Sugar Recognition Research Articles

Opening and closing of the periplasmic gate in lactose permease

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H(+) translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 --> Cys/Asn-245 --> Cys, Thr-45 --> Cys/Asn-245 --> Cys, and Ile-32 --> Cys/Asn-245 --> Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than approximately 15 A in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than approximately 15 A in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.

Viridans streptococcal carbohydrates and their salivary receptor proteins

Streptococcal colonization of damaged heart surfaces in infective endocarditis depends on the recognition of host lectin‐receptors by bacterial glycoprotein. Our group has studied the viridans streptococcal glycoproteins of 30 bacteremic patient isolates both by carbo‐HPLC and by endocarditis production in the rat model. Two strains, endocarditis virulent Streptococcus#15 (Strep 15)and endocarditis negative #4 (Strep 4) were used to measure salivary receptor protein titer for the bacterial sugars. Tests were run on 16 samples (96 tests). Mixed saliva was stimulated by chewing sterile paraffin, collected on ice, sterile filtered and stored at −70C until serially diluted for in vitro agglutination with washed, killed streps 4 and 15, suspended at 0.6 absorbance. Triplicate twofold dilutions of each saliva sample were incubated 24 hr with each fixed streptococcus, and aggregation was scored both visually and microscopically. Individual patients showed an agglutination titer representing the subject's salivary mucins and platelet surface sialic acids, and ranged from 1:8 for strep 4 to 1:2048 for strep15 with young, healthy subjects and 1:2 and 1:4 in other subjects. Although salivary titer differed significantly between subjects, the ratio of virulent to non‐virulent titer was constant between individuals. These experiments suggest that host recognition of bacterial sugars relates more to the bacterial sugar (and its virulence?) than to individual differences in salivary target lectins. This concept, if shown in a larger population, may provide important insights into the pathogenesis of infective endocarditis induced by oral streptococci.

Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.

Disaccharide recognition by binuclear copper(ii) complexes

The sugar recognition by binuclear copper(II) complexes in solution is strongly dependent on secondary interactions and cannot be predicted from the intermetallic Cu...Cu distance.

Synthesis of a trisulfated heparan sulfate disaccharide analog and its use as a template for preliminary molecular imprinting studies

A heparan sulfate disaccharide analog was synthesized by a multistep route. This synthesis was designed in such a way that one intermediate could be selectively deprotected to provide versatility during both synthesis and homologation of heparan sulfate related polysaccharides. Non-covalent imprinted polymers were prepared by using the synthesized disaccharide as a template and a primary amine functionalized acrylate as the key functional monomer suitable for specific sulfated sugar recognition. The binding of related sugars to the imprinted and non-imprinted polymers and the binding of template to the chemically modified polymers have been also investigated.

Sodium-Dependent Reorganization of the Sugar-Binding Site of SGLT1

The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that are involved in sugar recognition to the external environment. We constructed 15 individual Cys mutants in the four transmembrane helices (TMHs) that form the sugar binding and translocation domain. Each mutant was functionally characterized for transport kinetics and substrate specificity. Identification of interactions between mutated residues and hydroxyls on the pyranose ring was assessed by comparing the affinities of deoxy sugars to those of glucose. We determined conformation-dependent accessibility to the mutated residues by both a traditional substituted cysteine accessibility method (SCAM) and a new fluorescence binding assay. These data were integrated to orient the helices and construct a framework of residues that comprise the external sugar binding site. We present evidence that R499, Q457, and T460 play a direct role in sugar recognition and that five other residues are indirectly involved in transport. Arranging the four TMHs to account for Na-dependent accessibility and potential for sugar interaction allows us to propose a testable model for the SGLT1 sugar binding site.

The DOMON domains are involved in heme and sugar recognition

We expand the functionally uncharacterized DOMON domain superfamily to identify several novel families, including the first prokaryotic representatives. Using several computational tools we show that it is involved in ligand binding--either as heme- or sugar-binding domains. We present evidence that the DOMON domain along with the DM13 domain comprises a novel electron-transfer system potentially involved in oxidative modification of animal cell-surface proteins. Other novel versions might function as sugar sensors of histidine kinases of bacterial two component systems. Supplementary data are available at Bioinformatics online and also at ftp://ftp.ncbi.nih.gov/pub/aravind/domon/.

Control of Bacterial Aggregation by Thermoresponsive Glycopolymers

Thermoresponsive polymers with pendant glucose functionality form aggregates with bacterial cells that can be assembled/disassembled reversibly through temperature cycling. The polymers are chain extended at temperatures below 40 °C and display glucose moieties to solution, whereas above this temperature the globular state of the polymer “hides” the sugar groups. The specificity and reversibility of the glucose display is demonstrated chemically via a boronic acid−alizarin coupled assay and through cell aggregation assays utilizing bacteria with and without fimH sugar recognition sites.

Balanced on One Foot

Cd1 proteins bind lipid antigens and present them to T cells to initiate immune responses. Cd1d presents glycolipids with specific α-linked sugars to the natural killer T cells (NKT) for recognition by their T cell receptor (TCR) complex. Borg et al . report the crystal structure of Cd1 bound to α-galactosylceramide (α-GalCer) with the NKT TCR. They find that the TCR interaction is unlike that of TCRs with the peptide-presenting major histocompatibility proteins (pMHCs). In the case of the pMHC-TCR interaction, the TCR α and β chains both contact the pMHC (see Moody), revealing two binding site "footprints" on the surface of the pMHC. The crystal structures reported by Borg et al . show that NKT TCR only binds to the Cd1d-α-GalCer by the α subunit, with the β subunit showing limited interaction and mostly hanging over the side of Cd1d. Moody describes the two structures as the TCR standing with both feet contacting pMHC, whereas NKT TCR appears as a pirouette balanced on one foot at the Cd1d surface. The structure also shows that the α-linked galactose lies flat, optimizing the interaction surface between Cd1d and the NKT TCR, which may provide a basis for the selective recognition of α-linked sugars. This study reinforces the notion that glycolipid-antigen recognition is achieved through molecular mechanisms different from those for peptide-antigen recognition and cautions against casting the Cd1 lipid-presenting proteins as MHC-like. N. A. Borg, K. S. Wun, L. Kjer-Nielsen, M. C. J. Wilce, D. G. Pellicci, R. Koh, G. S. Besra, M. Bharadwaj, D. I. Godfrey, J. McCluskey, J. Rossjohn, CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor. Nature 448 , 44-49 (2007). [PubMed] D. B. Moody, How a T cell sees sugar. Nature 448 , 36-37 (2007). [PubMed]

Molecular recognition of sugars by lanthanide (III) complexes of a conjugate of N, N‐bis[2‐[bis[2‐(1, 1‐dimethylethoxy)‐2‐oxoethyl]amino]ethyl]glycine and phenylboronic acid

A novel conjugate of phenylboronic acid and an Ln(DTPA) derivative, in which the central acetate pendant arm was replaced by the methylamide of L-lysine, was synthesized and characterized. The results of a fit of variable (17)O NMR data and a (1)H NMRD profile show that the water residence lifetime of the Gd(III) complex (150 ns) is shorter than that of the parent compound Gd(DTPA)(2-) (303 ns). Furthermore, the data suggest that several water molecules in the second coordination sphere of Gd(III) contribute to the relaxivity of the conjugate. The Ln(III) complexes of this conjugate are highly suitable for molecular recognition of sugars. The interaction with various sugars was investigated by (11)B NMR spectroscopy. Thanks to the thiourea function that links the phenylboronic acid targeting vector with the DTPA derivative, the interactions are stronger than that of phenylboronic acid itself. In particular, the interaction with N-propylfructosamine, a model for the glucose residue in glycated human serum albumin (HSA), is very strong. Unfortunately, the complex also shows a rather strong interaction with hexose-free HSA (K(A) = 705 +/- 300).

A unique quinolineboronic acid-based supramolecular structure that relies on double intermolecular B-N bonds for self-assembly in solid state and in solution.

The boronic acid functional group plays very important roles in sugar recognition, catalysis, organic synthesis, and supramolecular assembly. Therefore, understanding the unique properties of this functional group is very important. 8-Quinolineboronic acid (8-QBA) is found to be capable of self-assembling in solid state through a unique intermolecular B-N bond mechanism reinforced by intermolecular boronic anhydride formation, π-π stacking, and hydrogen bond formation. NMR NOE and diffusion studies indicate that intermolecular B-N interaction also exists in solution with 8-QBA. In contrast, a positional isomer of 8-QBA, 5-quinolineboronic acid (5-QBA) showed very different behaviors in crystal packing and in solution and therefore different supramolecular network. Understanding the structural features of this unique 8-QBA assembly could be very helpful for the future design of new sugar sensors, molecular catalysts, and supramolecular assemblies.

A unique quinolineboronic acid-based supramolecular structure that relies on double intermolecular B–N bonds for self-assembly in solid state and in solution

The boronic acid functional group plays very important roles in sugar recognition, catalysis, organic synthesis, and supramolecular assembly. Therefore, understanding the unique properties of this functional group is very important. 8-Quinolineboronic acid (8-QBA) is found to be capable of self-assembling in solid state through a unique intermolecular B–N bond mechanism reinforced by intermolecular boronic anhydride formation, π–π stacking, and hydrogen bond formation. NMR NOE and diffusion studies indicate that intermolecular B–N interaction also exists in solution with 8-QBA. In contrast, a positional isomer of 8-QBA, 5-quinolineboronic acid (5-QBA) showed very different behaviors in crystal packing and in solution and therefore different supramolecular network. Understanding the structural features of this unique 8-QBA assembly could be very helpful for the future design of new sugar sensors, molecular catalysts, and supramolecular assemblies.

Recognition of sugars on surface-bound cap-shaped gold particles modified with a polymer brush

A dithiolated random copolymer with pendent phenylboronic acid residues (Cys-Poly(3-acrylamidophenylboronic acid- co-2-dimethylaminopropyl methacrylamide), Cys-Poly(APBA- co-DMAPMA)) obtained by photo-iniferter method was accumulated as a polymer brush on a cap-shaped gold particles deposited on a vacuum-evaporated gold film, and the usefulness of the polymer brush as a sensing element for glycoprotein, ovalbumin (OVA), was examined by using UV–vis spectroscopy with a help of surface plasmon resonance. A similar system was constructed with a dithiolated mannose-carrying polymer, dithiolated-poly(2-methacryloyloxyethyl- D-mannopyranoside) (DT-PMEMan), prepared by the atom transfer radical polymerization (ATRP). The brush composed of this polymer was examined as a sensing element for lectin, concanavalin A (Con A). The sensor cells modified with Cys-Poly(APBA- co-DMAPMA) and DT-PMEMan showed a concentration-dependent binding of OVA and Con A, respectively, with a comparable detection limit to those with a monolayer of polymer brush-coated gold particle deposited on a glass substrate. Using this system, it can be expected to open a new perspective to various functional polymer brushes fixed to the cap-shaped gold particle on a solid substrate.

Toward an alternative for specific recognition of sulfated sugars. Preparation of highly specific molecular imprinted polymers

Sulfated sugars are a class of complex naturally occurring compounds some of which play central biological roles in mammals. Among them, heparan sulfates are multifunctional cell regulators, whose biological activities are related to their sulfation pattern. Determination of fine structures of these sulfated sugars is a prerequisite for understanding their biological roles. We investigated the applicability of molecular imprinting technology for recognition of the biologically relevant 6- O-sulfate substitution on sugars by using glucose-6- O-sulfate as model. Our results show that molecular imprinted polymers can specifically recognize sulfated sugars by the introduction of primary amines at the polymer side. Imprinted polymers showed excellent selectivity with regard to the sulfate position, the sugar configuration, and the presence of N-acetyl groups. These factors are essential for specific recognition of heparan sulfates' sequences. Molecular imprinting technology promise a significant contribution to the selection of sulfated sugar fragments of biological relevance.

Carbohydrate molecular recognition: a spectroscopic investigation of carbohydrate–aromatic interactions

The physical basis of carbohydrate molecular recognition at aromatic protein binding sites is explored by creating molecular complexes between a series of selected monosaccharides and toluene (as a truncated model for phenylalanine). They are formed at low temperatures under molecular beam conditions, and detected and characterized through mass-selected, infrared ion depletion spectroscopy-a strategy which exploits the extraordinary sensitivity of their vibrational signatures to the local hydrogen-bonded environment of their OH groups. The trial set of carbohydrates, alpha- and beta-anomers of glucose, galactose and fucose, reflects ligand fragments in naturally occurring protein-carbohydrate complexes and also allows an investigation of the effect of systematic structural changes, including the shape and extent of 'apolar' patches on the pyranose ring, removal of the OH on the exocyclic hydroxymethyl group, and removal of the aglycon. Bound complexes invariably form, establishing the general existence of intrinsic intermolecular potential minima. In most of the cases explored, comparison between recorded and computed vibrational spectra of the bound and free carbohydrates in the absence of solvent water molecules reveal that dispersion forces involving CH-pi interactions, which promote little if any distortion of the bound carbohydrate, predominate although complexes bound through specific OH-pi hydrogen-bonded interactions have also been identified. Since the complexes form at low temperatures in the absence of water, entropic contributions associated with the reorganization of surrounding water molecules, the essence of the proposed 'hydrophobic interaction', cannot contribute and other modes of binding drive the recognition of sugars by aromatic residues. Excitingly, some of the proposed structures mirror those found in naturally occurring protein-carbohydrate binding sites.

Photo-isomerization of a phenylboronic acid bearing an azo group to switch sugar recognition

Photo-isomerization of a phenylboronic acid bearing an azo group to switch sugar recognition

「イオン交換と超分子」第２回 フェニルボロン酸を用いる糖認識センサーの開発

「イオン交換と超分子」第２回 フェニルボロン酸を用いる糖認識センサーの開発

Amino acid sequence and characterization of C-type lectin purified from the snake venom of Crotalus ruber

Galactoside-binding lectin was purified from the snake venom of Crotalus ruber by affinity chromatography on a lactose–agarose column, and the complete amino acid sequence was determined. The C. ruber venom lectin (CRL) showed a single band of 28 kDa by SDS-polyacrylamide electrophoresis under non-reducing conditions, but it showed a single band of 15 kDa under reducing conditions, indicating that CRL is a disulfide-linked homodimer of 15 kDa subunit. CRL specifically recognized beta-galactosides such as thiodigalactoside followed by N-acetylgalactosamine when examined with their inhibitory effects on CRL-induced hemagglutination. A CRL subunit was composed of 135 residues containing nine Cys residues and showed a high similarity to other C-type galactoside-binding lectins from snake venoms. C. atrox lectin (CAL) showed almost the same sequence except for eight amino acid residues. Neither CRL nor CAL induced platelet aggregation by itself or inhibited platelet aggregation mediated by von Willebrand factor or fibrinogen with agonists. CRL showed a similar oligomeric form and the sugar specificity as CAL, but it showed different divalent cation sensitivity such as Mn 2+ and Ni 2+. Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal–ion binding.

Rice chitinases: sugar recognition specificities of the individual subsites

Sugar recognition specificities of class III (OsChib1a) and class I (OsChia1cDeltaChBD) chitinases from rice, Oryza sativa L., were investigated by analyzing (1)H- and (13)C-nuclear magnetic resonance spectra of the enzymatic products from partially N-acetylated chitosans. The reducing end residue of the enzymatic products obtained by the class III enzyme was found to be exclusively acetylated, whereas both acetylated and deacetylated units were found at the nearest neighbor to the reducing end residue. Both acetylated and deacetylated units were also found at the nonreducing end residue and its nearest neighbor of the class III enzyme products. Thus, only subsite (-1) among the contiguous subsites (-2) to (+2) of the class III enzyme was found to be specific to an acetylated residue. For the class I enzyme, the reducing end residue was preferentially acetylated, although the specificity was not absolute. The nearest neighbor to the acetylated reducing end residue was specifically acetylated. Moreover, the nonreducing end residue produced by the class I enzyme was exclusively acetylated, although there was a low but significant preference for deacetylated units at the nearest neighbor to the nonreducing end. These results suggest that the three contiguous subsites (-2), (-1), and (+1) of the class I enzyme are specific to three consecutive GlcNAc residues of the substrate. In rice plants, the target of the class I enzyme might be a consecutive GlcNAc sequence probably in the cell wall of fungal pathogen, whereas the class III enzyme might act toward an endogenous complex carbohydrate containing GlcNAc residue.

Exo-β-d-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism

Catalytic residues and the mode of action of the exo-beta-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis were investigated using the wild-type and mutated enzymes. Mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q, and S468N/D469E) that were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted by sequence alignments of enzymes belonging to GH-2 family. When mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme, the nonreducing-end glucosamine unit was produced together with the transglycosylation products. The rate of hydrolysis of the disaccharide, 2-amino-2-deoxy-D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranose (GlcN-GlcNAc), was slightly lower than that of (GlcN)2, suggesting that N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. The time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)4] was quantitatively determined by high-performance liquid chromatography (HPLC) and used for in silico modeling of the enzymatic hydrolysis. The modeling study provided the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0, and -0.5 kcal/mol corresponding, respectively, to subsites (-2), (-1), (+1), (+2), (+3), and (+4). When chitosan polysaccharide was hydrolyzed by a binary enzyme system consisting of A. orientalis GlcNase and Streptomyces sp. N174 endochitosanase, the highest synergy in the rate of product formation was observed at the molar ratio 2:1. Thus, GlcNase would be an efficient tool for industrial production of glucosamine monosaccharide.