CHEMICAL BIOLOGY

Array-Based Techniques for Glycans

 

Oyindasola Oyelaran, Joseph C. Manimala, and Jeffrey C. Gildersleeve, Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, Frederick, Maryland

doi: 10.1002/9780470048672.wecb023

Advanced Article

Glycan arrays are powerful tools for high throughput analysis of carbohydrate-macromolecule interactions. A glycan array is composed of many different carbohydrate structures immobilized on a solid support in an orderly arrangement. This review describes the challenges and considerations in the development of a glycan array. Various array formats, immobilization techniques, and assay systems are discussed. In addition, several interesting applications of glycan array technology are described, such as assessing the specificities of carbohydrate-binding lectins and antibodies, profiling antiglycan antibodies in serum as biomarkers of disease, and evaluating carbohydrate-dependent cell binding.

 

DNA and protein microarray technology has revolutionized the way scientists study complex biological processes. These arrays consist of thousands of nucleic acids or proteins immobilized on a solid support. The array format allows one to rapidly evaluate interactions with a large number of molecules simultaneously. For example, one can examine the expression profiles of thousands of genes in a single experiment.

Glycan arrays are an equally powerful technology for the evaluation of carbohydrate-macromolecule interactions. Analogous to DNA and protein arrays, glycan arrays contain many different carbohydrates affixed to a solid support. This review will focus on development strategies, challenges, and applications of glycan arrays. Several other reviews have been published over the last few years (1-4).

 

Biological Background

Carbohydrates, biopolymers composed of monosaccharide units, play a central role in a wide range of biological processes such as protein folding, inflammation, and development. In addition, glycans undergo dramatic changes in expression during the onset and progression of many diseases such as rheumatoid arthritis and cancer. Unfortunately, progress toward defining the specific roles of most carbohydrates and understanding the relationships between structure and function has been frustratingly slow. Furthermore, efforts to exploit altered expression for therapeutic benefit have only been successful in a limited number of cases.

Molecular recognition is a fundamental element for both basic and applied carbohydrate research (see cross references: Glycan-glycan interactions, Glycan-protein interactions, and Sugar-lectin interactions in cell adhesion). Many important biological processes involve specific interactions between a carbohydrate-binding protein (lectin) and a glycan. For example, one early stage of inflammation involves interactions of selectins with carbohydrate ligands. Carbohydrate-protein interactions are also directly involved in diseases. For example, many pathogens such as the influenza virus, E. histolytica, and H. pylori bind carbohydrates on the surface of host cells as a key step of infection (5). For many other carbohydrate-binding proteins, however, the biological functions are still unknown. For example, most lectins used routinely as research reagents are isolated from plants. Although used in the laboratory for many years, the biological roles of many of these proteins are not well understood. As a result, there has been significant interest in identifying natural and unnatural ligands that can be used to modulate the activity of carbohydrate-binding proteins. Lectins and glycan-binding antibodies are also used extensively as research tools, diagnostics, and therapeutic agents. Information on the specificities of these proteins is critical for interpreting results and selecting the best clinical candidates.

Although analysis of carbohydrate-macromolecule interactions is crucial for glycobiology, it remains a challenging area of science. First, carbohydrates can be exceedingly difficult to obtain, especially in homogeneous form. With limited access to the ligands, one cannot easily assess recognition. Second, traditional methods used to evaluate carbohydrate-protein interactions such as monosaccharide and oligosaccharide inhibition studies, isothermal calorimetry (ITC), surface plasmon resonance (SPR), and enzyme-linked lectin assays (ELLAs) can be labor intensive or require large quantities of each carbohydrate. As a result, these methods are not well suited for high throughput evaluations. Finally, one must consider the issue of valency. In most cases, interactions between a single carbohydrate ligand and a single binding domain of a protein (referred to as monovalent binding) are very weak. The affinity of monovalent binding events is typically too low to withstand the washing involved in common biological assays such as enzyme-linked immunosorbent assays (ELISAs), Western blots, and immunohistochemical staining. However, most carbohydrate-binding proteins possess two or more binding sites or assemble into functional units with multiple binding sites. As a result, they can simultaneously bind two or more carbohydrate ligands (referred to as polyvalent binding) leading to a high overall affinity or “avidity.” Assays that probe interactions between carbohydrates and proteins must account for the unique aspects of polyvalent recognition. For example, the ligands should be presented in a polyvalent context. In addition, the spacing and orientation of ligands will affect the ability to form a polyvalent complex.

As a result of the difficulties mentioned above, researchers have been developing alternative methods to study carbohydrate-protein interactions. Array technology is specifically designed for high throughput evaluations of molecular recognition events. Miniaturization facilitates the process by allowing minimal use of reagents and other hard-to-obtain components. In the following sections, the development and application of glycan array technology are described.

 

Fabrication of Glycan Arrays

Fabrication of a glycan array involves several interrelated factors: choosing a solid support format, choosing a method to attach glycans to that solid support, and obtaining appropriately functionalized carbohydrates for immobilization.

 

Array formats

Antecedents of modern glycan arrays can be traced back to the method of separating a complex glycolipid mixture using thin-layer chromatography, then probing the “array” with a carbohydrate-binding protein (6). Recently, most glycan arrays are created on either microtiter plates or glass microscope slides (see Fig. 1).

 

 

Figure 1. Examples of formats that have been used for glycan arrays.

 

The microtiter plate array format involves immobilizing carbohydrates in wells of 96-, 384-, or 1536-well microtiter plates (see Fig. 1a). Each carbohydrate component is spatially separated from other components within the plate. Two of the primary advantages of a microtiter plate format are cost and simplicity. Carbohydrates can be distributed into wells using multichannel pipettors, and assay results can be measured using standard plate readers. Thus, the equipment and supplies needed for the array are relatively inexpensive and common. However, microtiter plates generally require larger amounts of each carbohydrate and can accommodate a smaller total number of components per support unit.

An alternative to the microtiter plate format is a glass microscope slide. Slides can come in various layouts. The array developed by Glycominds, Ltd. contains 200 microwells with a single carbohydrate in each well (7). The miniaturized wells use a smaller amount of material than a microtiter plate but retain spatial separation of components (see Fig. 1b). A second format involves spotting components directly onto the slides. One of the key differences is that all the carbohydrates are in the same “well” (see Fig. 1c). Consequently, recognition of each component is compared under identical assay conditions and well-to-well variation is minimized. In addition, microarray printers used for producing DNA microarrays can be used to print very small features (50-200-qm spots) with high precision allowing for tens of thousands of spots on each slide. As a result, much smaller quantities of material are required and the total capacity is considerably higher than a microtiter plate. However, a microarray printer and high resolution scanner are required. A glass slide can also be modified to create 2-16 macrowells on a glass slide (see Fig. 1d). In this format, an entire array is printed in each well.

Several three-dimensional (3-D) approaches to glycan arrays have also been published, including fiber optic bead-based glycan arrays (see Fig. 1e) (8) and hydrogel arrays (9).

 

Methods for immobilizing carbohydrates

The methods of attachment to the solid support can be divided into two broad categories: covalent and noncovalent. Several factors should be considered. First, the ligands should be stable to immobilization, storage, and assay conditions. Second, the immobilized ligands should be accessible to the receptors, which are usually in solution. Third, the method should be efficient.

One of the most straightforward methods for immobilizing ligands is noncovalent attachment (4, 9-18). This method involves adsorption onto surfaces using noncovalent forces such as hydrophobic interactions, charge-charge interactions, and charge-dipole interactions (see Fig. 2a). A key concern is that the immobilized ligands must withstand routine screening and assay conditions. In general, lower molecular weight hydrophilic molecules, such as monosaccharides and oligosaccharides, show poor retention on solid supports when directly attached by noncovalent methods. In contrast, polysaccharides, glycoproteins, and glycolipids are well retained and can be directly arrayed on modified solid supports.

 

 

Figure 2. Immobilization strategies. (a) A schematic representation of noncovalent adsorption. The lipid tail of a neo-glycolipid adheres to a hydrophobic surface via noncovalent interactions. (b) Strategies used for covalent immobilization of glycans.

 

In one of the first reports of a carbohydrate microarray, under-ivatized polysaccharides and glycoproteins were immobilized directly on nitrocellulose-coated glass slides (10). In a further demonstration of the feasibility of noncovalent immobilization of underivatized glycans, Willats et al. reported microarrays of glycoproteins, proteoglycans, and polysaccharides on oxidized black polystyrene slides (11). Modified neutral and anionic dextran polysaccharides have also been synthesized and printed on semicarbazide-coated glass slides (12).

One strategy for noncovalent attachment of smaller monosaccharides and oligosaccharides involves coupling them to a lipid tail or other hydrophobic moiety, which provides suitable retention on a solid support. This strategy was exemplified by arrays of synthetic and natural oligosaccharides as neoglycolipids or glycosylceramide derivatives affixed to nitrocellulose-coated slides (13) and microtiter plates (14). Alternative approaches include capture of azide-containing monosaccharides and disaccharides on immobilized activated lipid alkynes using a Cu(I)-catalyzed 1,3-dipolar cycloaddition (15); reductive amination reactions between underivatized oligosaccharides and immobilized aminolipids on microtiter plates (16); and urea formation between amino-derivatized sugars and tetradecyl isocyanate adsorbed on microtiter plates (17). Formation of hydrogel microarrays of amino-saccharides and polyacrylamide glycoconjugates has been reported. Here, glycans were printed on hydrophobic glass surfaces with polymerizable monomers, and then polymerization was induced by photoactivation to form 3-D gel elements comprising the glycans (4, 9).

An elegant alternative approach for noncovalent interaction relies on fluorous-fluorous interactions. A glycan array of monosaccharides and disaccharides bearing anomeric fluorous tags was noncovalently immobilized on fluorous-derivatized glass slides (19, 20). The attachment method is compatible with a wide range of functional groups and has been successfully used to probe carbohydrate-protein interactions.

Glycan arrays have also been developed to take advantage of the noncovalent, but extraordinarily strong, biotin-streptavidin interaction. The Consortium for Functional Glycomics developed a glycan array consisting of biotinylated oligosaccharides and amino acid glycoconjugates immobilized on streptavidin-coated 384-well microtiter plates (21, 22). Another biotin- streptavidin “sandwich” glycan microarray was produced by self-assembly of biotinylated alkylthiols on a gold substrate, followed by coating with streptavidin and printing of biotinylated polyacrylamide glycoconjugates (23).

An alternative to noncovalent approaches is covalent attachment of ligands to the surface. Methods for covalent attachment can be broadly categorized as follows: 1) condensation with the aldehyde of a reducing sugar, 2) nucleophilic addition to or displacement of a group on an activated surface or sugar, 3) cycloaddition, and 4) insertion reactions (see Fig. 2b). Some derivatization methods, for example, condensation and reductive amination, lead to ring opening of the reducing end sugar, whereas other methods result in indeterminate anomeric configurations, depending on the reaction conditions and point of attachment. The linker used to connect the sugar to the surface is an important consideration because it can affect recognition and accessibility of the ligands. In choosing an ideal linker, the issues to consider include the chemical nature and composition (hydrophobic vs. hydrophilic), stability, length, and flexibility of the linker.

Unmodified sugars have been attached to various hydrazide- and amino-derivatized slides by condensation or by reductive amination, which proceeds by reduction of the intermediate Schiff base (see Fig. 2b.1). This strategy was used for immobilization of free oligosaccharides onto hydrazide-derivatized plates (24) and various unmodified carbohydrates (monosaccharides, oligosaccharides, and polysaccharides) on hydrazide-coated glass slides (25). Later, immobilization of free oligosaccharides on hydrazide-derivatized self-assembled monolayer (SAM) of gold-coated glass slides was also reported (26). The formation of oximes between reducing end aldehydes of sugars and amino-oxy groups has been exploited in the fabrication of glycans arrays. This method was explored for the immobilization of monosaccharides, disaccharides, and oligosaccharides on amino-oxy glass slides and successfully used for making a functional glycan array of oligosaccharides on aminooxyacetyl-functionalized glass slides (27). However, stronger signals were observed for hydrazide-coated slides in comparison with aminooxy-coated slides (25). Several glycans arrays have also been reported where the simple condensation of aldehydes and amines was used to covalently attach the saccharides. Deaminated heparin oligosaccharides bearing aldehyde groups were attached to amine-coated glass slides as Schiff bases without further reduction (28), while aldehyde and amino functionalized monosaccharides and disaccharides were immobilized on amine- and aldehyde-coated slides, respectively (29, 30). It should be noted that reductive amination results in opening of the reducing end sugar whereas condensation can lead to changes in anomeric configuration or mixtures of anomers at the reducing end.

An alternative strategy used in the covalent attachment of glycans to solid supports is nucleophilic addition or displacement. In this method, the decision about where to install the nucleophile and the electrophile—on the sugar or on the solid support—rests with the researcher, but could be influenced by the ease of installation and availability of derivatized reaction partners. Glycan arrays involving the Michael addition of maleimide-linked carbohydrates and thiol-coated glass slides (see Fig. 2b.2) have been created and used to probe lectin-carbohydrate interactions (31, 32). The transposition of the reacting functional groups has also been reported where glycans arrays were fabricated using thiol-linked sugars on maleimide-functionalized glass slides (33, 34) and on self-assembled monolayers presenting maleimide groups (35).

Epoxide-opening reactions have also been used to covalently attach carbohydrates, glycoproteins, and neoglycoconju-gates to glass slides (see Fig. 2b.3). One report demonstrated the feasibility of immobilizing hydrazide-functionalized small molecules, including monosaccharides, on epoxide-coated slides and further showed that hydrazide-containing ligands react more rapidly with the epoxide-coated surface than thiols and amines (36). The use of epoxide-coated slides is relatively common for the fabrication of protein microarrays. One advantage of using epoxide-coated surfaces is the potential indiscriminate reactivity of epoxides to nucleophiles such as amines, thiols, and alcohols. However, it is important to note that the reactivity of these nucleophiles often depend on the pH conditions during printing. The development of glycan arrays of neoglycoconjugates and glycoproteins on epoxide-coated slides has been described (37, 38). In this report, neoglycoconjugates were synthesized by conjugating to lysine residues on serum albumin through activated carboxyl-bearing linkers, and then printed directly onto epoxide-functionalized glass slides.

The facile reaction of amines with N-hydroxysuccinimide (NHS)-activated carboxyl groups, and the concomitant formation of a stable amide bond, has been exploited in the fabrication of glycan arrays (see Fig. 2b.4). This method has been used for covalent attachment of amine-presenting glycans ranging in size from monosaccharides to glycoproteins on NHS-activated slides (39, 40). In one report, oligosaccharides were derivatized with photocleavable amine-bearing linkers used to attach the ligands to a porous silicon surface presenting NHS groups (41). Oligosaccharides labeled with fluorescent 2,6-diaminopyridine have also been printed on NHS-activated glass slides (42).

An array of monosaccharides and oligosaccharides was created by the reaction of p-aminophenyl-glycosides with cyano-chloride-activated linkers on wells of microtiter plates (7).

In the fabrication of bead-based glycan arrays, glycopeptides and bovine serum albumin neoglycoproteins were conjugated to the carboxyl-presenting beads using water-soluble carbodiimides. The beads were spatially arrayed into microwells for screening with lectins (8) or assayed for lectin binding as a suspension (43).

Covalent immobilization of glycans by cycloaddition (see Fig. 2b.5) and insertion reactions (see Fig. 2b.6 and 2b.7) has been used in the creation of arrays (44-47). Cyclopenta-diene-carbohydrates were arrayed on self-assembled monolayers alkanethiols presenting benzoquinione through penta-(ethylene)glycol linkers (44). An efficient Diels-Alder reaction between the modified glycan and the support and the reduction of nonspecific interactions by the use of the glycol linker are highlights of this report. One attachment method that portends the development of functional group-independent reactions where any underivatized molecule can be immobilized is the use of insertion reactions. In one report, various glycoproteins were printed on polymeric surfaces presenting aryltrifluo- romethyl diazirines. The surfaces were photoactivated resulting in formation of a covalent bond between the solid support and the printed ligand by carbene C-H insertion (46). In an alternative formal insertion strategy, glycans arrays were created by printing underivatized carbohydrates on SAMs presenting phthalimide groups, which, on photoactivation, undergo hydrogen atom abstraction and recombination of radicals to form a covalent bond (47).

 

Obtaining glycans for an array

Preferably, one would like access to a representative set of naturally occurring monosaccharides and oligosaccharides, polysaccharides, glycoproteins, and glycolipids and their synthetic analogs. However, obtaining a diverse set of carbohydrates in a format compatible with the immobilization strategy remains a major challenge for glycan array development. Current arrays contain around 10-300 different components (see Table 1). This level of diversity can provide useful information but represents only a small fraction of the total diversity of carbohydrate structures found in nature. Including glycans from animals, plants, and bacteria, there are over 10,000 different structures that have been identified and characterized to date. Therefore, one of the principal challenges for the future advancement of glycan array technology is developing strategies to obtain much larger sets of carbohydrate ligands.

One method that has been used to acquire carbohydrates is isolation and purification from natural sources such as human or animal tissue, milk, urine, plants, and bacteria (see cross reference: Isolation of glycans). Access to homogeneous carbohydrate structures can be challenging due to the difficulties in separation of complex mixtures, identification of carbohydrate(s) contained within each fraction, and preparation of sufficient quantities from the limited amounts present in a particular sample. Alternatively, mixtures of unknown composition can be used to survey a broad repertoire of the glycome. On identification of a mixture containing one or more members with interesting receptor-binding properties, the mixture can then be deconvoluted by further fractionation and separation by routine analytical techniques (13).

A second method that has been invaluable for accessing glycans is chemical synthesis (see cross references: Glycan synthesis, key reaction of; Glycan synthesis, key strategies for; Glycan synthesis, protection and deprotection steps of; and Glycopeptides and glycoproteins, synthesis of). This approach can provide both natural and unnatural structures that can be very useful for probing relationships between structure and function. Although there have been tremendous advances in our ability to synthesize carbohydrates both in solution and on solid phase, carbohydrate chemistry remains a challenging area of organic chemistry. Progress on the synthesis of carbohydrate libraries (48-50), automated carbohydrate synthesis (51), and one-pot multicomponent synthetic strategies (41, 52, 53) will substantially aid glycan array development.

Enzymes can be powerful tools for obtaining carbohydrates as well (54). Chemoenzymatic synthesis of oligosaccharides involves the use of glycosyltransferases or glycosidases for the regio- and steroselective formation (typically between unprotected reaction partners) or hydrolysis of glycosidic bonds, respectively. In some cases, one reaction partner is obtained by traditional solution or solid-phase chemical synthesis, followed by enzymatic glycosylation. In other cases, synthesis begins with enzymatic cleavage and continues with more traditional chemical synthesis methods.

 

Evaluation of Binding

Once a glycan array is constructed, one needs to develop a method to evaluate binding of proteins, cells, viruses, or other macromolecules to the array. The general process involves carrying out the assay, detecting signals on the array, and analyzing/processing the results. Although the overall process appears straightforward, assay development can be very challenging. Importantly, every step in the process from fabrication to assay conditions to detection must be successful to produce a signal. As a result, it can be difficult to determine which step or steps need to be optimized, especially when no signal is observed. Therefore, assay development typically requires systematic variation of many parameters. Additional considerations include sensitivity, signal-to-noise ratios, reproducibility, flexibility, and dynamic range.

The vast majority of array assays have used fluorescence as the detection method due to the high sensitivity and the availability of detectors such as fluorescent plate readers for microtiter plates and high resolution DNA microarray scanners for glass microarray slides. Therefore, the following discussion will focus on assay development coupled with fluorescent detection.

 

Nonspecific adsorption

One important general consideration is nonspecific binding. Proteins and other macromolecules can adsorb to surfaces leading to high background signals and low signal-to-noise ratios. One common approach to prevent nonspecific adsorption is to cover or block any “sticky” surface areas with a noninterfering protein or polymer that is unrelated to the receptor or secondary reagent such as bovine serum albumin (BSA), which simply involves incubating the array with a solution of BSA in an appropriate buffer (e.g., 3% BSA in phosphate buffered saline). An alternative approach involves using modified surfaces that resist nonspecific adsorption of proteins such as fluorous-coated slides (19, 20, 55).

 

Table 1. Summary of glycan arrays

 

Group or

Company

Composition

Format(s)

Number of

Componentsa

References

Consortium for

Oligosaccharides;

Plates; glass slides

264

[21, 22, 40, 64, 65,

Functional

neoglycoconjugates

 

68, 70-72, 74-76,

Glycomics

 

 

 

78-81, 97, 98]

Feizi, T.

Neoglycolipids

Glass slides

190

[13, 67, 69, 73, 77]

Gildersleeve, J.

Neoglycoproteins;

16-well glass slides

73

[37, 38]

 

glycoproteins

 

 

Glycominds

Oligosaccharides

200 microwells

37

[7, 66, 85, 90-93, 95]

 

 

1 sugar/well

 

 

Hsieh-Wilson, L.

Oligosaccharides

Glass slides

4

[29]

Koberstein, J.

Oligosaccharides;

Glass slides

10

[47]

 

polysaccharides

 

 

 

Ligler, F.

Monosaccharides

Glass slides

2

[82]

Malayer, J.

Lipopolysaccharides

Glass slides

9

[88]

Melnyk, O.

Polysaccharides

Glass slides

51

[12]

Mrksich, M.

Monosaccharides

Glass slides

10

[35, 44]

Pohl, N.

Mono-, disaccharides

Glass slides

8

[19, 20]

Rubina, A.

Oligosaccharides

Glass slides

6

[9]

Ruhl, S.

Neoglycoconjugates;

Nitrocellulose

28

[96]

 

glycoproteins

membrane

 

 

Schmidt, R.

Mono-, disaccharides

Glass slides

7

[30]

Seeberger, P.

Oligosaccharides

Glass slides;

14

[8, 28, 34, 39, 57-59,

 

 

Fiber optic

6

83, 94]

Shin, I.

Oligosaccharides

Glass slides

22

[25, 31, 32, 36]

Sprenger, N.

Oligosaccharides;

Glass slides

7

[46]

 

neoglycoconjugates;

glycoproteins

 

 

 

Sugahara, K.

Neoglycolipids

Nitrocellulose

14

[87]

 

 

membrane

 

 

Uzawa, H.

Anionic glycopolymers

Gold surface

3

[18]

Wang, D.

Polysaccharides;

Glass slides

51

[10, 47, 89]

 

glycoproteins;

glycosaminoglycans;

neoglycoconjugates

 

 

 

Wang, P.

Neoglycolipids

Plates

4

[16]

Willats, W.

Polysaccharides;

Black

23

[11]

 

proteoglycans;

polystyrene

 

 

 

neoglycoconjugates

slides

 

 

Wong, C.H.

Oligosaccharides;

Glass slides;

6

[14, 15, 17, 41, 45,

 

Neoglycolipids

Plates

21

84, 86, 99, 100]

Yamamoto, K.

Glycopeptides

Fiber optic

12

[43]

Zhi, Z.

Oligosaccharides

Glass slides

3

[26]

Zhou, J.

Oligosaccharides

Glass slides

10

[27]

Largest published array.

 

Directly labeled receptors

The most straightforward method for generating a fluorescent signal is directly labeling the protein or macromolecule of interest with a fluorophore (see Fig. 3a). Prior to incubating on the array, the receptor is conjugated to a fluorophore such as fluorescein, Cy3, or Cy5. One typically obtains 1-5 molecules of fluorophore per molecule of receptor. With a limited number of fluorophore molecules, this approach generally has the lowest sensitivity. In addition, covalent labeling of a protein can sometimes interfere with the functional properties of a protein. Nevertheless, this approach has been used successfully in many reports (19, 20, 32-34, 42, 46, 56-59).

 

 

Figure 3. Common assay strategies for evaluating binding to a glycan array.

 

Labeled secondary reagents

An alternative to direct labeling of a receptor involves the use of a secondary reagent that specifically binds the primary receptor of interest (see Fig. 3b). For example, a mouse monoclonal antibody can be detected with polyclonal anti-mouse antibody secondary reagents. In general, the array is incubated in the presence of the primary receptor such as a lectin or antibody. After washing away unbound receptor, the array is incubated with the secondary reagent. To generate a signal, the secondary reagent may be labeled with a fluorophore. One advantage of this method is that it is possible to get multiple secondary reagents binding to each molecule of primary receptor leading to increased sensitivity. Alternatively, the secondary reagent may be conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. To generate a signal, the array is incubated with a substrate that can be converted into a fluorescent product thus generating a large increase in fluorescent signal. Alternatively, a solution-phase fluorescent substrate is converted into a reactive intermediate that can covalently attach to the surface. In this case, soluble substrate is washed away and immobilized fluorophores are detected. The primary advantage of the enzyme-linked approach is sensitivity. As each molecule of enzyme can generate many molecules of fluorescent product, the signal is amplified. Moreover, the signal can be enhanced by increasing the length of time the substrate is incubated with the enzyme. In addition to enhanced sensitivity, use of a secondary reagent allows detection of receptors in the presence of complex mixtures of other proteins due to the specific binding between the primary and secondary proteins. For example, human serum contains a wide variety of different proteins and other components. With the aid of a secondary reagent, glycan-binding antibodies present in human serum can be profiled as biomarkers of disease (see below). Naturally, this approach is limited to situations where a secondary reagent is available.

 

Other methods of detection

Although labeled receptors and secondary reagents can be very useful, they are not suitable for all applications. Attachment of a label can disrupt binding, and secondary reagents are not available in all cases. Label-free assay systems have been used in protein microarrays to overcome these problems and are potentially useful in glycan array technology. Surface plasmon resonance imaging (60), mass spectrometry (61), oblique-incidence reflectivity difference (OI-RD) scanning microscope (62), and Kelvin nanoprobe (63) are some of the tools that have been adopted for label-free assay systems.

 

Processing of assay signals

Processing of signals and analysis of large datasets can present a major challenge for array technology. Several software programs have been used in combination with glycan arrays such as GenePix, Imagene, QuantArray, ArrayWorX, and ScanArray. As glycan arrays increase in size, the need for bioinformatics tools will increase significantly.

 

Applications of Glycan Arrays

Glycan arrays are designed for high throughput analysis of carbohydrate-macromolecule interactions. They are powerful tools for determining if a protein is a carbohydrate-binding protein, identifying ligands for carbohydrate-binding proteins, and evaluating the specificity of antibodies and lectins used as research tools, diagnostics, and therapeutic agents. In addition, they provide useful information for increasing our basic understanding of molecular recognition of carbohydrates. Glycan arrays are also emerging as useful tools for biomarker discovery.

 

Identification and characterization of carbohydrate-binding proteins

Glycan arrays have been used to evaluate the specificity of numerous plant, animal, and microbial lectins.

Plant lectins are used routinely as research tools, diagnostic agents, and therapeutic agents. Plant lectins are frequently used as an initial screen of a glycan array to verify that the glycans are accessible for binding and that the assay works. Numerous papers also describe the use of glycan arrays for more comprehensive analysis of plant lectin specificity (38, 40, 43, 64, 65). Although these proteins have been studied for years, array screening has uncovered novel binding properties illustrating the utility of high throughput, unbiased screening. In addition to published results, the Consortium for Functional Glycomics lists results for many lectins and other proteins on its website.

Animal lectins play an important role in many biological processes, and information on ligand binding is essential for development of agonists/antagonists. One interesting example is DC-SIGN, a calcium-dependent (C-type) lectin expressed on dendritic cells. DC-SIGN is known to bind HIV and facilitate infection of T cells. DC-SIGN has been profiled on glycan arrays and found to bind blood group antigens, galactose terminal sugars, and mannose oligosaccharides (22, 66). The related proteins SIGN-R1 through SIGN-R8 have also been evaluated (67, 68). Dectin-1, another C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens, was profiled using a glycolipid microarray (69). It was shown to bind clusters of 11-13 gluco-oligomers. The scavenger receptor C-type lectin (SRCL) was profiled on a glycan array and was found to bind LeX and related trisaccharides (70). A glycan array was also used to evaluate binding properties of the rat asialoglycoprotein receptor, Kupffer cell receptor, macrophage galactose lectin, and human scavenger receptor C-type lectin (71, 72). Based on their galactose-binding preferences, the lectins were classified into two categories: one group selective for Lewis antigens and the other showed broad selectivity for various Gal/GalNAc-containing residues. A glycan array consisting of LeX, SLeX, and their sulphated analogs was used to profile siglecs (73). This analysis revealed that the sulfate groups on the carbohydrate epitopes act as modulators of siglec binding. Through the use of a glycan array, mouse Siglec-F and human Siglec-8 (21) expressed on eosinophils were also shown to recognize 6'-sulfo-sialyl LeX (74). A polysaccharide microarray was used to study interactions between dextran polysaccharides and platelet-derived growth factor BB isoform (12). A chondroitin sulfate microarray was used as a screening tool to identify an antagonist for a therapeutically important proinflammatory cytokine, tumor necrosis factor-a (29). Other animal lectins profiled with glycan arrays include galectin-1 (75), galectin-4 (76), mannose 6-phosphate receptor (77), dectin-2 (78), Manila clam lectin (79), and Langerin (67).

Bacterial, viral, and microbial lectins are a third class of important proteins that have been screened on glycan arrays. One of the most interesting applications of glycan arrays involved profiling hemagglutinins from pathogenic strains of the influenza A virus including the pandemic 1918 strain (80, 81). Avian viruses were found to preferentially bind sialic acid with alpha 2-3 linkages while human viruses bound alpha 2-6 linkages. The difference in specificity provides a potential explanation for the variations in infectivity and pathogenicity. A group of bacterial toxins and cells, Salmonella typhimuriam, Listeria Monocytogenes, Escherichia coli, staphylococcal enterotoxinB, cholera toxin, and tetanus toxin, has also been profiled on a glycan array (82). Several lectins with potent antiHIV activity such as cyanovirin, scytovirin, and microvirin have been evaluated on glycan arrays (58, 83).

Another important application of glycan arrays is assessing the specificity of glycan-binding antibodies (37, 58, 84-87). Carbohydrate-binding antibodies are important research tools, diagnostics, and therapeutic agents. Information on specificity is critical for the proper interpretation of results or selection of clinical candidates. For example, antibody 2G12 is known to neutralize a broad range HIV-1 isolates in a carbohydrate-dependent manner. Profiling using a glycan array illustrated that the peptide backbone is not required for binding and that the antibody has a preference for Mana1-2Man linkages (58, 84). A glycan array comprising the tumor antigen Globo H and its fragments was used to profile two monoclonal antibodies against Globo H and serum from breast cancer patients (86). This array showed that the fucose residue was essential for recognition by the monoclonal antibodies but not for the serum antibodies. In another glycan array study, various antibodies and lectins that have been used for decades to monitor the expression of the tumor-associated Tn antigen were found to cross-react with other human glycans (37). The results suggest that information on the expression of the Tn antigen used for the development of diagnostics and vaccines may be inaccurate. The antimalaria antibody MG96 was evaluated in an effort to identify suitable carbohydrate antigens for a malaria vaccine (85).

 

Profiling serum antibodies as biomarkers for disease

Glycan arrays have also been used to measure levels of anticarbohydrate antibodies in patient serum as diagnostic or prognostic markers (10, 88-93). A glycan array was used to measure the anticarbohydrate antibody levels in eight Hodgkin’s lymphoma patients (92). From the study, elevated antibody levels against four carbohydrate epitopes were detected in the lymphoma patients. Blood samples from multiple sclerosis and Crohn’s disease patients were screened on a glycan array for elevated antibody expression (91). In another report, a glycan array was used to profile 107 multiple sclerosis patient sera (93). From this study, antibodies to Glca1-4Glc were identified as novel biomarkers for relapsing/remitting multiple sclerosis. A set of 72 Crohn’s disease patient sera was also profiled using a microarray (90). It was reported that antichitobioside and antilaminaribioside antibodies are novel serologic markers associated with Crohn’s disease.

 

Characterization of cell and virus binding

Cell-cell recognition can be a complex process involving multiple interactions between different proteins and glycans. In some cases, the specific proteins involved are not known or are not functional on their own. In addition, cell binding can trigger signaling cascades or other responses. As a result, evaluation of binding of whole cells to glycans can be very useful and several reports have demonstrated successful screening of cells on glycan arrays. Examples include screening of E. coli strains (25, 94), chicken hepatocytes and CD4+ human T-cells (95), and Helicobacter pylori (96). Viruses and viral capsids have also been screened on glycan arrays (97, 98).

 

Identification of enzyme substrates and inhibitors

Glycan arrays have also been used to identify inhibitors of enzymes. Inhibitors for the enzyme fucosyltransferase were discovered from a library of triazole-containing compounds using a glycan array (99). Due to the importance of aminoglycosides as therapeutic agents and the attractiveness of RNA as a drug target, a number of aminoglycoside glycan arrays have been developed. One aminoglycoside array was used for the high throughput analysis of glycan-RNA interactions (100). The molecular recognition between oligonucleotide mimics of aminoglycoside-binding sites in the bacterial 30 S ribosome and various aminoglycoside antibiotics has been studied by the use of a glycan array (57). Another aminoglycoside mimetic microarray was developed to assess the potency of these glycans as improved antibiotics (59). The interactions between aminoglycosides and two aminoglycoside acetyltransferases implicated in antibiotic resistance were analyzed. The array analysis showed that some of the aminoglycoside mimetics inhibit the aminoglycoside acetyltransferases, and thus have potential as improved antibiotics.

 

Conclusions

Glycan arrays have emerged as powerful tools for analysis of carbohydrate-macromolecule interactions. As research tools, they are being used to identify ligands for glycan-binding proteins and evaluate specificity of carbohydrate-binding proteins. In a clinical setting, glycan arrays hold enormous potential for the development of diagnostic and therapeutic agents. For example, several studies have demonstrated the utility of glycan arrays for profiling antiglycan antibodies in serum as diagnostic markers of disease. As the technology matures, the range of applications and capabilities will continue to expand and glycan array analysis is anticipated to become routine in chemistry, biology, and clinical laboratories.

 

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See Also

Glycan Synthesis, Key Reactions of

Glycan Synthesis, Key Strategies for

Glycan Synthesis, Protection and Deprotection Steps of

Glycan-Glycan Interactions

Glycan-Protein Interactions

Glycopeptides and Glycoproteins, Synthesis of

Isolation of Glycans

Sugar-Lectin Interactions in Cell Adhesion