N-Acetylglucosamine Recognition by a Family 32 Carbohydrate-Binding Module from Clostridium perfringens NagH

Many carbohydrate-active enzymes have complex architectures comprising multiple modules that may be involved in catalysis, carbohydrate binding, or protein-protein interactions. Carbohydrate-binding modules (CBMs) are a common ancillary module whose function is to promote the adherence of the complete enzyme to carbohydrate substrates. CBM family 32 has been proposed to be one of the most diverse CBM families classified to date, yet all of the structurally characterized CBM32s thus far recognize galactose-based ligands. Here, we report a unique binding specificity and mode of ligand binding for a family 32 CBM. NagHCBM32-2 is one of four CBM32 modules in NagH, a family 84 glycoside hydrolase secreted by Clostridium perfringens. NagHCBM32-2 has the β-sandwich scaffold common to members of the family; however, its specificity for N-acetylglucosamine is unusual among CBMs. X-ray crystallographic analysis of the module at resolutions from 1.45 to 2.0 Å and in complex with disaccharides reveals that its mode of sugar recognition is quite different from that observed for galactose-specific CBM32s. This study continues to unravel the diversity of CBMs found in family 32 and how these CBMs might impart the carbohydrate-binding specificity to the extracellular glycoside hydrolases in C. perfringens.     

Crystal Structure of the HA3 Subcomponent of Clostridium botulinum Type C Progenitor Toxin

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.     

UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase mediates the initial step in the formation of the methylphosphomannosyl residues on the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins

The Dictyostelium discoideum gene gpt1 encodes a protein XP_638036 with sequence similarity to the α/β subunits of mammalian UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase. We now demonstrate that extracts of D. discoideum clones with mutations in this gene transfer GlcNAc-P from UDP-GlcNAc to mannose residues at less than 5% the wild type value. Further, the lysosomal hydrolases of these mutant clones fail to bind to a cation-independent mannose 6-phosphate receptor affinity column, indicating a lack of methylphosphomannosyl residues on the high mannose oligosaccharides of these proteins. We conclude that the gpt1 gene product catalyzes the initial step in the formation of methylphosphomannosyl residues on D. discoideum lysosomal hydrolases.     

Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical  81-Å-long and  60-Å-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking “non-self” carbohydrate ligands and “self” carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.     

Glycan Specificity of the Vibrio vulnificus Hemolysin Lectin Outlines Evolutionary History of Membrane Targeting by a Toxin Family

Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0 Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

Three UDP-hexose 4-epimerases with overlapping substrate specificity coexist in E. coli O86:B7

The O-antigen gene cluster of Escherichia coli O86:B7 was sequenced previously in our lab. One UDP-hexose 4-epimerase gene (named gne2 in this paper) was found and later characterized to be able to catalyze the interconversion between UDP-GlcNAc/GalNAc and UDP-Glc/Gal with almost equal efficiency. However, sequencing of the flanking gene region upstream of the traditional O-antigen gene cluster revealed an open reading frame (gne1), sharing 100% identity with Gne from E. coli O55, previously identified as UDP-GlcNAc 4-epimerase. Furthermore, we also located the traditional galE gene in the gal operon of O86:B7, which can catalyze the interconversion of UDP-Glc to UDP-Gal. Thus, for the first time, three UDP-hexose 4-epimerases with overlapping substrate specificity were found to coexist in one bacterium. Deletion of gne1 and gne2 in O86:B7 produced two different LPS phenotypes: the gne1 mutant exhibited rough LPS, while the gne2 mutant showed semi-rough LPS phenotype. These findings provide new clues for understanding the mechanism of O-antigen biosynthesis.     

Crystal Structures of the Solute Receptor GacH of Streptomyces glaucescens in Complex with Acarbose and an Acarbose Homolog: Comparison with the Acarbose-Loaded Maltose-Binding Protein of Salmonella typhimurium

GacH is the solute binding protein (receptor) of the putative oligosaccharide ATP-binding cassette transporter GacFG, encoded in the acarbose biosynthetic gene cluster (gac) from Streptomyces glaucescens GLA.O. In the context of the proposed function of acarbose (acarviosyl-1,4-maltose) as a ‘carbophor,’ the transporter, in complex with a yet to be identified ATPase subunit, is supposed to mediate the uptake of longer acarbose homologs and acarbose for recycling purposes. Binding assays using isothermal titration calorimetry identified GacH as a maltose/maltodextrin-binding protein with a low affinity for acarbose but with considerable binding activity for its homolog, component 5C (acarviosyl-1,4-maltose-1,4-glucose-1,1-glucose). In contrast, the maltose-binding protein of Salmonella typhimurium (MalE) displays high-affinity acarbose binding. We determined the crystal structures of GacH in complex with acarbose, component 5C, and maltotetraose, as well as in unliganded form. As found for other solute receptors, the polypeptide chain of GacH is folded into two distinct domains (lobes) connected by a hinge, with the interface between the lobes forming the substrate-binding pocket. GacH does not specifically bind the acarviosyl group, but displays specificity for binding of the maltose moiety in the inner part of its binding pocket. The crystal structure of acarbose-loaded MalE showed that two glucose units of acarbose are bound at the same region and position as maltose. A comparative analysis revealed that in GacH, acarbose is buried deeper into the binding pocket than in MalE by exactly one glucose ring shift, resulting in a total of 18 hydrogen-bond interactions versus 21 hydrogen-bond interactions for MalE_acarbose. Since the substrate specificity of ATP-binding cassette import systems is determined by the cognate binding protein, our results provide the first biochemical and structural evidence for the proposed role of GacHFG in acarbose metabolism.     

Entamoeba histolytica: Effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.     

Crystal Structures of A. acidocaldarius Endoglucanase Cel9A in Complex with Cello-Oligosaccharides: Strong − 1 and − 2 Subsites Mimic Cellobiohydrolase Activity

Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with β-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 Å resolution and two complexes at 2.66 and 2.1 Å resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (α/α)₆-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites − 1 and − 2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides.     

Glycoproteomics: Past, present and future

This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.     

Desulfated galactosaminoglycans are potential ligands for galectins: evidence from frontal affinity chromatography

Galectins, a group of β-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of βGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.     

1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.     

Crystal structure of the GluR0 ligand-binding core from Nostoc punctiforme in complex with L-glutamate: structural dissection of the ligand interaction and subunit interface

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor (iGluR). We have solved the crystal structure of the ligand-binding core of NpGluR0 in complex with l-glutamate at a resolution of 2.1 Å. The structure exhibits a noncanonical ligand interaction and two distinct subunit interfaces. The side-chain guanidium group of Arg80 forms a salt bridge with the γ-carboxyl group of bound l-glutamate: in GluR0 from Synechocystis (SGluR0) and other iGluRs, the equivalent residues are Asn or Thr, which cannot form a similar interaction. We suggest that the local positively charged environment and the steric constraint created by Arg80 mediate the selectivity of l-glutamate binding by preventing the binding of positively charged and hydrophobic amino acids. In addition, the NpGluR0 ligand-binding core forms a new subunit interface in which the two protomers are arranged differently than the known iGluR and SGluR0 dimer interfaces. The significance of there being two different dimer interfaces was investigated using analytical ultracentrifugation analysis.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

Specificities of Ricinus communis agglutinin 120 interaction with sulfated galactose

Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA₁₂₀) recognizes non-reducing terminal β-d-galactose (Galβ) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galβ-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA₁₂₀. Our results showed that RCA₁₂₀ binding to Galβ1–4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galβ-binding protein but it cannot accommodate any sulfation on Galβ.     

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a ²S_o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.     

Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.     

The expanding roles of the Sd/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2

Background

The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sd^a antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups.

Scope of review

We describe the multiple and unsuspected aspects of the biology of the Sd^a antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.

Major conclusions

The immunodominant sugar of the Sd^a antigen is a β1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sd^a antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model.

General significance

The expression of the Sd^a antigen has a wide impact on the physiology and the pathology of different biological systems.