Carbohydrate microarrays: key developments in ... - Semantic Scholar

Article in press - uncorrected proof Biol. Chem., Vol. 390, pp. 647–656, July 2009 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2009.071

Review

Carbohydrate microarrays: key developments in glycobiology

Yan Liu, Angelina S. Palma and Ten Feizi* Glycosciences Laboratory, Division of Medicine, Imperial College London, Northwick Park and St. Mark’s Campus, Harrow HA1 3UJ, Middlesex, UK * Corresponding author e-mail: [email protected]

Abstract Carbohydrate chains of glycoproteins, glycolipids, proteoglycans, and polysaccharides mediate processes of biological and medical importance through their interactions with complementary proteins. The unraveling of these interactions is therefore a priority in biomedical sciences. Carbohydrate microarray technology is a new development at the frontier of glycomics that is revolutionizing the study of carbohydrate-protein interactions and the elucidation of their specificities in endogenous biological processes, microbe-host interactions, and immune defense mechanisms. In this review, we briefly refer to the principles of numerous platforms since the introduction of carbohydrate microarrays in 2002, and we highlight platforms that are beyond proof-of-concept and have provided new biological information. Keywords: carbohydrate-binding protein; glycolipid; glycoprotein; neoglycolipid; oligosaccharide; polysaccharide.

Introduction The diverse oligosaccharides (glycans) that ‘decorate’ glycoproteins, glycolipids, and proteoglycans, and those of polysaccharides are potentially a vast source of information, and harbor a ‘glycocode’ that is waiting to be deciphered in various contexts of biological and medical importance (Feizi and Chai, 2004; Taylor and Drickamer, 2006). There is increasing awareness of the important roles of carbohydrates in mediating a variety of physiological and pathological processes. Thus, ‘glycomics’ (the study of the diverse glycan repertoires of cells, tissues or organisms) is emerging as a frontier research field in the post-genomic era. The structural aspects of glycomics, namely the elucidation of the repertoires of glycan structures in cells and tissues, as well as their changes that occur during normal development, cell differentiation and in disease states, are being extensively addressed with the development of advanced profiling and structural characterization strategies, e.g., high-resolution chromatography methods coupled with exoglycosidase digestions (Campbell et al., 2008) and modern mass spectrometry (MS) analyses (Haslam et al., 2006),

and NMR (Petrescu et al., 2006). However, understanding the involvements of carbohydrates in diverse recognition systems (often through their interactions with effector proteins) that participate in cell-cell communication and signaling still remain challenging. Detailed analyses of carbohydrate-protein interactions present difficulties at all levels. First, only limited amounts of oligosaccharides (at submicromol levels) can typically be isolated from natural sources when released from proteins or lipids, and these are often highly heterogeneous. Second, the structural diversity of oligosaccharides leads to difficulties in their structural characterization; currently, there is a lack of an efficient means of automated assignment and the characterization is mainly reliant on expert interpretation by MS analyses. Third, the biosynthesis of oligosaccharides is not template driven, as for nucleic acids and proteins, and the diverse repertoire of oligosaccharides is difficult to access by chemical synthesis. Fourth, most carbohydrate-protein interactions are of low affinity, and there is a requirement of multivalent presentation of carbohydrate ligands for detection of binding in microscale screening analysis. Several aspects of these challenges are now being addressed with the advent of carbohydrate microarrays.

Overview of carbohydrate microarray technologies Carbohydrate microarray technologies are novel tools emerging at the frontier of glycomics that are revolutionizing studies of carbohydrate-protein interactions and the elucidation of carbohydrate ligands involved not only in endogenous receptor systems but also pathogen-host interactions (Paulson et al., 2006; Horlacher and Seeberger, 2008; Liu and Feizi, 2008; Liang et al., 2008). The main advantage of microarray analysis is that a broad range of glycans (tens or hundreds, and eventually, we hope, thousands) can be immobilized on solid matrices as minute spots and simultaneously interrogated. The multivalent display of arrayed saccharides can serve to mimic cell surface display and is ideal for detecting the generally very low affinities of interactions that involve carbohydrates. The miniaturization in microarrays is advantageous for making the most out of precious materials of both carbohydrates and protein analytes; and this, as well as the high-throughput feature of microarray technologies, is particularly well suited for investigations in glycomics, in that thousands of binding events can be assessed in parallel on a single chip containing only tiny amounts of natural glycans. In general, carbohydrate microarray methods fall into two categories: polysaccharide microarrays and oligosaccharide microarrays. Polysaccharide materials

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derived from natural sources can be readily and randomly immobilized on solid matrices based on hydrophobic physical absorption (Wang et al., 2002; Willats et al., 2002; Moller et al., 2007) or charge-based interaction (Shipp and Hsieh-Wilson, 2007) to generate polysaccharide microarrays, which are valuable for comparative antigenicity analyses. Oligosaccharide microarrays, on the other hand, are powerful tools to provide detailed information on structure-activity relationships in carbohydrate recognition events. The immobilization of oligosaccharides is, however, less straightforward due to their hydrophilic nature, and chemical derivatization procedures are normally required before arraying. Many different methodologies have been developed for constructing oligosaccharide microarrays, and these have been described in a number of reviews in some depth (Feizi et al., 2003a; Ratner et al., 2004; Shin et al., 2005; Culf et al., 2006; Laurent et al., 2008). One principle is to conjugate natural or chemically synthesized oligosaccharides to lipid by reductive amination to generate neoglycolipid (NGL) probes with amphipathic properties for arraying (Fukui et al., 2002; Feizi and Chai, 2004). The use of reductive amination has also been described for preparing fluorescent oligosaccharide derivatives which contain a primary amine for array generation (Xia et al., 2005; Song et al., 2008). Most other mono- or oligosaccharide probes generated for printing have been de novo synthesized chemically or chemo-enzymatically, requiring substantial chemical expertise for access to defined structures that incorporate specific functional groups, e.g., thiol (Adams et al., 2004; Ratner et al., 2004; Brun et al., 2006), maleimide (Park and Shin, 2002), amine (Blixt et al., 2004; Disney and Seeberger, 2004; Dyukova et al., 2005; de Paz et al., 2006; Shilova et al., 2008), azide (Fazio et al., 2002) and cyclopentadiene (Houseman and Mrksich, 2002) functionalities, or proteins as tags (Adams et al., 2004; Manimala et al., 2006) for covalent attachment to solid matrices. Oligosaccharides with lipid (Bryan et al., 2002) or fluorous tags (Mamidyala et al., 2006; Chen and Pohl, 2008; Zhu and Schmidt, 2009) for noncovalent immobilization have also been described. These methods are promising due to compatibilities with advanced chemical or enzymatic synthesis of oligosaccharides (Lee et al., 2007; Seeberger, 2008; Zhu and Schmidt, 2009). However, access to oligosaccharide libraries by current synthetic approaches is still limited (Seeberger and Werz, 2005, 2007). Multi-step manipulations in many of the microarray platforms limit applications to the small quantities of oligosaccharides that can be isolated from natural sources. As natural oligosaccharides are crucial for discoveries of hitherto unknown oligosaccharide ligands (Feizi et al., 2003a; Feizi and Chai, 2004), carbohydrate microarrays should ideally encompass oligosaccharides from both natural and synthetic sources. Approaches have been described for printing and covalent attachment of unmodified mono and short oligosaccharides onto aminooxy- or hydrazide-modified glass slides (Lee and Shin, 2005; Zhou and Zhou, 2006; Park et al., 2009) and heparin-derived sulfated oligosaccharides onto hydrazide-modified gold surfaces (Zhi et al., 2006). Potential disadvantages of direct covalent surface immobilization methods are the high concentrations of the sac-

charides required and the possible variation of immobilization efficiencies depending on the nature of saccharides (size and charge, for example). Fluorescence based measurement is, to date, the prevalent principle for detecting binding to carbohydrate microarrays. An alternative approach recently described by members of the UK Glycoarrays Consortium is a gold array platform (Karamanska et al., 2008; Zhi et al., 2008). This enables, in addition to the conventional fluorescence detection of binding, real-time and label-free analysis of protein interactions by surface plasmon resonance, as well as determination of glycoenzyme specificities using on-chip MS analysis. The aforementioned carbohydrate microarray strategies promise to contribute to knowledge on biological systems that operate through carbohydrate recognition. Many of the strategies thus far described have used as model proteins of known carbohydrate-binding specificities. We highlight below several oligosaccharide microarray systems that are beyond proof-of-concept and have provided new biological insights. We dwell in some detail on the NGL based oligosaccharide microarray platform with which we have firsthand experience.

Applications of carbohydrate microarrays in glycobiology Synthetic oligosaccharide microarrays – focused tools for studies of carbohydrate-mediated interactions Synthetic methods (chemical or chemo-enzymatic) provide access to homogeneous, sequence-defined oligosaccharide structures. Several array platforms containing relatively small repertoires of complex synthetic oligosaccharides, as well as their specific modified unnatural analogs, have been described as focused tools for probing carbohydrate-mediated interactions. Seeberger and colleagues who are developing automated carbohydrate synthesis have generated synthetic oligosaccharide microarrays (Figure 1A) to address several biological questions. For example, microarrays were constructed on maleimide-functionalized surfaces using seven thiol-containing synthetic high-mannose oligosaccharides for identification of human immunodeficiency virus (HIV) vaccine candidate antigens (Adams et al., 2004). The binding profiles were described for several proteins that interact with gp120 of HIV, including the receptor of the innate immune system known as DCSIGN, the antibody 2G12, and the bacterial proteins Cyanovirin-N and Scytovirin. The same array principle has been recently used in generation of parasite glycosylphosphatidylinositol (GPI) glycan arrays, which comprise seven synthetic GPI glycan fragments (Kamena et al., 2008). These arrays have been applied in studies of malaria-induced antibody responses, and provide more detailed insight than those obtained with conventional experimentation using GPI extracts from parasite preparations. Utilizing an alternative surface chemistry, amide formation, the Seeberger group has prepared microarrays displaying heparin oligosaccharides that are chemically synthesized; these were used to show differences

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Figure 1 Examples of synthetic oligosaccharide microarrays. (A) Microarrays of synthetic high-mannose oligosaccharides, GPI glycan fragments, and heparin oligosaccharides developed by the Seeberger group. (B) Glycan arrays of CFG generated by covalent immobilization of glycans (with different amino-terminal spacers) printed onto NHS-activated glass slides. The designation of the spacers, Sp0, Sp12, and Sp21 are as described on the CFG website (http://www.functionalglycomics.org).

in binding profiles of several fibroblast growth factors (de Paz et al., 2006; Noti et al., 2006) and chemokines (de Paz et al., 2007). Other applications of the amide-linked synthetic arrays include monosaccharide microarrays assembled for adhesion studies of pathogenic bacteria (Disney and Seeberger, 2004), as well as microarrays of aminoglycosides for the evaluation of the resistance mechanisms against different aminoglycoside antibiotics (Disney et al., 2004). A large collaborative effort towards developing oligosaccharide microarrays has been made by the Consortium for Functional Glycomics (CFG, a research initiative funded by the National Institute of General Medical Sciences; http://www.functionalglycomics.org), and two generations of microarrays have been developed (Alvarez and Blixt, 2006). The first is built on streptavidin-coated microtiter plates to which a library of biotinylated synthetic or natural glycans is attached (Blixt et al., 2003). This has now been replaced by the second generation, which is built using microarray printing technology to spot amine-terminating glycans on commercially available NHS-activated glass slides (Figure 1B), thus in a more high-throughput manner (Blixt et al., 2004). A growing library of more than 400 structurally defined mammaliantype glycans, predominantly synthetic, is included in the current version of CFG printed microarrays. The binding specificities of a wide variety of proteins, including C-type lectins, siglecs, galectins, anti-carbohydrate antibodies (including those present in human serum), and lectins from plants, have been examined (Blixt et al., 2004). In addition, glycan array analyses have been car-

ried out to compare and contrast the receptor-binding capabilities of natural and mutated human and avian influenza viruses, as recombinant hemagglutinins or as corresponding whole viruses produced by reverse genetics (Stevens et al., 2006, 2008; Yen et al., 2009). A sialoside analog array containing 34 synthetic sialyl trisaccharides having various 9-acyl substitutions on neuraminic acid residues has been described for recognition studies of receptors of the immune system known as siglecs (Blixt et al., 2008a). This study allowed screening for high affinity synthetic unnatural probes, which could be used as competitors with natural glycan ligands in the functional studies of siglecs. A recent development in this microarray platform is the preparation of glycan probes by one-step derivatization of free reducing glycans using a bi-functional spacer; this allows arraying natural glycans (Bohorov et al., 2006). By this means, Blixt and colleagues have assembled a Salmonella O-antigen microarray bearing both synthetic and lipopolysaccharide-derived carbohydrate antigens to assess antibody levels in human sera and to distinguish between infections with different strains (Blixt et al., 2008b). NGL-based oligosaccharide microarrays with provision for designer arrays for novel ligand discoveries Encouraged by the observations of Wang and colleagues (Wang et al., 2002) that polysaccharides and glycoproteins can be satisfactorily immobilized on nitrocellulose by noncovalent interaction, Feizi and colleagues adapted


their long-established and validated NGL technology (Tang et al., 1985; Feizi et al., 1994) to generate the first microarray system for complex oligosaccharides. These were arrayed as lipid-linked oligosaccharide probes (NGLs and glycosphingolipids) using nitrocellulose as the matrix (Fukui et al., 2002; Feizi and Chai, 2004; Galustian et al., 2004; Reddy et al., 2004). The NGL technology involves conjugating oligosaccharides by microscale reductive amination to an aminolipid, 1,2-dihexadecylsn-glycero-3-phosphoethanolamine (DHPE), as depicted in Figure 2. This allows minute amounts (nmol scale) of oligosaccharides released from O- and N-glycosylated proteins, glycosaminoglycans, and polysaccharides to be converted into lipid-linked probes (DH-NGLs). The amphipathic property conferred by introduction of the lipid tag enables immobilization in clustered display on solid matrices, such as silica-gel plates, plastic microtiter wells, and nitrocellulose. This clustered mode of display has served well for detecting binding signals of many carbohydrate-binding antibodies and receptors. The affinities of binding are generally low. Therefore, both the ligands and in most cases the carbohydrate binding sites of proteins need to be multimeric for detectable binding signals under the conditions of microarray analyses. Oligomericity in vivo is generally the mechanism of activation of carbohydrate recognition systems at the cell surface and extracellularly (Crocker and Feizi, 1996). In contrast with the covalently immobilized oligosaccharides which are fixed on the array surfaces, the noncovalently immobilized lipid-linked probes have potential lateral mobility; this is an advantage, as it might offer a suitable and optimal multivalent presentation to mimic the arrangement of clustered oligosaccharide structures at the cell surface. In addition, the NGL technology provides a way to resolve, by high performance thin layer chromatography (HPTLC), mixtures of oligosaccharide probes, and to perform carbohydrate-binding experiments on TLC plates in conjunction with oligosaccharide sequence determination by MS in situ (Chai et al., 1991). The NGL principle has led to discoveries of unsuspected oligosaccharide sequences on glycoproteins (Yuen et al., 1997), as well as new oligosaccharide ligands for carbohydrate-binding proteins (Yuen et al., 1992). Other examples are reviewed (Feizi et al., 1994, 2003b) and more recent examples are given below.

Other than ring-opening of the monosaccharide residues at the reducing ends, oligosaccharides remain intact in DH-NGLs. Those derived from tri- or larger oligosaccharides have performed well for the majority of carbohydrate-recognition systems which have the peripheral or backbone regions of oligosaccharides as recognition motifs (Feizi and Chai, 2004). However, ringopening of the monosaccharides at the reducing end may affect biological activities of short oligosaccharides. For instance, the DH-NGL of Lewisx (Lex) trisaccharide is not bound by anti-Lex antibodies (Streit et al., 1996), and that of sialyl-Lex tetrasaccharide is not bound by the selectins (Leteux et al., 1999). To overcome this limitation and enhance the applicabilities of NGL probes derived from short oligosaccharides which are the most accessible via chemical synthesis, a novel type of NGL with ringclosed monosaccharide cores has been introduced (Liu et al., 2006, 2007; see Figure 2). These are prepared from a variety of reducing oligosaccharides in conjugation with an aminooxy-functionalized DHPE (AOPE) via microscale oxime ligation (without reduction). The desirable features of these NGLs (AO-NGLs) have been demonstrated by microarray analyses: there is efficient presentation of short oligosaccharides, such as Lex trisaccharide to antiLex-antibodies and sialyllactose analogs to siglecs; the core-monosaccharide of a fucosylated N-glycan is also preserved as a ligand for the plant lectin from Pisum sativum (pea lectin) which recognizes the unmodified core. The NGL-based microarray platform is schematically presented in Figure 3. It currently contains approximately 600 robotically arrayed, sequence-defined oligosaccharide probes, which comprise NGLs derived from both natural and synthetic oligosaccharides, as well as natural and synthetic glycolipids, and are expanding in number. Included are: N-glycans (neutral and acidic, high-mannose and complex types), O-glycans and blood grouprelated sequences (A, B, H, Lea, Leb, Lex and Ley) on linear or branched backbones, and their sialylated and/ or sulfated analogs, gangliosides, glycosaminoglycans, homo-oligomers of sialic acid and oligosaccharide fragments of other polysaccharides, ranging in size from 2 to 20 monosaccharide units. This platform has all the attributes of the NGL technology with provision for generating ‘designer’ microarrays from targeted tissues and macromolecules (Osanai et al., 1996; Fukui et al., 2002;

Figure 2 Principles of the preparation of DH-NGL and AO-NGL probes from reducing oligosaccharides by reductive amination and oxime ligation, respectively. DHPE, 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine; AOPE, aminooxy-functionalized DHPE.


Figure 3 The NGL-based oligosaccharide microarray platform.

Palma et al., 2006). It is advantageous that there is the provision for ‘deconvolution’ by resolving arrays of NGL mixtures by HPTLC before ligand-binding experiments in conjunction with MS in situ for sequence assignment. This platform shows considerable promise as a novel approach to surveying entire glycomes and proteomes for the molecular definition of recognition systems. The first generation NGL-based microarray system using nitrocellulose membranes provided insights to the specificity of an antibody (CS-56) to chondroitin sulfates A and C (Fukui et al., 2002), and enabled the identification of ligands for receptors of the immune system known as SIGN-R1, SIGN-R3, and langerin (Galustian et al., 2004). Assignment could also be made of a carbohydrate-binding function to domain 5 of the cation-independent mannose 6-phosphate receptor (Reddy et al., 2004). In addition, the applicability of NGL based microarray to mixtures of oligosaccharides obtained from biological sources, such as cells, and a whole organ has been shown (Osanai et al., 1996; Fukui et al., 2002). With the advanced NGL-based microarrays, using nitrocellulose-coated glass slides, the influence of sulfation on carbohydrate recognition by siglecs has been shown (Campanero-Rhodes et al., 2006). For the micronemal protein-1 (MIC-1) of Toxoplasma gondii, this microarray system has revealed that an extremely broad range of sialyl motifs are recognized among N- and Oglycans and glycolipids (Blumenschein et al., 2007; see Figure 4). This finding provides an explanation for the unique adaptation of this protozoan parasite to infect a wide range of hosts, including virtually all warm-blooded animals and up to 50% of the world’s human population.

The flexibility of the NGL-based microarray platform to include natural glycolipids as well as NGLs derived from their oligosaccharide moieties, enabled identification of the N-glycolyl form of GM1 ganglioside, GM1(Gc) as the preferred receptor for the Simian virus 40 (SV40) (Campanero-Rhodes et al., 2007). The microarray analyses corroborated the previously documented binding of SV40 to N-acetyl GM1, GM1(Ac), which is found in mammals including humans. The stronger binding to GM1(Gc) ganglioside was shown to be mediated by the carbohydrate rather than the lipid moiety of the natural glycolipid. This was clinched by cell binding and infectivity assays using glycolipid-deficient cells supplemented with the respective natural gangliosides and the NGLs derived from their pentasaccharide moieties. GM1(Gc) is synthesized by simians and other mammals but not humans. This striking in vitro preference is likely to relate to in vivo preference and quite likely accounts for simians being the natural hosts for SV40. The ligands for Dectin-1, a receptor of the innate immune system directed at fungal pathogens, were found to be highly restricted; these were identified by generating designer microarrays from fungal polysaccharide glycomes (glucan polysaccharides) that Dectin-1 recognizes. The ligands were identified as linear b1–3-linked glucose oligomers, the minimum chain length required for recognition being a 10- or 11-mer (Palma et al., 2006). In a recent analysis (A.S. Palma, M. Campanero-Rhodes, Y. Liu, R.A. Childs and colleagues, unpublished) using a microarray of 327 probes, among them more than 270 mammalian-type sequences, binding was again detected to an 11mer and 13mer, but not to a 7mer of b1–3-linked glucose (Figure 5A). The mechanism by which Dectin-1 recognizes glucan sequences is not yet elucidated. The fold of the lectin-like domain of Dectin-1 is similar to those of the C-type (calciumdependent) lectin family (Weis et al., 1998), but it lacks the residues involved in the ligation of calcium; and Dectin-1 binds carbohydrates in a calcium-independent manner (Palma et al., 2006). The sizes of the oligosaccharides bound by Dectin-1 are unusually long for a Ctype lectin-like protein. In contrast to Dectin-1, carbohydrate-binding modules (CBMs) of bacterial glucan hydrolases can bind, in microarray analyses, to shorter glucan oligosaccharide probes (Y. Liu, A.S. Palma and colleagues, presented at the 2007 Annual Meeting of the Society for Glycobiology, Boston, MA, USA). The requirement for long oligosaccharide chain lengths by Dectin-1 has been corroborated by inhibition of binding experiments using oligosaccharides in solution. The phenomenon is likely to be related to a conformational motif displayed only by the long b1–3 oligosaccharide sequences. A recent ‘breakthrough’ of the NGL microarray system has been the identification of the endogenous ligand for malectin, a novel carbohydrate-binding protein, first identified in the Xenopus laevis pancreas, but later found to be highly conserved in animals (Schallus et al., 2008). This unique protein was initially shown by NMR to have a similarity in 3-dimensional structure to the aforementioned CBMs; and by isothermal titration calorimetry, it was shown to bind to glucose dimers including maltose,


Figure 4 Microarray analyses of the binding of the Toxoplasma gondii protein MIC-1. Among more than 200 sequence-defined lipid-linked oligosaccharide probes printed on nitrocellulose-coated slides, binding was observed only to terminally sialylated sequences. Numerical scores are shown for the binding signals of 59 sialyl oligosaccharide probes examined (69 positions are shown as 10* of the probes were printed at two positions); these are, plotted as fluorescence intensity, means of duplicate spots at 7 fmol/spot (with error bars), and are classified into seven groups (A–G) according to the backbone sequence. Selected probes are annotated, with designations of NeuAca2–3Gal linkage as pink; NeuAca2–6Gal, blue; NeuGca2–3Gal, green; and NeuAca2–8 linkage in yellow. NeuAca-3Galb-4Glcb-Cer(1) and NeuAca-3Galb-4Glcb-Cer(2) at positions 1 and 2 in the chart refer to hematoside and GM3, respectively; both are natural glycolipids obtained from different sources. Reprinted from Blumenschein et al. (2007) with permission from the EMBO Journal. *This is a correction of the number cited as ‘11’ in Blumenschein et al. (2007).

Glca1–4Glc (hence the name malectin) and also strongly to nigerose, Glca1–3Glc. Microarray analyses using the NGL probes prepared from glucan oligosaccharide fragments corroborated the glucan binding properties of malectin and the preference of malectin for maltose and nigerose among several glucan disaccharides. An indication towards the identification of the endogenous ligand was that malectin resides in the endoplasmic reticulum (ER) of mammalian cells (Schallus et al., 2008). This prompted us to populate our microarrays with ‘designer’ NGL probes derived from glucosylated N-glycans of the type that occur in the ER, namely, Glc1-, Glc2- and Glc3-

high mannose-N-glycans, which are intermediates in the early steps of protein N-glycosylation. Microarray analyses showed a unique selectivity of malectin binding to the Glc2-high mannose-N-glycan, among more than 330 oligosaccharide probes that had been included in the microarrays; of these, almost 270 were of mammaliantype (Figure 5B). Malectin is the first protein known to selectively bind to a diglucosyl high mannose N-glycan. The microarray findings now point to a role for malectin in the early steps of N-glycosylation, through its interaction with Glc2-high mannose N-glycans. The way is open for functional studies of this protein. The carbohy-


Figure 5 Microarray analyses of Dectin-1 and malectin. Oligosaccharide probes (lipid-linked) were printed in duplicate on nitrocellulose-coated glass slides at 2 and 7 fmol/spot. Binding signals at 7 fmol/spot are shown. (A) Analysis of the binding of murine Dectin-1 tested as an IgG-Fc chimera at 5 mg/ml. Binding was detected exclusively to b1–3-linked glucose sequences (11 and 13mers) derived from a glucan polysaccharide, in accordance with earlier data (Palma et al., 2006). (B) Analysis of the binding of hexaHis-tagged Xenopus laevis malectin tested at 5 mg/ml. High selectivity of malectin binding to the Glc2-N-glycan was revealed. Inset: expanded region of the microarray highlighting the selective binding of malectin to the Glc2-N-glycan probe in contrast with the broad binding profile of Concanavalin A. Abbreviations for the probes in the inset: G3N, Glc3Man7(D1)GlcNAc*; G2N, Glc2Man7(D1)GlcNAc; G1N, Glc1Man9GlcNAc2; M9N, Man9GlcNAc2; and M7N, Man7(D1)GlcNAc2. These five probes were arrayed at positions 162–165 and at position 168, respectively. Reprinted from Schallus et al. (2008) with permission from Molecular Biology of the Cell. *Glc3Man7(D1)GlcNAc, Glc2Man7(D1)GlcNAc and Man7(D1)GlcNAc2 refer to Man7-related N-glycans that contain the outer most mannose residue in the Mana1–2Mana1–2Mana1–3Man arm, namely the D1 mannose residue (Petrescu et al., 1997).

drate-recognition systems discussed above highlight the importance of being able to generate tailor-made probes of natural oligosaccharides for novel ligand discovery.

Perspectives Carbohydrate microarrays are coming of age as screening tools. Their advent is transforming studies of carbohydrate-protein interactions, leading to high-throughput

analyses of biomedically important systems that operate through carbohydrate recognition. There will almost certainly be increasing numbers of synthetic oligosaccharides in carbohydrate microarrays. It can be anticipated, however, that many new and hitherto unsuspected carbohydrate-binding proteins and their ligands will be identified by microarrays containing natural glycans. Although in most cases, to date, there has been no way of accurately knowing precisely how much sample is attached at each spot during microarray analysis, efforts are being


made to address this important issue (Song et al., 2008, 2009). Time is ripe for cross platform comparisons of the performance of carbohydrate microarrays. This is among topics to be addressed at a timely workshop in March of this year supported by the National Institutes of Health under the auspices of the CFG. It is hoped that in time carbohydrate microarrays will help in discoveries of clinically relevant biomarkers and be part of kits that incorporate markers of diagnostic and prognostic value.

Acknowledgments We gratefully acknowledge contributions of our colleagues in the Glycosciences Laboratory: Wengang Chai, Maria CampaneroRhodes, Robert Childs, Mark Stoll, Alex Lawson, Yibing Zhang, and Colin Hebert. The Glycosciences Laboratory acknowledges with gratitude collaborators over the years with whom our microarray probes were studied. For grant support, we acknowledge the U.K. Medical Research Council, the U.K. Research Councils Basic Technology Grant (GR/S79268, ‘Glycoarrays’) and the NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416). Y.L. is a recipient of a Welcome Trust Value in People Award and A.S.P. is a fellow of the ¸ ˜ o para a Cieˆncia e Tecnologia (SFRH/BPD/26515/2006, Fundaca Portugal).

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Received February 20, 2009; accepted March 31, 2009