipids and different types of glycoproteins. Two main classes of glycoprotein oligosaccharide chains are defined, depending on their linkage to either Asn (N-linked) or Ser/Thr (0-linked) amino acids. The blood group and related antigens occur at (distal) ends of both N- and 0-linked chains. In mucins, they can occur in multivalent form on many neighbouring 0-linked chains, which gives a cooperative effect for the interaction with antibodies or naturally occurring carbohydrate-binding proteins (lectins). Much glycoproteins found in the lining of the gastrointestinal and respiratory tracts are large, with molecular weight from 1000-15000 kDa, and often contain more than 50% carbohydrate, which gives rise to structural diversity and antigenic polyvalency. The protein backbones, coded by the MUC genes, contain tandem repeats of around 20 amino acid residues with a high content of Ser and Thr amino acids. On average, one in every three Ser or Thr residues may have GalNAc attached (the so-called T" antigen), which can be further substituted by other diverse monosaccharides to give eight different protein-to-oligosaccharide core regions [3, 81. Each of these can have additional Fuc and SA residues or can be elongated by backbone sequences which bear blood groups and other related antigens with or without additional SA (Fig. 3). The oligosaccharide chains are built up sequentially by glycosyltransferase enzymes in the Golgi apparatus, for example forming the colon cancer associated mucin antigens T", T, sialyl T" (ST"), sialyl T (ST), Lex, sia- 289 Glycoprotein changes in turnours: a renaissance in clinical applications GalNAc GalPlJGalNAc SAa2-CGalNAc T \ Glycoprotein (mucin) ST" SAa2-3Galpl-3GalNAc sr SAa2-6, 9aiNAc GalP1-3 ST SOr3GalP1-4, GleNAcPl+ Fueal-f FalNAc SAa2-3GalPl-3 DNA (MUC genes)=, mRNA- protein backbone T SulphoLe' Fig. 3. Common mucin core region and sialylated or sulphated oligosaccharide sequences lyl Le" (SLe") and SLe" etc. (Figs. 2 and 3). Mucins and mucin-type oligosaccharides are also found in serum and on cell surfaces, where they have been implicated in suppression of the immune response to protein tumour antigens [ll]. Additionally, mucins may specifically interact with antibodies directed to the same oligosaccharides on the tumour cell, thus inhibiting cell-mediated cytotoxicity at the tissue. Sialylated mucin oligosaccharides are the ligands for sialoadhesins, whereas in non-sialylated form they may respond to hyperproliferative stimuli. For example, the T antigen is unmasked in colon carcinoma [12], and this can bind to peanut and mushroom lectins. The former has been shown to cause proliferation of human colonic epithelium and colorectal cancer cell lines [12], whereas the latter causes suppression of proliferation. These studies highlight the fine differences in the specificity of GalP1-3GalNAc-binding lectins and antibodies, caused by the degree of clustering, tolerance of other substituents, amino acid sequence in the peptide to which GalNAc is attached, etc. Detection of alterations in antigen expression in tumours A variety of carbohydrate markers have been used to study the relevance of carbohydrate tumourassociated antigens (TAAs) in the clinical characterization of human carcinomas. The most frequent alteration of antigen expression in tumours is increased expression of naturally occurring carbohydrate TAA, with concomitant decreased expression of precursor sequence or alternate biosynthetic product. Sometimes aberrant expression may also be observed, which means that antigens not normally found appear. Less often, deletion of detectable expression occurs. Figure 4 illustrates how the expression of the antigenic structures is controlled. The protein backbone and the glycosyltransferases are both encoded by their respective genes. The transferases act by sequentially adding monosaccharides to the growing carbohydrate chain to build up the mucin. In colon carcinomas, a 10-fold increase in expression of the MUC protein back- DNA (transferase) - mRNA / protein enzymes Fig. 4. Relationship of M U C and glycosyltransferase genes to the expression of mucin carbohydrate TAAs bone has been reported, along with increased activity of the relevant transferases [13, 141, although this is not always the case. It is therefore suggested that several control mechanisms are involved in the observed structural changes. Eight MUC genes have now been characterized (Table 1). The functional implications of the changes in carbohydrate structure are not completely understood at present, but it is believed that the changes in cell surface glycoconjugates are reflected in the altered physiological properties and behaviour of the transformed cells. The potential of the new molecular biological methods for following alterations in mucin structure (i.e. at the MUC and glycosyltranferase gene levels) has not yet materialized. Traditionally, lectins were used to distinguish carbohydrate epitopes. Lectins are proteins or glycoproteins with specific affinity for oligo- or mono-saccharides, or parts of larger structures, and as such, their specificities may not be totally restricted to one antigen. The advent of polyclonal antisera increased the specificity of epitope detection, although it was not until the arrival of monoclonal antibodies (mAbs) that accurate epitope detection became possible. mAbs have often been found to be directed towards carbohydrate antigens [3, 9, lo], specific examples of which will be given in this review. Other mAbs have been shown to recognize conformational epitopes requiring both oligosaccharide and protein [ l l , 151. Today, improved analytical methods, such as MS or HPLC, enable us to determine carbohydrate structures from small amounts of sample. It is therefore possible to confirm the presence of a suggested oligosaccharide sequence on biopsies [12, 161. NMR spectroscopy has been able to characterize oligosaccharides in great detail from large amounts of mucin material, but MRS has the potential to correlate this information with data obtained directly from biopsies as discussed below. Table I. Characterized M U C genes MUC I MUC 2 MUC 3 MUC 4 MUC SAC MUC 58 MUC 6 MUC 7 MUC 8 Breast and colon cell surface episialin Colon and small intestine goblet cell secretion Intestinal tissue Trancheobronchialtract Respiratorytract and goblet cell secretion Submaxillary gland secretion Gastric gland secretion Salivary gland secretion Respiratorytract E. F. Hounsell et al. 290 Colon carcinoma Breast tumours Table 2 shows the carbohydrate TAA of special importance in colon cancer [13, 14, 17-24], together with examples of the mAbs (or for the T antigen the peanut lectin PNA) used to detect each antigen. The presence or absence of the antigen in normal tissue, hyperplastic polyps (HP), adenomatous polyps (AP) or carcinoma is shown. The ideal antigenic marker is present in AP (which is harmless by itself, but has the ability to turn into carcinoma) and carcinoma, but not in normal tissue or AP. SLe" and LeX are both expressed by normal tissue and HP as well as AP and carcinomas [18]. That means that these antigens are of limited value as markers. The LeXLeX, SLexLex and ST" antigens, which are all absent in normal tissues and HP, are therefore the markers of choice. A colonic carcinoma that had metastasized to the liver expressed a higher level of the FH6 antigen (Table 2) than the primary tumours [21]. The presence of ST" in mucinous colon carcinomas is often associated with advanced disease and has a poor prognosis [24,25]. Schwartz et al. [26] inhibited mucin 0-glycosylation with benzyl-N-acetyl-galactosamine (a competitive inhibitor of GalNAc-Sermhr transferase), giving reduced metastasis-related events such as adhesion and invasiveness. These studies suggested that, in addition to the increased expression of the unsialylated T antigen, there is an increase in both sialylated and also highly fucosylated oligosaccharides (LexLex, SLexLex) in the primary colon carcinoma and in metastatic sites. In some ways these are contradictory results, showing both an increase and decrease in sialylation levels, but also illustrating the subtleties of possible variations in oligosaccharide structure. MRS analysis of secreted glycoproteins from adenoma and carcinoma cells of increasing tumorigenicity show an increase in the number of different patterns of fucosylation [5]. The most tumorigenic cells had so-called crosspeaks IIa and IIb, which fit best [3, 41 with the diagnostic profile for separate Fucal-2 (e.g. blood group H) and Fucal-3 (Lex-type) linkages. Cross-peaks I11 and IV, also present in normal tissue, are most likely to represent the two fucoses of the Leb-type structure from their positions (chemical shifts) in the MRS and NMR spectra. After the colon, the breast is the most prevalent organ where mucin carbohydrate structural changes have been characterized as TAAs. Table 3 lists different antigens with the lectin (Table 4) or mAb used for identification, and an indication of the usefulness of the antigen as a marker in breast cancer. Each is described further below. In contrast to colon cancer, the structures T, ST, T" and ST" are all considered to be of little diagnostic value [27]. Lex has also been found to be an unsuitable marker in breast cancer, but a potential role in invasiveness has been suggested [28]. Lectin reactivity to breast cancer antigens have been extensively studied. Walker [29] included in his regime a selection of lectins and studied their value as markers of short-term prognosis in breast cancer. None could give prognostic information over and above that provided by histological evaluation. However, the lectin from the garden snail Helix pomutiu (HPA) that recognizes terminal GalNAc residues is more promising and has been investigated by several groups, but the results obtained are conflicting [30]. HPA-binding was observed to normal breast epithelium and to the majority of carcinomas, and it was later found that HPA recognizes a glycoprotein that is associated with metastasis and poor prognosis in breast cancer [31]. However, the International (Ludwig) Breast Cancer Study Group [32] reported no clinical predictive value of HPA; the work continues to characterize breast cancer glycans with terminal immunodominant GalNAc, which could be implicated in aggressive biological behaviour [33]. In general, histological studies are very dependent on the patients that are included and the methodology used. Discrepancies in results are also due to the tumour cells not staining evenly. Some cells are stained and some not. How many cells should be positive in order to conclude that the tumour is stained? These problems may be overcome by moving away from histological examination (with lectins or antibodies) and identifying oligosaccharides from previous lectin and antibody recognition studies as markers that can be targeted for MRS analysis. Fine-needle aspiration biopsy specimens have been used for diagnosis of breast and other carcinomas using mAb B72.3, but the antigenic determinant was Table 2. Glycoprotein markers in colonic tissues. N = normal, HP = hyperplastic polyps, AP = adenomatous polyps, C = carcinoma. Antigen SLea Lex Le'Le" SLe'Le" T" T ST" MAb/lectin N HP AP C Ref. MSWl 13 t t - t t t t - tt tt tt tt tt tt tt ttt ttt ttt ttt ttt ttt ttt [I81 [191 [201 SSEA-I FH4 FH6 MLS 128 PNA JTlk PI1 [221 ~ 3 1 ~ 4 1 29 I Glycoprotein changes in turnours: a renaissance in clinical applications Table 3. Glycoprotein markers in breast cancer Structure mAb/lectin Use T,ST,T",ST" Le" Carbohydrate GalNAc Epithelial mucin Epithelial mucin Non-sialylatedmucin Sialylatedglycoprotein Acidic glycoprotein Mucin, NeuSGc Structure t ? t It t t IgM Lectins HPA 872.3 (315-3, MCA 83D4 BTAA a 4 9 3E1.2 Ref. I text) .!- t t not characterized. The mAbs bl2/MCA and DF3/CA15-3, also uncharacterized, appear to be good prognostic indicators utilized in commercial tests [35]. Pancino et al. [36] characterized a 300-1000 kDa antigen of human breast carcinoma which reacted strongly with wheat germ agglutinin and peanut agglutinin, but gave no response to lentil or concanavalin A lectins (Table 4). The activity was strongly reduced by periodate oxidation, which is a method to destroy the carbohydrate, and also by trypsin digestion, which cleaves the protein chain. The activity was retained after neuraminidase treatment which results in the cleavage of sialic acids. These results indicate that a carbohydrate epitope is involved, but not SA. Pal et al. [37] purified a breast TAA (BTAA) with a molecular weight of 85 kDa that may be useful in diagnosis and prognosis. It was neuraminidase-, trypsin- and papain-sensitive, thermostable at high temperatures and stained by periodate/Schiff reagent for detecting carbohydrates. The authors concluded that breast TAA is a carbohydrate epitope involving SA. Another useful marker for stage IV breast cancer (CA549) was also found to recognize an acidic glycoprotein [38]. This study also tested mAbs raised against human milk fat globule membranes, which are known to be a rich source of fucosylated carbohydrate antigens that have also been characterized by NMR [31, 391. Also easily identified by NMR [4] is the difference between two members of the SA family, i.e. N-acetylneuraminic acid (NeuSAc), which we have been discussing so far as SA, and N-glycolylneuraminic acid (NeuSGc), which until recently was thought not to occur in humans. However, Devine et al. [40] reported the Table 4. The specificity of several of the lectins used in diagnosis Lectin Abbrevation Specificity Peanut agglutinin Wheat germ agglutinin ConcanavalinA Lentil Helix pornatia agglutinin PNA WGA Con A Lentil H PA /l-~-Gal( I-3)~-GalNAc D-GIcNAc,NeuSAc,o-GalNAc u-D-M~,a-D-Glc N-D-M~UI u-D-G~~NAc presence of a mucin carbohydrate epitope defined by antibody 3E1.2, which recognized an 0-linked glycan containing NeuSGc. This finding has been corroborated by Hanisch et al. [41] who have identified specific changes in glycosylation (glycoforms) of the MUC-1 protein from a breast tumour surgical resection and from T47D cells. These showed a lack of fucosylated Le blood-group-related antigens and the appearance of novel core-type ST antigen, Gal~l-3[Neu5Gca2-61GalNAc. Glycoprotein antigens in other neoplasms and general clinical implications Other studies have addressed changing carbohydrate structure in a range of tumours, including prostate, ovary, pancreas and lung. Table 5 shows some of those for the prostate and pancreas where the importance of mucin-type carbohydrate has been characterized [42-SO]. TURP-27 is related to the neural cell adhesion molecules recognized by the mAb HNK-1, the epitope for which is known to involve sulphated and polysialylated oligosaccharides such as those on neural cell adhesion molecules. Polysialic acid has also been shown to occur on 0-linked chains in breast cancer and leukaemia cell lines [51], whereas that on neural cell adhesion molecules is on N-linked chains. TAAs, which are glycoproteins where the carbohydrate chains are of the N-linked type, are found, such as the prostatespecific antigen (PSA) which is a glycosylated serine protease of the kallikrein family [52, 531. For the antigen 5T4 [15], a marker of several tumours, the carbohydrate and protein are involved together in antigenicity. 5T4 is involved in extravasation of the placenta, which can be considered as a model for metastasis and an oncodevelopmental paradigm. Similarly, oligosaccharides were first discovered as differentiation/developmental markers, particularly Lex and L e y [3, 9, 10, 441 and SLe" (CA19-9 [48]). It was shown that they had specific roles in morphogenesis by interacting with the carbohydrate-binding proteins, called selectins. Results have been obtained which indicate that SLex and SLea antigens Table 5. Glycoprotein markers in the prostate and pancreas. PAC, prostate carcinoma-associatedglycoprotein complex. Antigen Prostate Sialoglycoproteincomplex PAC Ld, H-type 2 Ley, di-Le" tri-Lex TAG-72 mucin Mucin sialylated oligosaccharides Pancreas Sialylated mucin SLea Sialylated mucin Lea/type I mucin mAb TUW-2 7 BI 83 142,431 B72.3, CC83, CC89 PR92 1451 ~461 DUPAN-2 CA19.9 Span-I, la3, Nd2 BW494 [47l ~481 MI WI 1501 292 E. F. Hounsell et al. on the surface of colon cancer cells function as receptors for E and P selectin expressed on vascular endothelium [54, 551. This may be the initial event before extravasation of cancer cells from the capillaries [56]. That many of the markers reoccur for different tumours points to a similar mechanism if they are involved in pathogenesis. For example, PSA also appears in lung and breast tumours [57]. Devine et al. [58] compared PSA with other more highly glycosylated antigens as a TAA in colon, breast, prostate, pancreas, lung, bladder and ovary. Changes in the lung have also been correlated with mRNA levels of several MUC genes [59], and the levels of mRNA of a number of other MUC genes have been found increased in different cancer tissues (recently reviewed in [60]). Elevated serum levels of CA15-3 [35] and other mucins have been reported in patients with lung, pancreatic, colon, breast and ovary cancer [61]. MRS studies have looked at ovary biopsies where the Fuc IIa and IIb cross-peaks, discussed above for colon studies, correlate with increased tumorigenesis [6]. Compared with NMR data [4, 8, 621, this pattern may be due to an increase in the CA19.9 antigen (also implicated in the pancreas [48]). The presence of SLe" and SLe" may have a reciprocal presentation with respect to T" and ST" [56], which is important because ST" can mediate inhibition to natural killer cell cytotoxicity [631. These clues as to mechanism are being approached with respect to new therapeutic regimes. For example, O'Boyle et al. [64] vaccinated 20 cancer patients with mucin containing T" and ST" determinants. A significant rise in the corresponding antibodies was observed, which has prompted further clinical trials. The use of synthetic peptides corresponding to the protein core of the polymorphic glycosylated epithelial mucin as a vaccine in cancer patients is being explored [65], and the high density of carbohydrate moieties peculiar to tumours has prompted the re-exploration of mAb directed towards them for use in stimulation of antibody-dependant cellular cytotoxicity [66]. Conjugates with mAb have been extensively investigated as possible drug-targeting systems. In a recent example using a radio-immunoconjugate against mucin antigens, Peterson et al. [67] cured six of seven mice breast tumours by treatment with tungsten-labelled mAb. The antigens were the major components of the human milk fat globule membrane, which expresses high levels of fucosylated and sialylated oligosaccharides [3, 9, 391. In summary, epithelial cells of the colon, breast, lung, ovary, prostate and pancreas express cell-surface much antigens that change during malignant transformation. There is a link between changes in the structure of cell carbohydrate and the abnormal properties of cancer cells. The structural changes in colon cancer are recognized by mAbs and confirmed by analytical methods. A large amount of attention has been given to these structural changes for possible utilization in the diagnosis of benign and malignant tumours, measurement of the effectiveness of treatment, and prognosis based on tumour maturity and metastatic potential. MRS offers a new technique with which to access tumour type and stage. By cross-matching to known oligosaccharide antigen NMR chemical-shift signatures, this will provide further knowledge with which to interpret pathogenic mechanisms in the light of our increasing understanding of endogenous carbohydrate-binding proteins and much biosynthesis. 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