Cell Wall Glycans Research Articles

Immunoprofiling reveals unique cell‐specific patterns of wall epitopes in the expanding Arabidopsis stem

The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

Analyses of Cell Wall Glycans Using Glycome Profiling in Two Commercially Important Lignocellulosic Fiber Raw Materials

Lignocellulosic fibers are plant-based bio-fibers that are sourced from terrestrial and non-terrestrial plants that also include agricultural by-products. In textile industry, being a renewable and sustainable resource, these fibers have attained market potential with their special qualities including biodegradability, applicability in green chemistry, ecofriendly properties and cost effectiveness in processing. Lignocellulosic fibers mainly comprise plant cell walls that are essentially composed of cellulose, lignin, pectin, hemicelluloses and waxes. Numerous studies have been conducted to explain the commercial performance, mechanical properties and sustainability of these lignocellulosic fibers. Although several work have been conducted on the cell wall compositional aspects of these fibers, majority of the studies have been focused on understanding the cellulose and lignin constituents. Hence, there is an increasing need for more studies to be conducted on non-cellulosic cell wall components in these fibers. In this study, we performed a novel immunological approach namely glycome profiling on two commercially important lignocellulosic fibers from aquatic and terrestrial plants namely duckweed and hemp, respectively. Our studies demonstrated differences in the extractability of cell wall glycans from biomass materials from these plants. Duckweed biomass had significantly higher abundance of extractable pectins in their walls. However, cell walls from biomass raw materials of terrestrial plant, hemp had lesser amounts of pectin with significantly higher amounts of xylans that are easily extractable. Duckweed fibers had significantly higher proportion of lignin-associated cell wall glycans in comparison to that of hemp. These results demonstrate that lignocellulosic fiber raw materials from varied plant sources are different in non-cellulosic cell wall glycan compositions and comprehending these variations could potentially be instrumental in commercial processing of these fibers.

Characterization of the recognition of Candida species by mannose-binding lectin using surface plasmon resonance

The interaction of mannose-binding lectins (MBLs) with Candida albicans has been analyzed previously by microscopy and flow cytometry. We recently demonstrated that serum MBL levels vary during infection with Candida spp. and that serum MBLs are capable of interacting with yeast cell wall components. The aim of this study was to use, for the first time, surface plasmon resonance (SPR) technology to characterize the interaction between living label-free yeasts and non-mutated MBL purified from human serum. Our preliminary results demonstrate the robustness of this tool, which revealed specific and differential reactivities between the principal Candida spp. of medical interest. This model offers new perspectives as a tool for the characterization of yeast strains carrying mutations in gene coding for the mannosylation of fungal cell wall glycans and will enable better characterization of the interactions between C-lectins and glycan motifs expressed on the surface of yeasts.

A survey of the natural variation in biomechanical and cell wall properties in inflorescence stems reveals new insights into the utility of Arabidopsis as a wood model

The natural trait variation in Arabidopsis thaliana (L.) Heynh. accessions is an important resource for understanding many biological processes but it is underexploited for wood-related properties. Twelve A. thaliana accessions from diverse geographical locations were examined for variation in secondary growth, biomechanical properties, cell wall glycan content, cellulose microfibril angle (MFA) and flowering time. The effect of daylength was also examined. Secondary growth in rosette and inflorescence stems was observed in all accessions. Organised cellulose microfibrils in inflorescence stems were found in plants grown under long and short days. A substantial range of phenotypic variation was found in biochemical and wood-related biophysical characteristics, particularly for tensile strength, tensile stiffness, MFA and some cell wall components. The four monosaccharides galactose, arabinose, rhamnose and fucose strongly correlated with each other as well as with tensile strength and MFA, consistent with mutations in arabinogalactan protein and fucosyl- and xyloglucan galactosyl-transferase genes that result in decreases in strength. Conversely, these variables showed negative correlations with lignin content. Our data support the notion that large-scale natural variation studies of wood-related biomechanical and biochemical properties of inflorescence stems will be useful for the identification of novel genes important for wood formation and quality, and therefore biomaterial and renewable biofuel production.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)(1,2). However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall(3). Furthermore, their composition is also modified during development(1), or in response to environmental cues(4). In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis(5). However, relatively few of the biosynthetic genes have been functionally characterized (4,5). Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types(6). Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained(7). Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)(8,9) have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously(9,10,11). Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)(10,11) that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems(12) and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Preventing Ralstonia solanacearum adhesion with glycans from cashew, cocoa, coffee, pumpkin, and tomato seed extract

Ralstonia solanacearum wilts many plants, causing heavy agricultural losses. Its pathogenic strain ATCC 11696 produces 2 hemagglutinating lectins: RSL and RS-IIL. These lectins may bind to terminal l-fucose-, d-arabinose-, and d-mannose-bearing seedling xylem cell wall glycans, thus enabling pathogen adhesion to them, with devastating infection establishment. Blocking the active sites of these lectins with seed embryo-surrounding oligo- and poly-saccharides hampers binding of the lectins to the embryos. The current study shows that seeds of cashew, cocoa, coffee, pumpkin, and tomato contain low and high molecular mass glycans that block RSL and RS-IIL (like its homologous Pseudomonas aeruginosa PA-IIL lectin). The blocking of the pathogen lectins, which is attributable to the documented composition of the oligo- and poly-saccharides of these seeds, is similar to that observed with animal glycoproteins of avian egg whites (protecting their embryos from infections) and of milk and royal jelly, which likewise protect mammal and bee neonates, respectively. RSL was most strongly inhibited by cashew seed glycans, and RS-IIL by coffee seed glycans. Western blot analyses with these lectins instead of antibodies revealed the hitherto undescribed presence of lectin-binding glycoproteins in the coffee, pumpkin, tomato, and cashew (but not cocoa) seeds. The use of these lectins for unveiling potent embryo-protecting seed glycans might be helpful for seedling-bioprotection projects similar to those planned for animal protection against antibiotic-resistant infections.

Current Challenges in Plant Cell Walls: Editorial Overview

composition of the plant cell wall. Here, powerful MS methods are detailed for the elucidation of the structure of oligosaccharides derived from hemicelluloses and pectins. The author illustrates how information on sequence, linkages, branching, and modifications can be obtained from characteristic fragmentation patterns. This method is of further note when combined with molecular genetics (mutant analysis) to elucidate the function of a target gene.Global transcript analyses based on a wealth of available micro-array datasets have revealed that genes that are in the same pathway are often transcribed in a coordinate manner. Here Ruprecht and Persson (2012) have summarized recent developments in software as well as combining transcriptional data from multiple plant spe-cies to learn about how particular cell wall processes are different, rather than the same. This exciting update provides successful examples of where expression profiling has helped to identify genes involved in the formation of cellulose, hemicelluloses, and lignin. In addition, they illustrate potential pitfalls and future perspectives. The broad employment of next generation sequencing for RNAseq experiments will surely expand the available data (as not all coding sequences are represented on a given microarray chip). This will require some software refinement, but also see further gains result-ing from coexpression profiling in the near future. The use of deep sequencing platforms like Illumina’s Genome Analyzer or ABI’s SOLiD was reviewed by Vidaurre and Bonetta (2012) in the context of map based cloning. Here, a path to rapidly accelerating forward genetics in the context of cell wall biosynthesis and deconstruc-tion was paved. Anyone who has laboriously mapped a missense mutation will greatly appreciate the ability to pinpoint a mutation selected from within an EMS (ethyl methyl suflonate) population in a single sequencing run. This combination of a classical genet-ics approach and cutting edge technology can holds immediate promise for gene identification.To assemble plant cell wall polysaccharides and glycan structures on glycoproteins, the plant needs extensive biosynthetic machin-ery, and it has been estimated that over 2000 gene products are involved in making and maintaining the wall. The Carbohydrate Active Enzyme (CAZy) database is an invaluable resource for glyco-biology and currently contains 45 glycosyltransferase (GT) families that are represented in plants. Hansen et al. (2012) describe the putative GTs that are not currently classified in the CAZy database. These families include proteins with domain of unknown func-tion (DUF). The evidence for certain members of the DUF class proteins being GTs and their possible roles in cell wall biosynthesis are discussed.

Combining polysaccharide biosynthesis and transport in a single enzyme: dual-function cell wall glycan synthases

Extracellular polysaccharides are synthesized by a wide variety of species, from unicellular bacteria and Archaea to the largest multicellular plants and animals in the biosphere. In every case, the biosynthesis of these polymers requires transport across a membrane, from the cytosol to either the lumen of secretory pathway organelles or directly into the extracellular space. Although some polysaccharide biosynthetic substrates are moved across the membrane to sites of polysaccharide synthesis by separate transporter proteins before being incorporated into polymers by glycosyltransferase proteins, many polysaccharide biosynthetic enzymes appear to have both transporter and transferase activities. In these cases, the biosynthetic enzymes utilize substrate on one side of the membrane and deposit the polymer product on the other side. This review discusses structural characteristics of plant cell wall glycan synthases that couple synthesis with transport, drawing on what is known about such dual-function enzymes in other species.

Recognition and Degradation of Plant Cell Wall Polysaccharides by Two Human Gut Symbionts

Author SummaryBacteria inhabiting the human gut are critical for digestion of the plant-derived glycans that compose dietary fiber. Enzymes produced by the human body cannot degrade these abundant dietary components, and without bacterial assistance they would go unused. We investigated the molecular strategies employed by two species belonging to one of the most abundant bacterial groups in the human colon (the Bacteroidetes). Our results show that each species has evolved to degrade a unique subset of glycans; this specialization is reflected in their respective genomes, each of which contains numerous separate gene clusters involved in metabolizing plant fiber polysaccharides or glycans present in secreted mucus. Each glycan-specific gene cluster produces a related series of membrane-associated proteins which together serve to bind and degrade a specific glycan. Expression of each glycan-specific gene cluster is controlled by an environmental sensor that responds to the presence of a unique molecular signature contained in the substrate that it targets. These results provide a view of how related bacterial species have diverged into different carbohydrate niches by evolving genes that sense and degrade unique suites of available polysaccharides, a process that likely applies to disparate bacteria from the gut and other habitats.

Biosynthesis of non-cellulosic polysaccharides of plant cell walls

Enzymes that make the polymer backbones of plant cell wall polysaccharides have proven to be recalcitrant to biochemical purification. Availability of mutational genetics and genomic tools paved the way for rapid progress in identifying genes encoding various cell wall glycan synthases. Mutational genetics, the primary tool used in unraveling cellulose biosynthesis, was ineffective in assigning function to any of the hemicellulosic, polymerizing glycan synthases. A combination of comparative genomics and functional expression in a heterologous system allowed identification of various cellulose synthase-like (Csl) sequences as being involved in the formation of β-1,4-mannan, β-1,4-glucan, and mixed-linked glucan. A number of xylose-deficient mutants have led to a variety of genes, none of which thus far possesses the motifs known to be conserved among polymerizing β-glycan synthases. Except for xylan synthase, which appears to be an agglomerate of proteins just like cellulose synthase, Golgi glycan synthases already identified suggest that the catalytic polypeptide by itself is sufficient for enzyme activity, most likely as a homodimer. Several of the Csl genes remain to be assigned a function. The possibility of the involvement of various Csl genes in making more than one product remains.

Down-regulation of UDP-glucuronic Acid Biosynthesis Leads to Swollen Plant Cell Walls and Severe Developmental Defects Associated with Changes in Pectic Polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

Linkage Specificity and Role of Properdin in Activation of the Alternative Complement Pathway by Fungal Glycans

ABSTRACTFungal cell walls are predominantly composed of glucans, mannans, and chitin. Recognition of these glycans by the innate immune system is a critical component of host defenses against the mycoses. Complement, an important arm of innate immunity, plays a significant role in fungal pathogenesis, especially the alternative pathway (AP). Here we determine that the glycan monosaccharide composition and glycosidic linkages affect AP activation and C3 deposition. Furthermore, properdin, a positive regulator of the AP, contributes to these functions. AP activation by glycan particles that varied in composition and linkage was measured by C3a generation in serum treated with 10 mM EGTA and 10 mM Mg2+ (Mg-EGTA-treated serum) (AP specific; properdin functional) or Mg-EGTA-treated serum that lacked functional properdin. Particles that contained either β1→3 or β1→6 glucans or both generated large and similar amounts of C3a when the AP was intact. Blocking properdin function resulted in 5- to 10-fold-less C3a production by particulate β1→3 glucans. However, particulate β1→6 glucans generated C3a via the AP only in the presence of intact properdin. Interestingly, zymosan and glucan-mannan particles (GMP), which contain both β-glucans and mannans, also required properdin to generate C3a. The β1→4 glycans chitin and chitosan minimally activated C3 even when properdin was functional. Finally, properdin binding to glucan particles (GP) and zymosan in serum required active C3. Properdin colocalized with bound C3, suggesting that in the presence of serum, properdin bound indirectly to glycans through C3 convertases. These findings provide a better understanding of how properdin facilitates AP activation by fungi through interaction with the cell wall components.

Developing a vaccine against aspergillosis

Although there is considerable experimental data to support the idea, bringing a fungal vaccine to fruition has been elusive. Moreover, vaccinating the immunocompromised, susceptible to an opportunistic disease such as invasive aspergillosis, seems formidable. However, pioneering studies using Aspergillus particulate forms or homogenates, and recently, recombinant proteins, have demonstrated feasibility. Moreover, T cell receptors also recognize glycotopes if presented in the appropriate MHC-binding context. The potential role of induced antibody has been appreciated only recently. Recent studies in our laboratory with heat-killed Saccharomyces (HKY) have raised the possibility of development of a panfungal vaccine. This yeast may be nature's experimental reagent, to show the way to a protective protein-carbohydrate conjugate vaccine. Subcutaneous HKY is an effective vaccine against Aspergillus, Coccidioides or Candida challenge. We have learned the protective moiety is in the cell wall, and proteins, glucan and lipid all seem important. We have also found the cell wall glycans alone, mannan or glucan, as a vaccine each provide significant protection. This leads to consideration of the importance of glycosylated proteins and glycan polymer-protein conjugates in vaccine development. We think the most productive route to a fungal-specific vaccine may be a conjugate vaccine that combines the optimally configured glycan with a specific immunogenic protein. Our work so far suggests that some proteins may be sufficiently cross-immunogenic, such that combined with the appropriate glycan, it may be possible to develop a pan-fungal vaccine.

A Comprehensive Toolkit of Plant Cell Wall Glycan-Directed Monoclonal Antibodies

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

Recent advances in mycobacterial cell wall glycan biosynthesis

The cell wall of mycobacteria, including the causative agents of the human diseases tuberculosis (Mycobacterium tuberculosis) and leprosy (M. leprae), is composed of an array of carbohydrate-containing molecules. These glycoconjugates are assembled by glycosyltransferases (GTs) that work in tandem through pathways that are only now beginning to be fully understood. Given the essentiality of cell wall glycans to mycobacterial viability, these enzymes represent novel targets for drug action. Summarized here are recent genetic and biochemical studies leading to the identification and characterization of mycobacterial GTs.

Novel Gal f-disaccharide mimics: synthesis by way of 1,3-dipolar cycloaddition reactions in water

Galactofuranose-disaccharide analogues incorporating a 1,4-dideoxy-1,4-imino- d-galactitol moiety were obtained by 1,3-dipolar cycloadditions of a galacto-configured cyclic nitrone with arabino- or galacto-furanosides carrying a C-vinyl or O-allyl substituent. Remarkably, the cycloadditions could be performed highly efficiently and stereoselectively in water using unprotected nitrone and sugar-derived dipolarophile as reaction partners. The resulting pseudo-disaccharides are analogues of the Gal f-Gal f motifs found in mycobacterial cell-wall glycans and therefore constitute mimics of the acceptor substrates of mycobacterial Gal f-transferases.

A signal transduction system in Streptomyces coelicolor that activates expression of a putative cell wall glycan operon in response to vancomycin and other cell wall‐specific antibiotics

This article was published in Mol Microbiol (2002) 44(5), 1199–1211. The authors wish to make the following correction. The panel labelled “ΔsigE” in Fig. 3 (B) was in error, and is in fact the data from a ΔcseA mutant, correctly published as such in Hutchings et al. (2006), and the ΔsigE panel has therefore never been published. To clarify, the complete data set, originally split between the two papers, is reproduced here. Figure. Induction of the sigE promoter (sigEp) in its native chromosomal context by vancomycin. S1 nuclease protection analysis of the sigE transcript in S. coelicolor M600 (wt), J2130 (ΔsigE), J2172 (ΔcseA), and J2142 (ΔcseB::hyg) grown to mid-late exponential phase in NMMP liquid medium containing 2 mM MgCl2 and exposed to 10 µg/ml vancomycin. RNA was extracted from samples taken immediately prior to the addition of vancomycin and at subsequent 30 min intervals. Relative signal strengths were quantified on a Phosphorimager; each signal is expressed as a percentage of the strongest signal.

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles.

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.

High‐throughput mapping of cell‐wall polymers within and between plants using novel microarrays

We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from multiple organs or tissues with the generation of microarrays, which are probed with monoclonal antibodies (mAbs) or carbohydrate-binding modules (CBMs) with specificities for cell-wall components. The profiles generated provide a global snapshot of cell-wall composition, and also allow comparative analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant cell walls, and demonstrate the potential of CoMPP as a component of systems-based approaches to cell-wall biology.

High-throughput microarray analysis of pectic polymers by enzymatic epitope deletion

Pectic polysaccharides are abundant plant cell wall glycan polymers and are also widely used as functional food ingredients. The fine structure of pectins, and especially the degree and pattern of methyl-esterification of the homogalacturonan (HG) backbone, play a major part in determining functional properties. We have developed a high-throughput method of analysing pectic polymers by combining microarray-technology, pectin lyase (PL) and anti-HG monoclonal antibodies (mAbs). The technique demonstrates that pectins immobilised in a microarray format are effective substrates for PL, and that epitope loss or degradation can be tracked by the deletion of mAb binding.