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Mats Ohlin - One of the best experts on this subject based on the ideXlab platform.
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Novel xylan-binding properties of an engineered family 4 Carbohydrate-Binding Module
The Biochemical journal, 2007Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Eva Nordberg Karlsson, Harry J. Gilbert, Richard B Tunnicliffe, Mike P Williamson, Mats OhlinAbstract:Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (Carbohydrate-Binding Modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered Module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding Module with different binding properties to those displayed by CBMs available in Nature.
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Novel xylan-binding properties of an engineered family 4 Carbohydrate-Binding Module
Biochemical Journal, 2007Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Harry J. Gilbert, Richard B Tunnicliffe, Mike P Williamson, Eva Nordberg Karlsson, Mats OhlinAbstract:Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into Carbohydrate-Binding Modules has not been extensively explored. Here, we report the engineering of a Carbohydrate-Binding Module, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared to the wild type protein, this engineered Module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylooligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific Carbohydrate-Binding Modules in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding Module with different binding properties to those displayed by CBMs available in Nature.
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Evolution of a carbohydrate binding Module into a protein-specific binder.
Biomolecular engineering, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Eva Nordberg Karlsson, Olle Holst, Linda Dexlin, Mats OhlinAbstract:A carbohydrate binding Module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.
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Engineered xyloglucan specificity in a Carbohydrate-Binding Module
Glycobiology, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Harry Brumer, Eva Nordberg Karlsson, Qi Zhou, Mats OhlinAbstract:The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the Carbohydrate-Binding Module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding Modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.
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Molecular engineering of a thermostable Carbohydrate-Binding Module
Biocatalysis and Biotransformation, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Eva Nordberg Karlsson, Mats Andersson, Olle Holst, Mats OhlinAbstract:Structure-function studies are frequently practiced on the very diverse group of natural Carbohydrate-Binding Modules in order to understand the target recognition of these proteins. We have taken a step further in the study of Carbohydrate-Binding Modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable Carbohydrate-Binding Module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities. (Less)
Kazuo Sakka - One of the best experts on this subject based on the ideXlab platform.
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A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides
Applied Microbiology and Biotechnology, 2020Co-Authors: Puangpen Limsakul, Makiko Sakka, Kazuo Sakka, Paripok Phitsuwan, Rattiya Waeonukul, Patthra Pason, Chakrit Tachaapaikoon, Kanokwan Poomputsa, Akihiko Kosugi, Khanok RatanakhanokchaiAbstract:Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 Carbohydrate-Binding Module, two fibronectin type-3 domains, and a family 3 Carbohydrate-Binding Module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli . Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite − 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. Key points • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus . • PcAA10A showed broad substrate specificity on β-1,4 glycosidic linkage substrates . • Non-catalytic domains are important for degrading complex polysaccharides . • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation .
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Function of a laminin_G_3 Module as a Carbohydrate-Binding Module in an arabinofuranosidase from Ruminiclostridium josui.
FEBS letters, 2018Co-Authors: Makiko Sakka, Tetsuya Kimura, Emi Kunitake, Kazuo SakkaAbstract:Laminin_G_3 Modules can exist together with family-43 catalytic Modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 Module and a GH43 Module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A-Abf43B-Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43-1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 32 -α-l-arabinofuranosyl-(1,5)-α-l-arabinotriose but not for α-1,5-linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α-1,5-linked arabinooligosaccharides. RjGH43-1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 Module in RjGH43-1 plays an important role in substrate hydrolysis as a Carbohydrate-Binding Module.
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Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled Carbohydrate-Binding Module.
Journal of microbiological methods, 2015Co-Authors: Yoshihiro Ojima, Asep Suparman, Minh Hong Nguyen, Makiko Sakka, Kazuo Sakka, Masahito TayaAbstract:A fused protein composed of a Carbohydrate-Binding Module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs.
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Probing of exopolysaccharides with green fluorescence protein-labeled Carbohydrate-Binding Module in Escherichia coli biofilms and flocs induced by bcsB overexpression
Journal of Bioscience and Bioengineering, 2014Co-Authors: Minh Hong Nguyen, Yoshihiro Ojima, Makiko Sakka, Kazuo Sakka, Masahito TayaAbstract:Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a Carbohydrate-Binding Module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.
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Essential role of a family-32 Carbohydrate-Binding Module in substrate recognition by Clostridium thermocellum mannanase CtMan5A.
FEBS letters, 2014Co-Authors: Kimiya Mizutani, Makiko Sakka, Tetsuya Kimura, Kazuo SakkaAbstract:The family-5 glycoside hydrolase domain (GH5) and the family-32 Carbohydrate-Binding Module (CBM32) of Clostridium thermocellum mannanase CtMan5A, along with their genetically inactivated derivatives, were collectively or separately expressed. Their catalytic and substrate-binding abilities were measured to investigate importance of CBM32 in substrate recognition by CtMan5A. Characterization of the truncated derivatives of CtMan5A and isothermal calorimetry analysis of the interaction between the inactivated proteins and mannooligosaccharides suggested that GH5 and CBM32 collectively formed a substrate-binding site capable of accommodating a mannotetraose unit in CtMan5A. This suggested that CBM32 directly participated in the substrate recognition required for catalytic action.
Alisdair B. Boraston - One of the best experts on this subject based on the ideXlab platform.
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An unusual mode of galactose recognition by a family 32 Carbohydrate-Binding Module.
Journal of molecular biology, 2013Co-Authors: Julie M. Grondin, Alisdair B. Boraston, Elizabeth Ficko-blean, Seth Chitayat, S. Houliston, Cheryl H. Arrowsmith, Steven P. SmithAbstract:Carbohydrate-Binding Modules (CBMs) are ancillary Modules commonly associated with carbohydrate-active enzymes (CAZymes) that function to mediate the adherence of the parent enzyme to its carbohydrate substrates. CBM family 32 (CBM32) is one of the most diverse CBM families, whose members are commonly found in bacterial CAZymes that modify eukaryotic glycans. One such example is the putative μ-toxin, CpGH84A, of the family 84 glycoside hydrolases, which comprises an N-terminal putative β-N-acetylglucosaminidase catalytic Module and four tandem CBM32s. Here, we report a unique mode of galactose recognition by the first CBM32, CBM32-1 from CpGH84A. Solution NMR-based analyses of CpGH84A CBM32-1 indicate a divergent subset of residues, located in ordered loops at the apex of the CBM, conferring specificity for the galacto-configured sugars galactose, GalNAc, and LacNAc that differs from those of the canonical galactose-binding CBM32s. This study showcases the impressive variability in ligand binding by this CBM family and offers insight into the growing role of these Modules in the interaction of CAZymes with eukaryotic glycans.
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Quantitative approaches to the analysis of Carbohydrate-Binding Module function.
Methods in enzymology, 2012Co-Authors: D. Wade Abbott, Alisdair B. BorastonAbstract:Abstract Carbohydrate-Binding Modules (CBMs) are important components of carbohydrate-active enzymes. Their primary functions are to assist in substrate turnover by targeting appended catalytic Modules to substrate and concentrating appended catalytic Modules on the surface of substrate. Presented here are four well-established methodologies for investigating and quantifying the CBM–polysaccharide binding relationship. These methods include: (1) the solid state depletion assay, (2) affinity gel electrophoresis, (3) UV difference and fluorescence spectroscopy, and (4) isothermal titration calorimetry. In addition, entropy-driven CBM–crystalline cellulose binding events and differential approaches to calculating stoichiometry with polyvalent polysaccharide ligands are also discussed.
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restricted access of proteins to mannan polysaccharides in intact plant cell walls
Plant Journal, 2010Co-Authors: Susan E. Marcus, Anthony W Blake, Thomas A S Benians, Kieran J D Lee, Callum Poyser, Lloyd Donaldson, Olivier Leroux, Artur Rogowski, Henriette L Petersen, Alisdair B. BorastonAbstract:How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a Carbohydrate-Binding Module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific Carbohydrate-Binding Module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.
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N-Glycosidase–Carbohydrate-Binding Module fusion proteins as immobilized enzymes for protein deglycosylation
Protein engineering design & selection : PEDS, 2005Co-Authors: Emily Kwan, Douglas G. Kilburn, Alisdair B. Boraston, Bradley W. Mclean, R. Antony J. WarrenAbstract:A Carbohydrate-Binding Module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-b-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM–EndoF1 and CBM–PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after productionbyEscherichiacoli.CBM–EndoF1isaseffective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM–PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for atleast1monthat4 � C.Thehexahistidinetagcanberemoved
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Structural and thermodynamic dissection of specific mannan recognition by a carbohydrate binding Module, TmCBM27
Structure, 2003Co-Authors: Alisdair B. Boraston, Timothy J. Revett, Catherine M Boraston, Didier Nurizzo, Gideon J. DaviesAbstract:The C-terminal 176 amino acids of a Thermotoga maritima mannanase (Man5) constitute a carbohydrate binding Module (CBM) that has been classified into CBM family 27. The isolated CBM27 domain, named TmCBM27, binds tightly (K as 105-106 M-1) to β-1, 4-mannooligosaccharides, carob galactomannan, and konjac glucomannan, but not to cellulose (insoluble and soluble) or soluble birchwood xylan. The X-ray crystal structures of native TmCBM27, a TmCBM27-mannohexaose complex, and a TmCBM27-63,64-α-D-galactosyl-mannopentaose complex at 2.0 Å, 1.6 Å, and 1.35 Å, respectively, reveal the basis of TmCBM27's specificity for mannans. In particular, the latter complex, which is the first structure of a CBM in complex with a branched plant cell wall polysaccharide, illustrates how the architecture of the binding site can influence the recognition of naturally substituted polysaccharides.
Harry J. Gilbert - One of the best experts on this subject based on the ideXlab platform.
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a novel carbohydrate binding Module from sugar cane soil metagenome featuring unique structural and carbohydrate affinity properties
Journal of Biological Chemistry, 2016Co-Authors: B M Campos, Harry J. Gilbert, Igor Polikarpov, M V Liberato, Thabata M Alvarez, Leticia Maria Zanphorlin, Gabriela C G Ematsu, Hernane Barud, Roberto Ruller, Ana Carolina De Mattos ZeriAbstract:Abstract Carbohydrate-Binding Modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, discovered through a metagenomics approach, which is the founding member of a novel CBM family, CBMxx. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked beta-1,4-beta-1,3-glucans, xyloglucan, avicel and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical beta-sandwich fold comprising two beta sheets. The planar ligand binding site, observed in a parallel orientation with the beta strands, is a typical feature of Type A CBMs. On the other hand, affinity for bacterial crystalline cellulose was not detected, a ligand recognized by Type A CBMs, while binding to soluble glucans was enthalpically driven, typical of Type B Modules. These unique properties of CBM_E1 are at the interface between Type A and Type B CBMs.
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Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a Carbohydrate-Binding Module.
FEBS letters, 2015Co-Authors: Mercedes C. Hernandez-gomez, Artur Rogowski, Carlos M. G. A. Fontes, Aurore Labourel, Maja G. Rydahl, Carl Morland, Alan Cartmell, Lucy I. Crouch, William G. T. Willats, Harry J. GilbertAbstract:Type A non-catalytic Carbohydrate-Binding Modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan (XG), a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed.
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Carbohydrate-Binding Module assisting glycosynthase-catalysed polymerizations
The Biochemical journal, 2015Co-Authors: Victoria Codera, Harry J. Gilbert, Magda Faijes, Antoni PlanasAbstract:Carbohydrate-Binding Modules (CBMs) are found within multi-modular polysaccharide degrading enzymes [glycoside hydrolases (GHs)]. CBMs play a critical role in the recognition of plant cell-wall polysaccharides and enhance the hydrolase activity of their cognate catalytic domains by increasing enzyme substrate proximity. Mimicking their role in Nature, we, in the present study, propose that CBMs may assist in vitro glycosynthase-catalysed polymerization reactions to produce artificial polysaccharides. Glycosynthases are GHs that have been engineered to catalyse glycoside bond formation for the synthesis of oligosaccharides, glycoconjugates and glycans. The degree of polymerization (DP) of the glycans generated is limited by the solubility of the polymeric product. In the present study, we have targeted the synthesis of artificial 1,3-1,4-β-glucans with a regular sequence using the glycosynthase E(134)S derived from a Bacillus licheniformis lichenase. We show that the addition of CBM11, which binds mixed-linked β-glucans, either as an isolated protein or fused to the glycosynthase E(134)S, has an effect on the DP of the polysaccharide products that is dependent on the rate of polymerization. The mechanism by which CBM influences the DP of the synthesized glycans is discussed.
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Overproduction, purification, crystallization and preliminary X-ray characterization of the C-terminal family 65 Carbohydrate-Binding Module (CBM65B) of endoglucanase Cel5A from Eubacterium cellulosolvens.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2013Co-Authors: I. Venditto, Arnaud Baslé, A.s. Luis, Max J. Temple, Luís M. A. Ferreira, Carlos M. G. A. Fontes, Harry J. Gilbert, Shabir NajmudinAbstract:The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated Carbohydrate-Binding Module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second Carbohydrate-Binding Module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P65 and tetragonal space group P43212, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.
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Novel xylan-binding properties of an engineered family 4 Carbohydrate-Binding Module
The Biochemical journal, 2007Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Eva Nordberg Karlsson, Harry J. Gilbert, Richard B Tunnicliffe, Mike P Williamson, Mats OhlinAbstract:Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (Carbohydrate-Binding Modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered Module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding Module with different binding properties to those displayed by CBMs available in Nature.
Lavinia Cicortas Gunnarsson - One of the best experts on this subject based on the ideXlab platform.
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Novel xylan-binding properties of an engineered family 4 Carbohydrate-Binding Module
The Biochemical journal, 2007Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Eva Nordberg Karlsson, Harry J. Gilbert, Richard B Tunnicliffe, Mike P Williamson, Mats OhlinAbstract:Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (Carbohydrate-Binding Modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered Module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding Module with different binding properties to those displayed by CBMs available in Nature.
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Novel binding specificities engineered into the scaffold of a carbohydrate binding Module
2007Co-Authors: Lavinia Cicortas GunnarssonAbstract:The growing field of biotechnology is in constant need of proteins that can function as recognition tools for separational, analytical and therapeutic purposes. Different molecular engineering approaches are applied on natural proteins in order to create variants with desired properties. This thesis is based on five original papers that deal with selection, characterisation and application of novel binding specificities engineered into the scaffold of a carbohydrate binding Module that originates from a xylanase in the thermophilic bacterium Rhodothermus marinus. Molecular evolution studies on this scaffold allowed for the generation of variants that bind specifically to the carbohydrate targets xylan, Avicel?, mannan and xyloglucan. In addition, the scaffold employed in this work was also able to adopt specific protein recognition to a human IgG4 molecule. Apart from high binding specificities, the engineered proteins have additional properties such as high thermal stability and ease of production in Escherichia coli, which are advantageous in most applications. One of the papers in this thesis demonstrates the potential use of the created xylan-binding variants for detection of their target in wood fibres and plant sections. Also, generated variants with other binding specificities have the potential to find similar use as bioanalytical tools. In conclusion, the scaffold of the carbohydrate binding Module engaged in the engineering studies of this thesis proved to be suitable for carrying diversity and has thus allowed for the creation of novel variants with diverse binding specificities useful in biotechnological applications.
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Evolution of a carbohydrate binding Module into a protein-specific binder.
Biomolecular engineering, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Eva Nordberg Karlsson, Olle Holst, Linda Dexlin, Mats OhlinAbstract:A carbohydrate binding Module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.
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Engineered xyloglucan specificity in a Carbohydrate-Binding Module
Glycobiology, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Cedric Montanier, Harry Brumer, Eva Nordberg Karlsson, Qi Zhou, Mats OhlinAbstract:The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the Carbohydrate-Binding Module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding Modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.
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Molecular engineering of a thermostable Carbohydrate-Binding Module
Biocatalysis and Biotransformation, 2006Co-Authors: Lavinia Cicortas Gunnarsson, Eva Nordberg Karlsson, Mats Andersson, Olle Holst, Mats OhlinAbstract:Structure-function studies are frequently practiced on the very diverse group of natural Carbohydrate-Binding Modules in order to understand the target recognition of these proteins. We have taken a step further in the study of Carbohydrate-Binding Modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable Carbohydrate-Binding Module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities. (Less)