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.     

Affinity electrophoresis used for determination of binding constants for antibody-antigen reactions.

The use of affinity electrophoresis in agarose gels for determination of binding constants for the interaction of antigens with monoclonal antibodies is exemplified for monoclonal anti-human serum albumin and anti-alpha 1-fetoprotein antibodies. The calculated binding constants are verified by independent binding assays. The electrophoretic separation of antigen-antibody complexes of different stoichiometry is also demonstrated. Thus, affinity electrophoresis represents an alternative method for both qualitative and quantitative assessment of antigen-antibody interactions.     

The structural basis for the ligand specificity of family 2 carbohydrate-binding modules

The interactions of proteins with polysaccharides play a key role in the microbial hydrolysis of cellulose and xylan, the most abundant organic molecules in the biosphere, and are thus pivotal to the recycling of photosynthetically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). The largest prokaryotic CBM family, CBM2, contains members that bind cellulose (CBM2a) and xylan (CBM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate interaction is rotated by 90 degrees compared with its position in CBM2a (thus matching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Arg-262-->Gly), a CBM2b xylan-binding module completely loses its affinity for xylan and becomes a cellulose-binding module. The structural effect of the mutation has been revealed using NMR spectroscopy, which confirms that Trp-259 rotates 90 degrees to lie flat against the protein surface. Except for this one residue, the mutation only results in minor changes to the structure. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the recognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.     

Enzyme and storage protein electrophoresis in varietal identification of sugar beet

Summary Different methods of classification, based on total protein patterns as well as on specific isoenzyme patterns, were compared in order to establish an identification system for sugar beet varieties and lines. Single seed patterns and bulk extractions of total and fractionated proteins were compared on SDS-PAGE. Due to important intra-populational variation contrasting with similarity at the varietal level no method proved to be sufficiently discriminatory. Twelve sugar beet lines have been genotypically fingerprinted on the basis of five allozyme systems. The allele frequencies of each variety have been measured by using 60–100 individuals. From the data, genetic distance coefficients have been calculated in order to group the different entries by cluster analysis. In addition, a comparison has been made between two seed lots independently obtained from the same parental lines, to test the stability over years. Seeds from the same parental lines, but produced in different localities (Denmark, Italia and USA), were compared to test the influence of the environment on the classification. It has been concluded that isozymes could provide a useful tool for cultivar distinction. The variability at the level of allele frequencies within localities was small. The stability of different generations of the strains was relatively constant. Different strains originating from the same seed firm were less distinct than strains originating from different seed firms.     

Divergent modes of glycan recognition by a new family of carbohydrate-binding modules

The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.     

Separation, identification, and characterization of microorganisms by capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.     

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

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

Novel family of carbohydrate-binding modules revealed by the genome sequence of Spirochaeta thermophila DSM 6192

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.     

Insight into ligand diversity and novel biological roles for family 32 carbohydrate-binding modules

Family 32 carbohydrate-binding modules (CBM32s) are found in a diverse group of microorganisms, including archea, eubacteria, and fungi. Significantly, many members of this family belong to plant and animal pathogens where they are likely to play a key role in enzyme toxin targeting and function. Indeed, ligand targets have been shown to range from insoluble plant cell wall polysaccharides to complex eukaryotic glycans. Besides a potential direct involvement in microbial pathogenesis, CBM32s also represent an important family for the study of CBM evolution due to the wide variety of complex protein architectures that they are associated with. This complexity ranges from independent lectin-like proteins through to large multimodular enzyme toxins where they can be present in multiple copies (multimodularity). Presented here is a rigorous analysis of the evolutionary relationships between available polypeptide sequences for family 32 CBMs within the carbohydrate active enzyme database. This approach is especially helpful for determining the roles of CBM32s that are present in multiple copies within an enzyme as each module tends to cluster into groups that are associated with distinct enzyme classes. For enzymes that contain multiple copies of CBM32s, however, there are differential clustering patterns as modules can either cluster together or in very distant sections of the tree. These data suggest that enzymes containing multiple copies possess complex mechanisms of ligand recognition. By applying this well-developed approach to the specific analysis of CBM relatedness, we have generated here a new platform for the prediction of CBM binding specificity and highlight significant new targets for biochemical and structural characterization.     

Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.     

Molecular basis for the selectivity and specificity of ligand recognition by the family 16 carbohydrate-binding modules from Thermoanaerobacterium polysaccharolyticum ManA

Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward beta-1,4-linked sugars, they can engage both cello- and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.     

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers

Novel molecular probes have been developed for the analysis and detection of polysaccharides in plant cell walls using carbohydrate-binding modules (CBMs) derived from modular glycoside hydrolases belonging to families 2a, 6, and 29. Recombinant forms of these proteins containing his-tags, in conjunction with anti-his-tag detection, provide a flexible system that utilizes CBMs as molecular probes in a range of applications. Assays for the rapid analysis of the binding of CBMs to polysaccharides and oligosaccharides using nitrocellulose-based CBM macroarrays and microtiter plate-based CBM capture and competitive-inhibition assays are described. We also demonstrate the use of CBMs with his-tags for the localization of their target ligands in planta. The generation of molecular probes from other families of CBMs will dramatically increase the repertoire of molecular probes available to determine the developmental and functional aspects of plant cell walls.     

Affinity capillary electrophoresis for the screening of novel antimicrobial targets

The increasing number of multiantibiotic-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), requires the development of novel chemotherapies that are structurally distinct and exempt from current resistance mechanisms. Bioinformatics data mining of microbial genomes has revealed numerous previously unexploited essential open reading frames (ORFs) of unknown biochemical function. The potential of these proteins as screening targets is not readily apparent because most screening technologies rely on knowledge of biological function. To address this problem, the authors employed affinity capillary electrophoresis (ACE) to identify antimicrobial compounds that bound the novel target YihA. Screening a small-molecule library of 44,000 compounds initially identified 115 binders, of which 76% were confirmed. Furthermore, the ACE assay distinguished diverse compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates. These data validate ACE as a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

Development of screening methods for detection of carbohydrate-binding proteins by use of soluble glycosylated polyacrylamide-based copolymers.

Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.     

Affinity and stoichiometry of calcium binding by arsenazo III

Arsenazo III forms a 1:1 complex with calcium. The affinity constant of arsenazo III for calcium (pK_Ca) has been determined by titrating purified arsenazo III with standard calcium solutions. The method of evaluation used allows one to determine correct pK_Ca values even in the presence of micromolar amounts of contaminating calcium. The pK_Ca is influenced by the following factors: (a) in the neutral pH range the apparent pK_Ca increases strongly with pH; (b) alkali ions bind weakly to arsenazo III and millimolar concentrations cause a decrease in the apparent pK_Ca; (c) the magnesium affinity of arsenazo III, although much lower than the calcium affinity, increases strongly with pH in the neutral range (at pH 7.0 the calcium affinity of arsenazo III is not appreciably altered by up to 2 mm magnesium); (d) strontium and barium form weaker complexes with arsenazo III than calcium, but much stronger complexes than magnesium; (e) the apparent pK_Ca decreases with increasing buffer concentration in the millimolar range. The pK_Ca of arsenazo III is so high that, unless the arsenazo III concentration greatly exceeds the calcium concentration, a considerable fraction of the total arsenazo III is in the calcium complexed form. Because of this, arsenazo III responds nonlinearly to all but the lowest calcium concentrations; however, quantitation of the calcium concentration can readily be done from the mass action law provided that the pK_Ca is determined under the actual experimental conditions. Arsenazo III is a reliable calcium indicator if the experimental conditions, particularly pH, are well controlled.