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Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research*

Open AccessPublished:September 17, 2012DOI:https://doi.org/10.1074/jbc.M112.396598
      Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

      Introduction

      Glycans are crucial for plant life and are used for storage, defense, and signaling and as structural cell wall components (
      • Bacic A.
      • Harris A.J.
      • Stone B.A.
      ,
      • De Lorenzo G.
      • Ferrari S.
      Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi.
      ,
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      Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells.
      ,
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      Starch: its metabolism, evolution, and biotechnological modification in plants.
      ,
      • Lee K.J.
      • Marcus S.E.
      • Knox J.P.
      Cell wall biology: perspectives from cell wall imaging.
      ). Plant oligo- and polysaccharides are also important components of food and feed and have numerous industrial applications. Starch is the most common carbohydrate in the human diet, whereas plant cell walls provide bulk materials including timber, paper, and cloth, as well as fine chemicals, food ingredients, and biofuel feedstocks (
      • Bacic A.
      • Harris A.J.
      • Stone B.A.
      ,
      • Lee K.J.
      • Marcus S.E.
      • Knox J.P.
      Cell wall biology: perspectives from cell wall imaging.
      ,
      • Willats W.G.
      • Knox J.P.
      • Dalgaard Mikkelsen J.
      Pectin: new insights into an old polymer are starting to gel.
      ,
      • Pauly M.
      • Keegstra K.
      Plant cell wall polymers as precursors for biofuels.
      ). The complexity and diversity of plant polysaccharides underpin their biological roles and many of their industrially important characteristics, but also produce challenges for research and optimal utilization. A detailed knowledge of the structures, functions, interactions, and occurrence of plant glycans is essential for understanding their complex contributions to plant life and to fully exploit their commercial potential. However, unlike proteins and nucleotides, complex carbohydrates are not readily amenable to sequencing or synthesis, and existing biochemical techniques for glycan analysis, although powerful, are usually low throughput (
      • Fry S.C.
      Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells.
      ,
      • Albersheim P.
      • Darvill A.
      • Roberts K.
      • Sederoff R.
      • Staehelin A.
      ).
      The development of rapid genome sequencing methods and improvements in protein expression techniques enable the production of large numbers of carbohydrate-active enzymes and carbohydrate-binding proteins including carbohydrate-binding modules (CBMs).
      The abbreviations used are: CBM
      carbohydrate-binding module
      HTP
      high throughput
      AGP
      arabinogalactan protein
      RGI
      rhamnogalacturonan I.
      There has been an exponential increase in the number of entries of these proteins in the Carbohydrate-Active enZYmes (CAZy) database (
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      The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics.
      ), but this has not been matched by structural analysis or determination of their biochemical activities (
      • Gilbert H.J.
      The biochemistry and structural biology of plant cell wall deconstruction.
      ). Similarly, monoclonal antibodies (mAb) are immensely valuable molecular probes for carbohydrate research, but their usefulness is dependent on knowledge of the epitopes they recognize (
      • Knox J.P.
      The use of antibodies to study the architecture and developmental regulation of plant cell walls.
      ,
      • Willats W.G.
      • McCartney L.
      • Mackie W.
      • Knox J.P.
      Pectin: cell biology and prospects for functional analysis.
      ,
      • Knox J.P.
      Revealing the structural and functional diversity of plant cell walls.
      ,
      • Pattathil S.
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      • Baldwin D.
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      ). The rate-limiting step in CBM, mAb, and enzyme production is often a lack of efficient methods for screening their specificities. There is therefore a clear need in plant biology for high throughput (HTP) and high resolution techniques for the analysis of carbohydrate-active proteins including enzymes.
      Microarray technology has underpinned the development of multiplexed assays that have revolutionized the HTP analysis of nucleotides, proteins, and increasingly, carbohydrates (
      • Schena M.
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      Quantitative monitoring of gene expression patterns with a complementary DNA microarray.
      ,
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      ,
      • McWilliam I.
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      Inkjet printing for the production of protein microarrays.
      ). Using microarrays, the abundance of, and interactions between, hundreds or thousands of molecules can be assessed simultaneously using very small amounts of analytes (
      • Schena M.
      • Shalon D.
      • Davis R.W.
      • Brown P.O.
      Quantitative monitoring of gene expression patterns with a complementary DNA microarray.
      ,
      • Ekins R.
      • Chu F.W.
      Microarrays: their origins and applications.
      ,
      • McWilliam I.
      • Chong Kwan M.
      • Hall D.
      Inkjet printing for the production of protein microarrays.
      ). Carbohydrate microarrays were first produced in 2002, and a variety of approaches has been developed for the printing and immobilization of oligo- and polysaccharides (
      • Feizi T.
      Progress in deciphering the information content of the 'glycome'–a crescendo in the closing years of the millennium.
      ,
      • Fukui S.
      • Feizi T.
      • Galustian C.
      • Lawson A.M.
      • Chai W.
      Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.
      ,
      • Wang D.
      • Liu S.
      • Trummer B.J.
      • Deng C.
      • Wang A.
      Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells.
      ,
      • Willats W.G.
      • Rasmussen S.E.
      • Kristensen T.
      • Mikkelsen J.D.
      • Knox J.P.
      Sugar-coated microarrays: a novel slide surface for the high-throughput analysis of glycans.
      ,
      • Blixt O.
      • Head S.
      • Mondala T.
      • Scanlan C.
      • Huflejt M.E.
      • Alvarez R.
      • Bryan M.C.
      • Fazio F.
      • Calarese D.
      • Stevens J.
      • Razi N.
      • Stevens D.J.
      • Skehel J.J.
      • van Die I.
      • Burton D.R.
      • Wilson I.A.
      • Cummings R.
      • Bovin N.
      • Wong C.H.
      • Paulson J.C.
      Printed covalent glycan array for ligand profiling of diverse glycan-binding proteins.
      ,
      • Feizi T.
      • Chai W.
      Oligosaccharide microarrays to decipher the glyco code.
      ,
      • Park S.
      • Lee M.R.
      • Shin I.
      Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.
      ,
      • Smith D.F.
      • Song X.
      • Cummings R.D.
      Use of glycan microarrays to explore specificity of glycan-binding proteins.
      ). However, the representation of glycomes on these arrays is generally far less comprehensive than is the coverage of transcriptomes/genomes and proteomes by nucleotide and protein arrays, respectively. The primary reason for this is the lack of facile methods for the production of sets of homogeneous, sequence-defined plant oligosaccharide structures (
      • Feizi T.
      • Fazio F.
      • Chai W.
      • Wong C.H.
      Carbohydrate microarrays — a new set of technologies at the frontiers of glycomics.
      ,
      • Liu Y.
      • Palma A.S.
      • Feizi T.
      Carbohydrate microarrays: key developments in glycobiology.
      ). In contrast, partially defined polysaccharides are relatively easy to obtain, and microarrays and enzyme-linked immunosorbent assays (ELISAs) populated with such samples have shown potential for HTP screening (
      • Sørensen I.
      • Willats W.G.
      Plant cell walls: new insights from an ancient species.
      ,
      • Klopffleisch K.
      • Phan N.
      • Augustin K.
      • Bayne R.S.
      • Booker K.S.
      • Botella J.R.
      • Carpita N.C.
      • Carr T.
      • Chen J.G.
      • Cooke T.R.
      • Frick-Cheng A.
      • Friedman E.J.
      • Fulk B.
      • Hahn M.G.
      • Jiang K.
      • Jorda L.
      • Kruppe L.
      • Liu C.
      • Lorek J.
      • McCann M.C.
      • Molina A.
      • Moriyama E.N.
      • Mukhtar M.S.
      • Mudgil Y.
      • Pattathil S.
      • Schwarz J.
      • Seta S.
      • Tan M.
      • Temp U.
      • Trusov Y.
      • Urano D.
      • Welter B.
      • Yang J.
      • Panstruga R.
      • Uhrig J.F.
      • Jones A.M.
      Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.
      ,
      • Sørensen I.
      • Willats W.G.
      Screening and characterization of plant cell walls using carbohydrate microarrays.
      ). However, most plant polysaccharides and especially those from plant cell walls are complex heteropolymers and, even if pure, will typically accommodate a range of smaller oligosaccharide substructures (or epitopes), and so, polysaccharide-based assays, whether they be microarrays or ELISAs, lack analytical resolution (
      • Klopffleisch K.
      • Phan N.
      • Augustin K.
      • Bayne R.S.
      • Booker K.S.
      • Botella J.R.
      • Carpita N.C.
      • Carr T.
      • Chen J.G.
      • Cooke T.R.
      • Frick-Cheng A.
      • Friedman E.J.
      • Fulk B.
      • Hahn M.G.
      • Jiang K.
      • Jorda L.
      • Kruppe L.
      • Liu C.
      • Lorek J.
      • McCann M.C.
      • Molina A.
      • Moriyama E.N.
      • Mukhtar M.S.
      • Mudgil Y.
      • Pattathil S.
      • Schwarz J.
      • Seta S.
      • Tan M.
      • Temp U.
      • Trusov Y.
      • Urano D.
      • Welter B.
      • Yang J.
      • Panstruga R.
      • Uhrig J.F.
      • Jones A.M.
      Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.
      ,
      • Sørensen I.
      • Willats W.G.
      Screening and characterization of plant cell walls using carbohydrate microarrays.
      ,
      • Moller I.
      • Marcus S.E.
      • Haeger A.
      • Verhertbruggen Y.
      • Verhoef R.
      • Schols H.
      • Ulvskov P.
      • Mikkelsen J.D.
      • Knox J.P.
      • Willats W.
      High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles.
      ).
      We have developed a new generation of glycan microarrays for plant research based on defined oligosaccharide structures produced either by isolation from polysaccharides or by de novo chemical synthesis. Once coupled to bovine serum albumin (BSA), these neoglycoprotein sets are highly versatile, and microarrays can be printed on a variety of slides and membranes. Most of the oligosaccharides we describe here are derived from, or based on, cell wall polysaccharides that are among the most complex in nature and present particular challenges for HTP analysis, but we have also included starch-related oligosaccharides and novel synthesized structures.

      DISCUSSION

      It is generally recognized that there is a widening gap between our ability to discover genes and proteins and to understand their roles in plant glycobiology. For example, it is estimated that we can safely predict the activities of no more than 20% of the proteins within the CAZy database (
      • Gilbert H.J.
      The biochemistry and structural biology of plant cell wall deconstruction.
      ). We show here that carbohydrate microarrays can make a valuable contribution to the HTP analysis of a variety of carbohydrate-protein interactions, and although oligosaccharide microarrays have been described for use in medical animal and microbial research, equivalent technology has not previously been developed for plant research.
      The use of defined oligosaccharides rather than polysaccharides is important for obtaining detailed information about carbohydrate-interacting proteins. A library of cell wall-derived oligosaccharides is a significant resource in itself, and once coupled to protein, it can be used to produce microarrays on diverse surfaces. The versatility of being able to print microarrays on both nitrocellulose and slides is important. Although slide-based microarrays can only be analyzed using specialized scanning equipment, membrane-based arrays can be used by non-experts and scanned using an ordinary office scanner and are thus ideal for wider distribution to researchers. Several linkers have been developed for the covalent and noncovalent attachment of oligosaccharides to substrates. For example, coupling to lipids has also been shown to be a highly effective method for oligosaccharide microarray production (
      • Feizi T.
      Progress in deciphering the information content of the 'glycome'–a crescendo in the closing years of the millennium.
      ,
      • Fukui S.
      • Feizi T.
      • Galustian C.
      • Lawson A.M.
      • Chai W.
      Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.
      ,
      • Park S.
      • Lee M.R.
      • Shin I.
      Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.
      ,
      • Smith D.F.
      • Song X.
      • Cummings R.D.
      Use of glycan microarrays to explore specificity of glycan-binding proteins.
      ,
      • Palma A.S.
      • Liu Y.
      • Muhle-Goll C.
      • Butters T.D.
      • Zhang Y.
      • Childs R.
      • Chai W.
      • Feizi T.
      Multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin.
      ). One reason for choosing BSA as a carrier molecule was because BSA-based neoglycoproteins are a multifunctional resource that can be used not just for microarray production but also as immunogens and as components of other assays for which immobilization is required. Nevertheless, any coupling procedure that involves modification of reducing ends is likely to interfere with the activity of reducing end-acting probes or enzymes, and this may be exacerbated by the large size of the BSA molecule. We found that BSA-coupled oligosaccharides arrayed as described previously (see “Experimental Procedures”) were effective substrates for several exo-acting glycosyl hydrolases but not for endo-acting enzymes (data not shown). Presumably, the exo-acting enzymes were nonreducing-end acting, and the lack of activity of the endo-acting enzymes was a result of steric hindrance from the BSA.
      This study highlighted some important technical aspects of oligosaccharide microarray production including the relative merits of different microarray robot printers. The pin-based MicroGrid II printer was suited for the production of microarrays on nitrocellulose membrane with larger spot sizes. However, we found that array quality often decreased with longer print runs such that some spots were missing or not properly printed, and this is likely to result from the inevitable wear of the pins that occurs with contact printing. This drawback is avoided with noncontact printers such as the Arrayjet Sprint that dispel samples by a highly reproducible piezo-actuation process. Another major advantage of the Arrayjet Sprint is its much greater speed, which is important not just to increase throughput but because the evaporation of sample buffer with a concomitant concentration of samples can be highly problematic during long microarray print runs. Importantly, by printing arrays on multipad slides (as shown in supplemental Fig. S1), such that each pad is isolated by a gasket during probing, it is possible to simultaneously assess the binding of large numbers of mAbs, CBMs, etc. against many immobilized samples. For example, using ten 16-pad slides, it is possible to simultaneously screen 160 antibodies, each against 400 immobilized glycans.
      The primary goal of this work was to develop plant oligosaccharide microarray technology per se, but we also obtained new epitope-level information about mAb specificities. For example, mAbs LM14 and JIM14 have previously been described as binding to unknown epitopes occurring on AGPs (
      • Moller I.
      • Marcus S.E.
      • Haeger A.
      • Verhertbruggen Y.
      • Verhoef R.
      • Schols H.
      • Ulvskov P.
      • Mikkelsen J.D.
      • Knox J.P.
      • Willats W.
      High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles.
      ,
      • Yates E.A.
      • Valdor J.F.
      • Haslam S.M.
      • Morris H.R.
      • Dell A.
      • Mackie W.
      • Knox J.P.
      Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies.
      ). The oligosaccharide microarrays demonstrated that both mAbs bind with high specificity to glucoronyl-(1→2)-α-(1→4)-β-d-xylotriose (structure 42) (Fig. 3G), which is a constituent of glucoronoxylan and glucoronoarabinoxylans, and this finding is therefore interesting because it implies that these two mAbs may bind to an epitope not usually associated with AGPs in addition to binding to glucoronyl residues decorating arabinogalactan structures. The synthetic galactosyl structures 20, 21, and 22 are not known to occur on any plant cell wall polysaccharide, and it was therefore surprising that mAb LM16 bound strongly to 6′-β-d-galactosyl-(1→4)-β-d-galactotriose (structure 21). LM16 has been described previously as binding to an epitope occurring on sugar beet RGI that is generated by arabinofuranosidase treatment and is galactosidase-labile (
      • Verhertbruggen Y.
      • Marcus S.E.
      • Haeger A.
      • Verhoef R.
      • Schols H.A.
      • McCleary B.V.
      • McKee L.
      • Gilbert H.J.
      • Knox J.P.
      Developmental complexity of arabinan polysaccharides and their processing in plant cell walls.
      ). Sugar beet arabinan side chains can in some cases be attached to RGI backbones via short galactosyl motifs, and it is possible that LM16 recognizes this structure once exposed by arabinofuranosidase (
      • Verhertbruggen Y.
      • Marcus S.E.
      • Haeger A.
      • Verhoef R.
      • Schols H.A.
      • McCleary B.V.
      • McKee L.
      • Gilbert H.J.
      • Knox J.P.
      Developmental complexity of arabinan polysaccharides and their processing in plant cell walls.
      ). It has been shown that such short galactan stubs can be substituted with ferulic acid at the C6 position, but substitution with another sugar has not been reported (
      • Ralet M.C.
      • André-Leroux G.
      • Quéméner B.
      • Thibault J.F.
      Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall.
      ). LM16 does not bind to galactosyl residues per se because it does not bind to linear galactan, galactomannan, or galactoxyloglucans. The strong binding of LM16 to both structure 21 and native sugar beet pectin therefore raises the intriguing possibility of a novel RGI epitope.
      The importance of obtaining detailed information about epitope structures was clearly illustrated by the anti-xyloglucan mAbs LM15, LM24, and LM25. Oligosaccharide array analysis revealed subtle differences in the binding profiles of these mAbs, which most likely would not have been discriminated using previous polysaccharide-based ELISA or microarrays. Immunolabeling of tobacco sections with these mAbs showed that despite their relatively small differences in structure, the epitopes recognized had distinct cellular locations. Although the biological significance of these findings is unclear at present, they show that a detailed evaluation of epitope structures that oligosaccharide arrays can provide is important for the subsequent interpretation of data produced in antibody studies.

      Acknowledgments

      We thank Mohammed Saddik Motawie for oligosaccharide 72.

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