Laura von Schantz

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

Background: Microarrays of plant-derived oligosaccharides are potentially powerful tools for the high throughput discovery and screening of antibodies, enzymes, and carbohydrate-binding proteins. Results: Oligosaccharide microarrays were produced, and their utility was demonstrated in several applications. Conclusion: A new generation of oligosaccharide microarrays will make an important contribution to plant glycomic research. Significance: High throughput screening technology enables the more effective production of carbohydrate active enzymes and molecular probes. 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.

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

BackgroundMolecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.ResultsTwo modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections.ConclusionWe conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

Structural Basis for Carbohydrate-Binding Specificity--A Comparative Assessment of Two Engineered Carbohydrate-Binding Modules.

Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and β-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.

The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections.

Neutron crystallographic studies reveal hydrogen bond and water-mediated interactions between a carbohydrate-binding module and its bound carbohydrate ligand.

Carbohydrate-binding modules (CBMs) are key components of many carbohydrate-modifying enzymes. CBMs affect the activity of these enzymes by modulating bonding and catalysis. To further characterize and study CBM-ligand binding interactions, neutron crystallographic studies of an engineered family 4-type CBM in complex with a branched xyloglucan ligand were conducted. The first neutron crystal structure of a CBM-ligand complex reported here shows numerous atomic details of hydrogen bonding and water-mediated interactions and reveals the charged state of key binding cleft amino acid side chains.

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH‐Π interactions

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands. (Less)Engineering of novel carbohydrate‐binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein‐carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi‐specific carbohydrate‐binding module (CBM) X‐2 L110F bound to a branched oligomer of xyloglucan (XXXG). X‐2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β‐glucans. The structural observations of the present study compared with previously reported structures of X‐2 L110F in complex with linear oligomers, show that the π‐surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X‐2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH‐π interactions can influence binding specificity and that flexibility is a key feature for the multi‐specificity displayed by X‐2 L110F, allowing for the accommodation of branched ligands. Proteins 2014; 82:3466–3475.

Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules

Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses.

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

BackgroundDerivatized celluloses, such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), are of pharmaceutical importance and extensively employed in tablet matrices. Each batch of derivatized cellulose is thoroughly characterized before utilized in tablet formulations as batch-to-batch differences can affect drug release. The substitution pattern of the derivatized cellulose polymers, i.e. the mode on which the substituent groups are dispersed along the cellulose backbone, can vary from batch-to-batch and is a factor that can influence drug release.ResultsIn the present study an analytical approach for the characterization of the substitution pattern of derivatized celluloses is presented, which is based on the use of carbohydrate-binding modules (CBMs) and affinity electrophoresis. CBM4-2 from Rhodothermus marinus xylanase 10A is capable of distinguishing between batches of derivatized cellulose with different substitution patterns. This is demonstrated by a higher migration retardation of the CBM in acrylamide gels containing batches of MC and HPMC with a more heterogeneous distribution pattern.ConclusionsWe conclude that CBMs have the potential to characterize the substitution pattern of cellulose derivatives and anticipate that with use of CBMs with a very selective recognition capacity it will be possible to more extensively characterize and standardize important carbohydrates used for instance in tablet formulation.

Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase.

Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate-protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A of Rhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm(3)) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement using phenix.refine showed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein-carbohydrate binding interactions.

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Π interactions: Xyloglucan Binding in Carbohydrate Binding Modules

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands. (Less)Engineering of novel carbohydrate‐binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein‐carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi‐specific carbohydrate‐binding module (CBM) X‐2 L110F bound to a branched oligomer of xyloglucan (XXXG). X‐2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β‐glucans. The structural observations of the present study compared with previously reported structures of X‐2 L110F in complex with linear oligomers, show that the π‐surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X‐2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH‐π interactions can influence binding specificity and that flexibility is a key feature for the multi‐specificity displayed by X‐2 L110F, allowing for the accommodation of branched ligands. Proteins 2014; 82:3466–3475.