Glycans stand out from all classes of biomolecules because of their unsurpassed structural complexity. This is generated by variability in anomeric status of the glycosidic bond and its linkage points, ring size, potential for branching and introduction of diverse site-specific substitutions. What poses an enormous challenge for analytical processing is, at the same time, the basis for the fingerprint-like glycomic profiles of glycoconjugates and cells. What's more, the glycosylation machinery is sensitive to disease manifestations, earning glycan assembly a reputation as a promising candidate to identify new biomarkers. Backing this claim for a perspective in clinical practice are recent discoveries that even seemingly subtle changes in the glycan structure of glycoproteins, such as a N-glycan core substitution by a single sugar moiety, have far-reaching functional consequences. They are brought about by altering the interplay between the glycan and (i) its carrier protein and (ii) specific receptors (lectins). Glycan attachment thus endows the protein with a molecular switch and new recognition sites. Co-ordinated regulation of glycan display and presentation of the cognate lectin, e.g. in cancer growth regulation exerted by a tumour suppressor, further exemplifies the broad functional dimension inherent to the non-random shifts in glycosylation. Thus studies on glycobiomarkers converge with research on how distinct carbohydrate determinants are turned into bioactive signals.

Why glycobiomarkers?

Onset and progression of a disease are manifested by molecular deviations from the normal range of physiological parameters. Monitoring the clinical status aids in selecting the optimal therapeutic modality. It is towards this aim that there is an intense quest for the identification of biomarkers, driven by the need for reliable diagnostic information. At best, such markers should already be applicable at early stages with high specificity and selectivity, with potential to reflect the course of disease, to include changes due to therapeutic interventions, and with prognostic value. For monitoring several types of malignancy, a group of glycoproteins, α-fetoprotein, carcinoembryonic antigen, CA19–9 (CA is cancer antigen), CA125 or the prostate-specific antigen among them, have already entered clinical practice, their application indicating an apparent requirement for improving marker specificity and/or selectivity. Since ongoing screening by transcriptomic microarrays/proteomics in cancer is not uncovering a confirmed specific qualitative change in a particular gene product, the activity in this field is increasingly devoted to efforts to exploit the presence of post-translational modifications as markers. In this respect, glycosylation figures prominently and is attracting increasing attention, for several reasons.

Glycosylation is the enzymatic attachment of glycans to proteins and lipids as carriers, followed by intricate structural tailouring. It is a common process, with inherent capacity for generation of a wide variety of sugar chains that can result in a fingerprint-like profile of the glycome (the equivalent of the proteome or genome on the level of glycans) for distinct cells [1]. N-glycans, for example, can be carried by more than 10% of all proteins; whether glycosylation sites defined by the N-X-S/T sequon are occupied and which carbohydrate units constitute the respective chain can vary, opening the door to a large number of glycoforms sharing the carrier backbone, but differing in the glycan part [1]. In contrast with phosphorylation, certainly a valuable indicator for the status of proliferation, information on glycosylation naturally goes far beyond measuring its presence on a carrier. In fact, the production process of the glycan chains and the chemical properties of the building blocks (sugar alphabet) meet all criteria for versatile and flexible information coding.

Since the assembly of glycan chains is not a template-dependent process, but instead reflects the current composition of enzymes, transporters and substrates in the production line, glycan synthesis is a complex, dynamically regulatable process. The occurrence of diverse branch points within the core regions of N- and O-glycans and variegated fine-structural tailoring of branch ends makes a large panel of protein- and lipid-bound oligosaccharides possible [26], and this potential is realized. Not only can different organisms be separated by their characteristic glycan signatures [7], but also cells at different stages of development or disease states, such as malignancy, present phenotypic changes in glycosylation [1,810]. For N-glycans, there changes can concern shifts between the three major classes, as well as the introduction of substitutions to their core, i.e. core fucosylation, presence of the bisecting GlcNAc (N-acetylglucosamine) moiety and branching, and to terminal positions, here particularly fucosylation and sialylation. Frequency and truncation are respective parameters susceptible to changes of mucin-type O-glycans. As these examples attest, the structural analysis of glycans is much more demanding than that of nucleic acids and proteins. It must cover, beyond the sequence, the anomeric linkage, the linkage points, the ring size and presence of any substitutions (e.g. sulfation, phosphorylation or O-acetylation) [1]. Given the enormous potential for structural diversity, the task of fully defining all structural details of each glycan chain has called for a new level of technical sophistication in examining post-translational modifications. Owing to the groundbreaking advances in analytical precision and sensitivity, especially by MS, the glycome of serum/tissue samples and of distinct glycoproteins can now be defined in detail in molecular terms [1016].

In this context, the application of carbohydrate-determinant-selective/specific probes (lectins, antibodies; see [1] for an overview on the characteristics and modes of binding of sugar epitopes by these proteins) has proven valuable to enrich glycoproteins with common glycan characteristics from extracts/serum and to select for the presence of certain sugar-encoded ligands from glycopeptide mixtures (for examples, please see [17,18]). In these reports, increased sialylation and fucosylation in the serum glycoproteins complement C3, histidine-rich glycoprotein and kininogen-1 for colorectal cancer and enhanced extent of core substitutions in tissue periostin for ovarian cancer were described. The strategic combination of structural glycan analysis with glycoproteomics, flanked by a glycan-type-selective detection, is thus capable of tracking down disease-associated alterations on the level of individual gene products. To answer the question on the nature of suitable probes for the candidate regions sensitive to disease-associated alterations, Table 1 lists a collection of lectins. They represent reliable sensors for investigations focusing on N-glycan core substitutions and the status of sialylation, as demonstrated recently by lectin histochemical staining for cell markers [19].

Table 1
Example of a panel of plant/human lectins useful for glycophenotyping of structural aspects of N-glycans
Latin name (common name) Abbreviation Monosaccharide specificity Potent oligosaccharide 
Phaseolus vulgaris erythroagglutinin (kidney bean) PHA-E Bisected complex-type N-glycans: Galβ4GlcNAcβ2Manα6(GlcNAcβ2-Manα3)(GlcNAcβ4)Manβ4GlcNAc 
Phaseolus vulgaris leucoagglutinin (kidney bean) PHA-L Tetra- and tri-antennary N-glycans with β6-branching 
Viscum album (mistletoe) VAA Gal Galβ2(3)Gal, Galα3(4)Gal, Galβ3(4)GlcNAc without/with α2,6-sialylation, Fucα2Gal 
Sambucus nigra (elderberry) SNA Gal/GalNAc Neu5Acα6Gal/GalNAc, clustered Tn-antigen, 9'-O-acetylation tolerated 
Siglec-2 CD22 Neu5Acα6Galβ3(4)GlcNAc, 9'-O-acetylation blocks binding 
Dolichos biflorus (horse gram) DBA GalNAc GalNAcα3GalNAcα3Galβ4Galβ4Glc 
Maackia amurensis I (leucoagglutinin) MAA I Neu5Acα3Galβ4GlcNAc/Glc, 3'-sulfation tolerated 
Latin name (common name) Abbreviation Monosaccharide specificity Potent oligosaccharide 
Phaseolus vulgaris erythroagglutinin (kidney bean) PHA-E Bisected complex-type N-glycans: Galβ4GlcNAcβ2Manα6(GlcNAcβ2-Manα3)(GlcNAcβ4)Manβ4GlcNAc 
Phaseolus vulgaris leucoagglutinin (kidney bean) PHA-L Tetra- and tri-antennary N-glycans with β6-branching 
Viscum album (mistletoe) VAA Gal Galβ2(3)Gal, Galα3(4)Gal, Galβ3(4)GlcNAc without/with α2,6-sialylation, Fucα2Gal 
Sambucus nigra (elderberry) SNA Gal/GalNAc Neu5Acα6Gal/GalNAc, clustered Tn-antigen, 9'-O-acetylation tolerated 
Siglec-2 CD22 Neu5Acα6Galβ3(4)GlcNAc, 9'-O-acetylation blocks binding 
Dolichos biflorus (horse gram) DBA GalNAc GalNAcα3GalNAcα3Galβ4Galβ4Glc 
Maackia amurensis I (leucoagglutinin) MAA I Neu5Acα3Galβ4GlcNAc/Glc, 3'-sulfation tolerated 
*

No monosaccharide known as ligand; for application of this lectin panel in histochemical analysis and further strategic considerations, see [19].

Table 2
Examples of the effect of structural changes in N-glycans on properties of cell-surface receptors with focus on the epidermal growth factor receptor

EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular-signal-regulated kinase; FAK, focal adhesion kinase; FucT-I/VIII: α1,2-fucosyltransferase-I/α1,6-fucosyltransferase-VIII; GnT-III/V: N-acetylglucosaminyltransferases-III/V; HEK, human embryonic kidney. For information on other cell-surface glycoprotein receptors, see [28,29].

Experimental system Altered glycosylation Functional consequence 
Overexpression of GnT-III (Mgat3) in transfected human cervical adenocarcinoma HeLaS3 cells Increased level of bisecting GlcNAc Reduced EGF binding (low-affinity sites), increased internalization and ERK phosphorylation [47
FucT-VIII overexpression in transfected HEK-293 cells Enhanced core fucosylation Enhanced EGF-dependent signalling and cell growth, increased sensitivity to gefitinib (an EGFR inhibitor) [48
Stable FucT-VIII knockdown by short hairpin RNA in HEK-293 cells and human lung cancer A549 cells Reduced core fucosylation Decreased EGF-dependent cell growth and sensitivity to gefitinib [48
Down-regulation of GnT-V (Mgat5) by siRNA in metastatic and invasive human breast carcinoma MDA-MB231 cells and breast carcinoma cells from GnT-V (Mgat5)-null mice Reduced level of N-linked β1,6-branching Suppression of FAK/paxillin dephosphorylation via decreasing EGF-dependent activation of tyrosine phosphatase SHP2 despite unaltered EGF binding, reduced ERK signalling, tumour cell invasiveness and growth [29,49
FucT-I overexpression in human ovarian cancer RMG-I cells Increased level of Ley Increased EGFR phosphorylation and downstream signalling [50
Knockdown of α-mannosidase IB in human ovarian epidermoid carcinoma A431 cells/enzymatic unmasking of terminal GlcNAc in N-glycans in lysates Reduced/increased level of terminal GlcNAc Level of inhibition of EGFR phosphorylation by ganglioside GM3 reduced/increased (for a review, see [33]) 
Experimental system Altered glycosylation Functional consequence 
Overexpression of GnT-III (Mgat3) in transfected human cervical adenocarcinoma HeLaS3 cells Increased level of bisecting GlcNAc Reduced EGF binding (low-affinity sites), increased internalization and ERK phosphorylation [47
FucT-VIII overexpression in transfected HEK-293 cells Enhanced core fucosylation Enhanced EGF-dependent signalling and cell growth, increased sensitivity to gefitinib (an EGFR inhibitor) [48
Stable FucT-VIII knockdown by short hairpin RNA in HEK-293 cells and human lung cancer A549 cells Reduced core fucosylation Decreased EGF-dependent cell growth and sensitivity to gefitinib [48
Down-regulation of GnT-V (Mgat5) by siRNA in metastatic and invasive human breast carcinoma MDA-MB231 cells and breast carcinoma cells from GnT-V (Mgat5)-null mice Reduced level of N-linked β1,6-branching Suppression of FAK/paxillin dephosphorylation via decreasing EGF-dependent activation of tyrosine phosphatase SHP2 despite unaltered EGF binding, reduced ERK signalling, tumour cell invasiveness and growth [29,49
FucT-I overexpression in human ovarian cancer RMG-I cells Increased level of Ley Increased EGFR phosphorylation and downstream signalling [50
Knockdown of α-mannosidase IB in human ovarian epidermoid carcinoma A431 cells/enzymatic unmasking of terminal GlcNAc in N-glycans in lysates Reduced/increased level of terminal GlcNAc Level of inhibition of EGFR phosphorylation by ganglioside GM3 reduced/increased (for a review, see [33]) 

In view of the observed complexity of the structure of glycans, in stark contrast with the constancy of structural parameters of their protein/ceramide carriers, raises the question as to whether the structural changes arise randomly as ‘whim of Nature’ or can be considered as a ‘multipurpose tool’ [1,20]. Naturally, carbohydrates surpass all other classes of biomolecules in their capacity to code information, so that it would mean missing a wealth of opportunities if they were not recruited as bioactive signals to cell society [21]. Experimental backing of this reasoning can be obtained by studying disease-associated alterations. Salient arguments against emergence of glycodiversity simply by random fluctuations would be established if the activity of oncogenes/tumour suppressors led to specific alterations and if functional consequences of these changes for the tumour behaviour could indeed be delineated.

Glycobiomarkers as bioactive signals

The analysis of modulation of glycosylation by defined genetic manipulations, first by promoting increased oncogene expression, has indeed tracked down changes in the glycome, most notably increases in N-glycan branching, chain length and branch-end sialylation (for a review, see [8]). Oncogene activation can affect the enzymatic machinery via different means, e.g. redistribution of glycosyltransferases in the Golgi/endoplasmic reticulum compartment or transcriptional up-regulation of glycosyltransferase genes in the case of the tyrosine kinase src oncogene [2224]. Proteins with suppressor activity, too, elicit changes on the level of glycans in a manner specific for the effector (i.e. p16INK4a, activin type 2 receptor, absent in melanoma 2 and transforming growth factor-β type 2 receptor), as detected by transcriptomics of glycosyltransferase genes and lectin binding using plant/human agglutinins [25,26]. Figure 1 illustrates the conspicuous effect of the tumour suppressor p16INK4a on cell-surface α2,6-sialylation in a model for pancreatic carcinoma, detected by a human lectin and a plant agglutinin listed in Table 1. That a human lectin senses such differences inspires the concept for a functional correlation [21]. In principle, the increased extent of tumour cell reactivity illustrated in Figure 1 and the general changes noted above can make their presence felt via two routes: acting (i) as cis-effector on the carrier protein, and (ii) as trans-effector by serving as a docking site for a lectin in intermolecular interplay. It is now apparent that defects and aberrations in glycosylation cause malfunctions on the level of the organism which underscore the medical significance of glycan integrity [2729].

Regulation of sialylation by a tumour suppressor

Figure 1
Regulation of sialylation by a tumour suppressor

The restitution of positive tumour suppressor status in human Capan-1 pancreatic carcinoma cells is associated with marked changes in lectin reactivity detected by FACScan analysis (quantitative data in percentage of positive cells and mean fluorescence intensity are given and the negative control in the absence of lectin shown as a grey area) [25]. Binding sites for the pro-anoikis effector galectin-1 (Gal-1) are masked by sialylation in mock-treated cells (without p16INK4a expression) and are up-regulated by tumour suppressor expression, whereas the presence of α2,6-sialylation (detected by SNA; for details on its sugar specificity, see Table 1) is down-regulated (for functional consequences, see Figure 2). The alterations caused by mild sialidase treatment underline the role of sialylation for cell reactivity to both lectins.

Figure 1
Regulation of sialylation by a tumour suppressor

The restitution of positive tumour suppressor status in human Capan-1 pancreatic carcinoma cells is associated with marked changes in lectin reactivity detected by FACScan analysis (quantitative data in percentage of positive cells and mean fluorescence intensity are given and the negative control in the absence of lectin shown as a grey area) [25]. Binding sites for the pro-anoikis effector galectin-1 (Gal-1) are masked by sialylation in mock-treated cells (without p16INK4a expression) and are up-regulated by tumour suppressor expression, whereas the presence of α2,6-sialylation (detected by SNA; for details on its sugar specificity, see Table 1) is down-regulated (for functional consequences, see Figure 2). The alterations caused by mild sialidase treatment underline the role of sialylation for cell reactivity to both lectins.

The first mode of glycan functionality is its effect on properties of the carrier protein, with consequences for instance on the regulation of an enzymatic activity and ligand binding or of proteolytic degradation and oligomerization, which are already triggered by attachment of a single sugar moiety, i.e. the O-GlcNAcylation equaling phosphorylation in efficacy [5]. The different types of O-glycosylation and certainly N-glycosylation are active in this respect. As an instructive case study from the class of cell-surface glycoprotein receptors acting as tyrosine kinases, examples for the correlation between introducing a particular malignancy-relevant N-glycan structure to the glycoprotein and selected functional consequences are presented in Table 2. As it turned out, additions of core or branch-end substitutions into N-glycans are capable of changing receptor properties and downstream signalling. These observations suggest the induction of structural rearrangements of the N-glycan antennae, altering the interplay with the protein part. Computational chemistry is a means to test the implicit concept comparing core substitutions with molecular switches. Indeed, significant shifts in conformational equilibria of N-glycans upon entering core substitutions have been measured and would explain the experimentally detected effects [3032]. Explicitly, the core substitutions appear to act as long-range modulators of the shape of N-glycans, a topological factor underlying the glycans' impact on the activity of the growth factor receptor. Further probing into the subtleties of this intra-glycoprotein interplay has also revealed tuning/inhibitory effects that apply to terminal chain modification (Table 2), and these are even more pronounced following truncation of complex-type N-glycan termini. The presentation of GlcNAc residues at branch ends in receptor N-glycans promotes a switch to a second effector pathway operating via intermolecular interactions. In this case, the ganglioside GM3, long been known to inhibit tyrosine kinase activity by interfering with growth factor binding, is likely to be engaged in carbohydrate–carbohydrate interactions with these truncated N-glycans [33]. These contacts establish a further level of regulation of receptor activity (similar responsiveness has also been shown for the insulin receptor or vascular endothelial growth factor receptor-2 on vascular endothelium) by dynamic remodelling of ganglioside abundance on the cell surface, e.g. by sialidases [4,33,34].

The second mode of glycan functionality, alluded to above for the ganglioside and also a human lectin, is mediated by the reactivity of the glycan with complementary sites. It leads to associations via carbohydrate–carbohydrate/protein recognition. In this case, the glycans of the glycoprotein harbour structural information essential for binding, the first step in decoding by the counter-receptor. In other words, the glycan chains can engage in recognition processes. In the case of lectins that can cross-link glycans by virtue of bi- or oligo-valency, an ensuing post-binding signalling translates the sugar-encoded information into cellular responses [35]. The number of proteins able to host sugar ligands will be a clear indication of the range of physiological relevance of carbohydrate–protein interactions, and this answer is very clear: a large panel of protein folds with at least 14 different types has developed which accommodate carbohydrates without any enzymatic processing, impressively underscoring the versatility of this intermolecular recognition mode [1,21]. Epitomizing this aspect of the sugar code, the flow of information from glycans to effects via lectins underlies various cellular activities. At the level of cells, several lectin families induce signalling for growth and engender cell contacts (Table 3). This compilation signifies a high level of inherent specificity in the way a tissue lectin finds its way to the target site among the glycomic diversity. Despite this multitude of glycoconjugates on cell surfaces, presenting the typical termini with substituted β-galactosides, endogenous lectins can apparently single out a limited set of counter-receptors, by sensing fine-structural changes and topological factors of ligand presentation. Six levels of affinity regulation are operative to accomplish the accurate translation of the information encoded in glycans into biological responses [21]. Even among homologous proteins, structural variations of the ligand, such as presence of α2,3-sialylation, can make a large impact on cell binding, N-glycan core substitutions are capable of fine-tuning lectin reactivity to soluble and cell-membrane lectins and the degree of saturation of multivalent glycans substantially affects affinity constants, these three examples strongly emphasizing the relevance of testing natural glycoconjugates for physiological considerations [14,3032,3639].

Table 3
Examples of involvement of human/animal lectins in regulation of cell adhesion/growth and recognition of aberrant glycosylation

CRP, C-reactive protein; N-CAM, neuronal cell-adhesion molecule; RHAMM, receptor for hyaluronic acid-mediated motility.

Activity Example of lectin 
Cell growth control, induction of apoptosis/anoikis and axonal regeneration Galectins, C-type lectins, amphoterin-like protein, hyaluronic acid-binding proteins, cerebellar soluble lectin, CD22 (siglec-2), MAG (siglec-4) 
Cell migration and routing Galectins, selectins and other C-type lectins, I-type lectins, hyaluronic acid-binding proteins (RHAMM, CD44, hyalectans/lecticans) 
Cell–cell interactions Selectins and other C-type lectins such as DC-SIGN, galectins, I-type lectins (siglecs, N-CAM, P0 or L1), gliolectin 
Cell–matrix interactions Galectins, heparin- and hyaluronic acid-binding lectins including hyalectans/lecticans, calreticulin 
Matrix network assembly Proteoglycan core proteins (C-type CRD and G1 domain of hyalectans/lecticans), galectins (e.g. galectin-3/hensin), non-integrin 67 kDa elastin/laminin-binding protein 
Recognition of foreign or aberrant glycosignatures on cells (including endocytosis or initiation of opsonization or complement activation) Collectins, ficolins, C-type macrophage and dendritic cell lectins, CR3 (CD11b/CD18, Mac-1 antigen), α/θ-defensins, pentraxins (CRP, limulin), RegIIIγ (HIP/PAP), siglecs, tachylectins 
Activity Example of lectin 
Cell growth control, induction of apoptosis/anoikis and axonal regeneration Galectins, C-type lectins, amphoterin-like protein, hyaluronic acid-binding proteins, cerebellar soluble lectin, CD22 (siglec-2), MAG (siglec-4) 
Cell migration and routing Galectins, selectins and other C-type lectins, I-type lectins, hyaluronic acid-binding proteins (RHAMM, CD44, hyalectans/lecticans) 
Cell–cell interactions Selectins and other C-type lectins such as DC-SIGN, galectins, I-type lectins (siglecs, N-CAM, P0 or L1), gliolectin 
Cell–matrix interactions Galectins, heparin- and hyaluronic acid-binding lectins including hyalectans/lecticans, calreticulin 
Matrix network assembly Proteoglycan core proteins (C-type CRD and G1 domain of hyalectans/lecticans), galectins (e.g. galectin-3/hensin), non-integrin 67 kDa elastin/laminin-binding protein 
Recognition of foreign or aberrant glycosignatures on cells (including endocytosis or initiation of opsonization or complement activation) Collectins, ficolins, C-type macrophage and dendritic cell lectins, CR3 (CD11b/CD18, Mac-1 antigen), α/θ-defensins, pentraxins (CRP, limulin), RegIIIγ (HIP/PAP), siglecs, tachylectins 

Overall, the potential for structural complexity of glycans and the dynamic regulation by remodelling explains the feasibility for changes to occur dynamically in glycan display. Of note, they may not be global but affect primarily certain glycoconjugates, as e.g. glycoproteomic analysis of serum constituents has revealed [17,18]. In this sense, it is self-evident that endogenous lectins such as the adhesion/growth-regulatory galectins [35] target few high-affinity ligands on a cell often endowed with cell-type-selective glycosylation features (Table 4). The combination of the presence of distinct glycans, a suited topological presentation and low degree of saturation with lectin adds up to binding specificity, the cell being destined to react in a distinct manner by virtue of its glycans. Physiologically, the underlying glycosignature has biosignal activity in the interplay with endogenous lectins, thus qualifying as a functional glycobiomarker. This glycan–lectin interaction can have negative or positive consequences, as will now be outlined with examples on glycoproteins and glycolipids.

Table 4
Glycoconjugate ligands of the adhesion/growth-regulatory galectins-1 and -3
Galectin Ligand 
Galectin-1 Ovarian carcinoma antigen CA125, CD2, CD3, CD4, CD7, CD43, CD45, CD95(Fas), carcinoembryonic antigen (CEA), fibronectin (tissue), gastrointestinal mucin, hsp90-like glycoprotein, β1-integrin (CD29), α1/α4/α5/α7β1- and α4β7-integrins, cell adhesion molecule L1, laminin, lamp-1, Mac-2-binding protein, nephrin, neuropilin-1, receptor protein-tyrosine phosphatase (RPTPβ), thrombospondin, Thy-1, tissue plasminogen activator, chondroitin sulfate proteoglycan, distinct neutral glycolipids, ganglioside GM1 
Galectin-3 CD7, CD11b of CD11b/CD18 (Mac-1 antigen, CR3), CD13 (aminopeptidase N), CD32, CD43, CD45, CD66a,b, CD71, CD95, CD98, CEA, colon cancer mucin and MUC1-D (N-glycan at Asn36), cubilin, C4.4A (member of Ly6 family), epidermal growth factor receptor, haptoglobin β-subunit (after desialylation), hensin (DMBT-1), glycoform of IgE, β1-integrin (CD29) and α4/α5β1-integrins, LI-cadherin, laminin, lamp-1/-2, Mac-2-binding protein, Mac-3, MAG, MP20 (tetraspanin), NG2 proteoglycan, TCR complex, tenascin, tissue plasminogen activator, transforming growth factor-β receptor, vascular cell adhesion molecule-1, ganglioside GM1 
Galectin Ligand 
Galectin-1 Ovarian carcinoma antigen CA125, CD2, CD3, CD4, CD7, CD43, CD45, CD95(Fas), carcinoembryonic antigen (CEA), fibronectin (tissue), gastrointestinal mucin, hsp90-like glycoprotein, β1-integrin (CD29), α1/α4/α5/α7β1- and α4β7-integrins, cell adhesion molecule L1, laminin, lamp-1, Mac-2-binding protein, nephrin, neuropilin-1, receptor protein-tyrosine phosphatase (RPTPβ), thrombospondin, Thy-1, tissue plasminogen activator, chondroitin sulfate proteoglycan, distinct neutral glycolipids, ganglioside GM1 
Galectin-3 CD7, CD11b of CD11b/CD18 (Mac-1 antigen, CR3), CD13 (aminopeptidase N), CD32, CD43, CD45, CD66a,b, CD71, CD95, CD98, CEA, colon cancer mucin and MUC1-D (N-glycan at Asn36), cubilin, C4.4A (member of Ly6 family), epidermal growth factor receptor, haptoglobin β-subunit (after desialylation), hensin (DMBT-1), glycoform of IgE, β1-integrin (CD29) and α4/α5β1-integrins, LI-cadherin, laminin, lamp-1/-2, Mac-2-binding protein, Mac-3, MAG, MP20 (tetraspanin), NG2 proteoglycan, TCR complex, tenascin, tissue plasminogen activator, transforming growth factor-β receptor, vascular cell adhesion molecule-1, ganglioside GM1 

The first case study starts from observations of enhanced incorporation of α1,2/4-linked fucose moieties into N-glycans. As a consequence of this increased branch-end fucosylation, clusters of Lea/Leb epitopes are produced on colon cancer glycoproteins. The ones on the glycoprotein carcinoembryonic antigen of SW1116 cells have conspicuous reactivity for a C-type lectin, which is a sensor for adapting the activation status of dendritic cells, i.e. DC-SIGN (dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin) [40]. These glycan clusters present on primary cancer colon epithelia, but not non-malignant control tissue, are assumed to attract DC-SIGN, with negative consequences on the afflicted dendritic cells to mount an antitumour response. Conversely, such characteristic clusters can also signal an aberration tied to malignancy to the innate host-defence system, a surveillance mechanism described in Table 4. In fact, the correspondingly glycosylated glycoproteins CD26/CD98hc are reactive with the collectin mannan-binding lectin, an activator of the complement cascade [41].

The linking of glycosylation and lectin expression to trigger effects synergistically is illustrated by the co-regulation of increases in cell-surface presentation of ganglioside GM1 and the growth effector galectin-1 upon differentiation of human SK-N-MC neuroblastoma cells (and also the communication between regulatory/effector T-cells in autoimmune suppression), proper microdomain organization being essential for the high affinity [4245]. Disrupting microdomain integrity by cell treatment with either the macrolide filipin III or 2-hydroxypropyl-β-cyclodextrin markedly lowered affinity of galectin binding [45]. The connection between glycan remodelling, in this case by a cell-surface ganglioside sialidase, and an endogenous lectin is further strengthened by unveiling the master-regulator activity of the tumour suppressor p16INK4a. The enhanced reactivity of the engineered Capan-1 tumour cells for galectin-1 shown in Figure 1 is already an indication for a co-ordination of glycan-lectin presentation, en route to establishing susceptibility for anoikis. Indeed, the transcriptional up-regulation of galectin-1 and its marked association with the fibonectin receptor has been documented [25,46]. The galectin-1-dependent glycoprotein cross-linking, made feasible by the reprogramming of the glycosylation machinery, eventually activates caspase 8 and, apparently optimizing pro-anoikis signal intensity, the anti-anoikis effector galectin-3, a competitor of pro-anoikis galectin-1, is down-regulated post-transcriptionally [46]. The way these different described effects work together for anoikis induction under the tumour suppressor's guidance is summarized in Figure 2. The glycosylation of the fibronectin receptor (and/or a tightly associated binding partner) is thus controlled by the tumour suppressor (see Figure 1), reflecting restored susceptibility to anoikis, and can therefore be identified as a functional glycobiomarker. Its reactivity to a human effector (i.e. galectin-1) provides a precedent for co-ordinated glycan–lectin expression, which accounts for the cellular response (Figure 2).

Glycobiological route to anoikis induction by a tumour suppressor

Figure 2
Glycobiological route to anoikis induction by a tumour suppressor

The tumour suppressor p16INK4a is the master regulator to enhance signalling via the fibronectin receptor (α5β1-integrin) in human Capan-1 pancreatic carcinoma cells in vitro. Galectin-1 (Gal-1) is the pro-anoikis effector. The extent of its binding to the cells' surface is increased (Figure 1). Orchestrated up-regulation of presentation of Gal-1 and the integrin, combined with the enhanced reactivity of glycosylation to the lectin, favours caspase 8 activation [25,46]. Gal-3, a competitor of Gal-1 with additional intracellular anti-anoikis potential, is down-regulated in parallel [46].

Figure 2
Glycobiological route to anoikis induction by a tumour suppressor

The tumour suppressor p16INK4a is the master regulator to enhance signalling via the fibronectin receptor (α5β1-integrin) in human Capan-1 pancreatic carcinoma cells in vitro. Galectin-1 (Gal-1) is the pro-anoikis effector. The extent of its binding to the cells' surface is increased (Figure 1). Orchestrated up-regulation of presentation of Gal-1 and the integrin, combined with the enhanced reactivity of glycosylation to the lectin, favours caspase 8 activation [25,46]. Gal-3, a competitor of Gal-1 with additional intracellular anti-anoikis potential, is down-regulated in parallel [46].

Conclusions

The unsurpassed structural complexity of cellular glycans has long hampered fine-structural analysis. Its feasibility, even on the level of the glycoproteome, has brought us into a new era in the quest to define glycobiomarkers. Capitalizing on the great strides taken in analytical glycan chemistry, any sample can now be scrutinized for its glycomic profile to spot disease-associated deviations. They should not be dismissed as a random event but appear to be functionally relevant, by virtue of cis- and trans-activities. In fact, the given structural complexity is the basis for a high-density mode of biological information coding with far-reaching functional consequences [1]. It thus comes as no surprise that glycosylation is under the control of master regulators, in certain cases even along with effector lectins, teaching us instructive lessons on the pathways sugar coding and translation into bioeffects take. At the same time, this work pinpoints glycan structures with a mission in disease manifestation, tied to a relevant process and thus of potentially predictive power as markers and as targets for therapeutic considerations. The apparent combination of carbohydrate structure with topology of presentation to generate high lectin affinity and selectivity gives research aimed at developing bioinspired tools for these two medical applications a clear direction.

Glycomarkers for Disease: An Independent Meeting held at the Conference Centre of the Polish National Academy of Sciences, Wierzba, Poland, 12–16 September 2010. Organized by Sviatlana Astrautsova (Grodno, Belarus), Cathy Merry (Manchester, U.K.), Tony Merry (Manchester, U.K.), Jean-Claude Michalski (Lille, France), Grażyna Palamarczyk (Warsaw, Poland) and Krzysztof Zwierz (Białystok, Poland). Edited by Tony Merry.

Abbreviations

     
  • CA

    cancer antigen

  •  
  • DC-SIGN

    dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin

  •  
  • GlcNAc

    N-acetylglucosamine

Funding

This work is connected to the European Commission GlycoHIT research programme.

References

References
1
Gabius
H.-J.
The Sugar Code
Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
2
Brockhausen
I.
Schachter
H.
Gabius
H.-J.
Gabius
S.
Glycosyltransferases involved in N- and O-glycan biosynthesis
Glycosciences: Status and Perspectives
1997
London
Chapman & Hall
(pg. 
79
-
113
)
3
Spiro
R.G.
Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds
Glycobiology
2002
, vol. 
12
 (pg. 
43R
-
56R
)
4
Kopitz
J.
Gabius
H.-J.
Glycolipids
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
177
-
198
)
5
Patsos
G.
Corfield
A.
Gabius
H.-J.
O-Glycosylation: structural diversity and function
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
111
-
137
)
6
Zuber
C.
Roth
J.
Gabius
H.-J.
N-Glycosylation
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
98
-
110
)
7
Wilson
I.B.H.
Paschinger
H.
Rendic
D.
Gabius
H.-J.
Glycosylation of model and ‘lower’ organisms
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
139
-
154
)
8
Hakomori
S.-I.
Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens
Adv. Cancer Res.
1989
, vol. 
52
 (pg. 
257
-
331
)
9
Brockhausen
I.
Schutzbach
J.
Kuhns
W.
Glycoproteins and their relationship to human disease
Acta Anat.
1998
, vol. 
161
 (pg. 
36
-
78
)
10
An
H.J.
Kronewitter
S.R.
de Leoz
M.L.A.
Lebrilla
C.B.
Glycomics and disease markers
Curr. Opin. Chem. Biol.
2009
, vol. 
13
 (pg. 
601
-
607
)
11
Arnold
J.N.
Saldova
R.
Hamid
U.M.A.
Rudd
P.M.
Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation
Proteomics
2008
, vol. 
8
 (pg. 
3284
-
3293
)
12
Dai
Z.
Zhou
J.
Qiu
S.-J.
Liu
Y.-K.
Fan
J.
Lectin-based glycoproteomics to explore and analyze hepatocellular carcinoma-related glycoprotein markers
Electrophoresis
2009
, vol. 
30
 (pg. 
2957
-
2966
)
13
Nakagawa
H.
Gabius
H.-J.
Analytical aspects: analysis of protein-bound glycans
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
71
-
83
)
14
Uematsu
R.
Shinohara
Y.
Nakagawa
H.
Kurogochi
M.
Furukawa
J.
Miura
Y.
Akiyama
M.
Shimizu
H.
Nishimura
S.-I.
Glycosylation specific for adhesion molecules in epidermis and its receptor revealed by glycoform-focused reverse genomics
Mol. Cell. Proteomics
2009
, vol. 
8
 (pg. 
232
-
244
)
15
Vanderschaeghe
D.
Festjens
N.
Delanghe
J.
Callewaert
N.
Glycome profiling using modern glycomics technology: technical aspects and applications
Biol. Chem.
2010
, vol. 
391
 (pg. 
149
-
161
)
16
Amano
M.
Yamaguchi
M.
Takegawa
Y.
Yamashita
T.
Terasima
M.
Furukawa
J.-i.
Miura
Y.
Shinohara
Y.
Iwasaki
N.
Minami
A.
Nishimura
S.-I.
Threshold in stage-specific embryonic glycotypes uncovered by a full portrait of dynamic N-glycan expression during cell differentiation
Mol. Cell. Proteomics
2010
, vol. 
9
 (pg. 
523
-
537
)
17
Qiu
Y.
Patwa
T.H.
Xu
L.
Shedden
K.
Misek
D.E.
Tuck
M.
Jin
G.
Ruffin
M.T.
Turgeon
D.K.
Synal
S.
, et al. 
Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot
J. Proteome Res.
2008
, vol. 
7
 (pg. 
1693
-
1703
)
18
Abbott
K.L.
Lim
J.M.
Wells
L.
Benigno
B.B.
McDonald
J.F.
Pierce
M.
Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis
Proteomics
2010
, vol. 
10
 (pg. 
470
-
481
)
19
Lohr
M.
Kaltner
H.
Schwartz-Albiez
R.
Sinowatz
F.
Gabius
H.-J.
Towards functional glycomics by lectin histochemistry: strategic probe selection to monitor core and branch-end substitutions and detection of cell-type and regional selectivity in adult mouse testis and epididymis
Anat. Histol. Embryol.
2010
, vol. 
39
 (pg. 
481
-
483
)
20
Reuter
G.
Gabius
H.-J.
Eukaryotic glycosylation: whim of nature or multipurpose tool?
Cell. Mol. Life Sci.
1999
, vol. 
55
 (pg. 
368
-
422
)
21
Gabius
H.-J.
Glycans: bioactive signals decoded by lectins
Biochem. Soc. Trans.
2008
, vol. 
36
 (pg. 
1491
-
1496
)
22
Pierce
M.
Arango
J.
Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAcβ(1,6)Manα(1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells
J. Biol. Chem.
1986
, vol. 
261
 (pg. 
10772
-
10777
)
23
Buckhaults
P.
Chen
L.
Fregien
N.
Pierce
M.
Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
19575
-
19581
)
24
Gill
D.J.
Chia
J.
Senewiratne
J.
Bard
F.
Regulation of O-glycosylation through Golgi-to-ER relocation of initiation enzymes
J. Cell Biol.
2010
, vol. 
189
 (pg. 
843
-
858
)
25
André
S.
Sanchez-Ruderisch
H.
Nakagawa
H.
Buchholz
M.
Kopitz
J.
Forberich
P.
Kemmner
W.
Böck
C.
Deguchi
K.
Detjen
K.M.
, et al. 
Tumor suppressor p16INK4a: modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells
FEBS J.
2007
, vol. 
274
 (pg. 
3233
-
3256
)
26
Patsos
G.
André
S.
Roeckel
N.
Gromes
R.
Gebert
J.
Kopitz
J.
Gabius
H.-J.
Compensation of loss of protein function in microsatellite-unstable colon cancer cells (HCT116): a gene-dependent effect on the cell surface glycan profile
Glycobiology
2009
, vol. 
19
 (pg. 
726
-
734
)
27
Hennet
T.
Gabius
H.-J.
Diseases of glycosylation
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
365
-
383
)
28
Honke
K.
Taniguchi
N.
Gabius
H.-J.
Animal models to delineate glycan functionality
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
385
-
401
)
29
André
S.
Kopitz
J.
Kaltner
H.
Villalobo
A.
Gabius
H.-J.
Gabius
H.-J.
Glycans as functional markers in malignancy?
The Sugar Code. Fundamentals of Glycosciences
2009
Weinheim, Germany
Wiley-VCH
(pg. 
419
-
432
)
30
André
S.
Unverzagt
C.
Kojima
S.
Frank
M.
Seifert
J.
Fink
C.
Kayser
K.
von der Lieth
C.-W.
Gabius
H.-J.
Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo
Eur. J. Biochem.
2004
, vol. 
271
 (pg. 
118
-
134
)
31
André
S.
Kozár
T.
Schuberth
R.
Unverzagt
C.
Kojima
S.
Gabius
H.-J.
Substitutions in the N-glycan core as regulators of biorecognition: the case of core-fucose and bisecting GlcNAc moieties
Biochemistry
2007
, vol. 
46
 (pg. 
6984
-
6995
)
32
André
S.
Kozár
T.
Kojima
S.
Unverzagt
C.
Gabius
H.J.
From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans
Biol. Chem.
2009
, vol. 
390
 (pg. 
557
-
565
)
33
Lopez
P.H.
Schnaar
R.L.
Gangliosides in cell recognition and membrane protein regulation
Curr. Opin. Struct. Biol.
2009
, vol. 
19
 (pg. 
549
-
557
)
34
Sonnino
S.
Aureli
M.
Loberto
N.
Chigorno
V.
Prinetti
A.
Fine tuning of cell functions through remodeling of glycosphingolipids by plasma membrane-associated glycohydrolases
FEBS Lett.
2010
, vol. 
584
 (pg. 
1914
-
1922
)
35
Villalobo
A.
Nogales-Gonzalés
A.
Gabius
H.-J.
A guide to signaling pathways connecting protein-glycan interaction with the emerging versatile effector functionality of mammalian lectins
Trends Glycosci. Glycotechnol.
2006
, vol. 
18
 (pg. 
1
-
37
)
36
Unverzagt
C.
André
S.
Seifert
J.
Kojima
S.
Fink
C.
Srikrishna
G.
Freeze
H.
Kayser
K.
Gabius
H.-J.
Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional α2,3/α2,6-sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake
J. Med. Chem.
2002
, vol. 
45
 (pg. 
478
-
491
)
37
Wu
A.M.
Wu
J.H.
Liu
J.-H.
Singh
T.
André
S.
Kaltner
H.
Gabius
H.-J.
Effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the Galβ1-terminated core saccharides on the binding of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)
Biochimie
2004
, vol. 
86
 (pg. 
317
-
326
)
38
Dam
T.K.
Gabius
H.-J.
André
S.
Kaltner
H.
Lensch
M.
Brewer
C.F.
Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants
Biochemistry
2005
, vol. 
44
 (pg. 
12564
-
12571
)
39
Solís
D.
Maté
M.J.
Lohr
M.
Ribeiro
J.P.
López-Merino
L.
André
S.
Buzamet
E.
Cañada
F.J.
Kaltner
H.
Lensch
M.
, et al. 
N-domain of human adhesion/growth-regulatory galectin-9: preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution
Int. J. Biochem. Cell. Biol.
2010
, vol. 
42
 (pg. 
1019
-
1029
)
40
Nonaka
M.
Ma
B.Y.
Murai
R.
Nakamura
N.
Baba
M.
Kawasaki
N.
Hodohara
K.
Asano
S.
Kawasaki
T.
Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells
J. Immunol.
2008
, vol. 
180
 (pg. 
3347
-
3356
)
41
Kawasaki
N.
Kawasaki
T.
Recognition of endogenous ligands by C-type lectins: interaction of serum mannan-binding protein with tumor-associated oligosaccharide epitopes
Trends Glycosci. Glycotechnol.
2010
, vol. 
22
 (pg. 
141
-
151
)
42
Kopitz
J.
von Reitzenstein
C.
Burchert
M.
Cantz
M.
Gabius
H.-J.
Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
11205
-
11211
)
43
André
S.
Kaltner
H.
Lensch
M.
Russwurm
R.
Siebert
H.-C.
Fallsehr
C.
Tajkhorshid
E.
Heck
A.J.R.
von Knebel-Döberitz
M.
Gabius
H.-J.
Kopitz
J.
Determination of structural and functional overlap/divergence of five proto-type galectins by analysis of the growth-regulatory interaction with ganglioside GM1 in silico and in vitro on human neuroblastoma cells
Int. J. Cancer
2005
, vol. 
114
 (pg. 
46
-
57
)
44
Wang
J.
Lu
Z.H.
Gabius
H.-J.
Rohowsky-Kochan
C.
Ledeen
R.W.
Wu
G.
Cross-linking of GM1 ganglioside by galectin-1 mediates regulatory T cell activity involving TRPC5 channel activation: possible role in suppressing experimental autoimmune encephalomyelitis
J. Immunol.
2009
, vol. 
182
 (pg. 
4036
-
4045
)
45
Kopitz
J.
Bergmann
M.
Gabius
H.-J.
How adhesion/growth-regulatory galectins-1 and -3 attain cell specificity: case study defining their target on neuroblastoma cells (SK-N-MC) and marked affinity regulation by affecting microdomain organization of the membrane
IUBMB Life
2010
, vol. 
62
 (pg. 
624
-
628
)
46
Sanchez-Ruderisch
H.
Fischer
C.
Detjen
K.M.
Welzel
M.
Wimmel
A.
Manning
J.C.
André
S.
Gabius
H.-J.
Tumor suppressor p16INK4a: downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model
FEBS J.
2010
, vol. 
277
 (pg. 
3552
-
3563
)
47
Sato
Y.
Takahashi
M.
Shibukawa
Y.
Jain
S.K.
Hamaoka
R.
Miyagawa
J.
Yaginuma
Y.
Honke
K.
Ishikawa
M.
Taniguchi
N.
Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
11956
-
11962
)
48
Matsumoto
K.
Yokote
H.
Arao
T.
Maegawa
M.
Tanaka
K.
Fujita
Y.
Shimizu
C.
Hanafusa
T.
Fujiwara
Y.
Nishio
K.
N-glycan fucosylation of epidermal growth factor receptor modulates receptor activity and sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor
Cancer Sci.
2008
, vol. 
99
 (pg. 
1611
-
1617
)
49
Guo
H.-B.
Randolph
M.
Pierce
M.
Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes. Inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
22150
-
22162
)
50
Liu
J.-J.
Lin
B.
Hao
Y.-Y.
Li
F.F.
Liu
D.W.
Qi
Y.
Zhu
L.C.
Zhang
S.L.
Iwamori
M.
Lewis(y) antigen stimulates the growth of ovarian cancer cells via regulation of the epidermal growth factor receptor pathway
Oncol. Rep.
2010
, vol. 
23
 (pg. 
833
-
841
)