Mechanism Of Carbohydrate Recognition Research Articles

C-type lectin 17A and macrophage-expressed receptor genes are magnified by fungal β-glucan after Vibrio parahaemolyticus infection in Totoaba macdonaldi cells

C-type lectins are a principal carbohydrate recognition mechanism as glucans on cell surfaces. This study identified and investigated molecular characterization and immune roles of a novel c-type lectin 17A from Totoaba macdonaldi (TmCLEC17A), which were described in head-kidney leukocytes after immunostimulation with fungal β-glucan 197A and Vibrio parahaemolyticus infection. This nucleotide sequence from totoaba was acquired using NGS and bioinformatics tools. Its full-length cDNA sequence consisted of 1128 bp (including the stop codon) and an open reading frame (ORF) of 771 bp encoding a 256 amino acid protein, 5´-UTR of 48 bp and 3´-UTR of 309 bp. The TmCLEC17A protein revealed a C-terminal-C-type lectin (CTL, also named carbohydrate-recognition domain, CRD), a N-terminal trans-membrane domain and a coiled coil motif, showing the highest similarity (80%) and identity (96%) with Larimichthys crocea. Fungal β-glucan 197A plus V. parahaemolyticus enhanced transcriptions of CLEC17A and TLR2 significantly besides the macrophage receptors, such as macrophage mannose receptor 1 and macrophage colony-stimulating factor 1 receptor 2. In addition, natural resistance-associated macrophage protein 2 was significantly up-regulated in leukocytes challenged with live V. parahaemolyticus. Overall, these results indicated that CLEC17A might be implicated in T. macdonaldi innate immunity as a pattern recognition receptor; fungal β-glucan 197A could stimulate cellular immune mechanisms in head-kidney leukocytes; and it could be used as potential immunostimulant in fish aquaculture.

Computational analysis of carbohydrate recognition based on hybrid QM/MM modeling: a case study of norovirus capsid protein in complex with Lewis antigen.

Norovirus is a major pathogen of nonbacterial acute gastroenteritis in humans and animals. Carbohydrate recognition between norovirus capsid proteins and Lewis antigens is considered to play a critical role in initiating infection of eukaryotic cells. In this article, we first report a detailed atomistic simulation study of the norovirus capsid protein in complex with the Lewis antigen based on ab initio QM/MM combined with MD-FEP simulations. To understand the mechanistic details of ligand binding, we analyzed and compared the carbohydrate recognition mechanism of the wild-type P domain protein with a mutant protein. Small structural differences between two capsid proteins are observed on the weak interaction site of residue 389, which is located on the solvent exposed surface of the P domain. To further clarify affinity differences in ligand binding, we directly evaluated free energy changes of the ligand binding process. Although the mutant protein loses its interaction energy with the Lewis antigen, this small amount of energy penalty is compensated for by an increase in the solvation stability, which is induced by structural reorganization at the ligand binding site on the protein surface. As a sum of these opposite energy components, the mutant P domain obtains a slightly enhanced binding affinity for the Lewis antigen. The present computational study clearly demonstrated that a detailed free energy balance of the interaction energy between the capsid protein and the surrounding aqueous solvent is the mechanistic basis of carbohydrate recognition in the norovirus capsid protein.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication.

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Structural basis of laminin binding to the LARGE glycans on dystroglycan.

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin G-like (LG) domains 4-5 of laminin α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a novel mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.

Alteration of the Carbohydrate-Binding Specificity of a C-type Lectin CEL-I Mutant with an EPN Carbohydrate-Binding Motif

CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

Carbohydrate recognition mechanism of HA70 from Clostridium botulinum deduced from X-ray structures in complexes with sialylated oligosaccharides

Clostridium botulinum produces the botulinum neurotoxin, forming a large complex as progenitor toxins in association with non-toxic non-hemagglutinin and/or several different hemagglutinin (HA) subcomponents, HA33, HA17 and HA70, which bind to carbohydrate of glycoproteins from epithelial cells in the infection process. To elucidate the carbohydrate recognition mechanism of HA70, X-ray structures of HA70 from type C toxin (HA70/C) in complexes with sialylated oligosaccharides were determined, and a binding assay by the glycoconjugate microarray was performed. These results suggested that HA70/C can recognize both α2–3- and α2–6-sialylated oligosaccharides, and that it has a higher affinity for α2–3-sialylated oligosaccharides.

Carbohydrate-Recognition and Hemolytic Mechanism of the Hemolytic Lectin from Marine Invertebrate

CEL-III is a Ca2+-dependent hemolytic lectin isolated from the sea cucumber Cucumaria echinata. X-ray crystallographic analysis revealed that CEL-III is composed of two ricin-like carbohydrate-binding domains (domain 1/2) and an oligomerization domain (domain 3). After binding to the specific carbohydrate chains on erythrocyte membrane, CEL-III undergoes conformational change, which leads to formation of membrane pores composed of its oligomers. Carbohydrate-recognition mechanism of CEL-III and its relationship with self-oligomerization mediated by domain 3 are discussed.

Structures of the Carbohydrate Recognition Domain of Ca2+-independent Cargo Receptors Emp46p and Emp47p

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca 2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for α-galactosides than for β-galactosides, while other C. echinata lectins show preferential binding of β-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.

Terminal Sialic Acid Residues on Human Glycophorin A Are Recognized by Porcine Kupffer Cells

We have previously shown that recognition of human erythrocytes by porcine Kupffer cells is mediated by a carbohydrate-dependent mechanism. The present study explores the possible ligands existing on human glycophorin A and tests their ability to inhibit erythrocyte rosette formation. Human erythrocytes were tested for ABO and MN specificity and used as targets in a 51Chromium quantitative erythrocyte rosette assay. Monosaccharides present on human glycophorin A, neuraminyl lactoses, bovine and porcine submaxillary mucins (BSM and PSM), and hyaluronic acid as well as proteoglycan N-linked glycosidase F(PNGaseF)- and sialidase A-treated human erythrocyte glycoproteins (hEGP) and human erythrocytes were all tested for inhibitory potential in the rosetting assay. Porcine Kupffer-cell recognition of human erythrocytes was insensitive to differences in blood groups A, B, O, or MN. At 30 mM, the monosaccharide, N-acetylneuraminic acid, and the trisaccharide mixture, neuraminyl lactoses, disrupted human erythrocyte recognition by 25% and 30%, respectively. A dilution of BSM but not PSM inhibited the rosetting assay by 17% (.2 mg/mL), 33% (1 mg/mL), and 53% (2 mg/mL). The same dilution of hyaluronic acid had no effect on rosetting. Removal of N-linked oligosaccharides from hEGP with PNGaseF did not impair its ability to inhibit the rosetting assay. In contrast, removal of sialic acid completely abrogated its inhibitory ability. Treatment of whole human erythrocytes with sialidase A likewise prevented recognition by porcine Kupffer cells. Terminal sialic acid on human erythrocytes is a target recognized by porcine Kupffer cells, suggesting a role for a sialic-acid receptor in innate cellular recognition of xenogeneic epitopes. Inasmuch as this work reveals a carbohydrate-recognition mechanism for cellular rejection, we shed light on a potential new boundary that will need to be overcome within xenotransplantation.

Identification of Residues Essential for Carbohydrate Recognition by the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.

A Novel Mechanism of Carbohydrate Recognition by the C-type Lectins DC-SIGN and DC-SIGNR: SUBUNIT ORGANIZATION AND BINDING TO MULTIVALENT LIGANDS

DC-SIGN and DC-SIGNR are cell-surface receptors that mediate cell-cell interactions within the immune system by binding to intercellular adhesion molecule-3. The receptor polypeptides share 77% amino acid sequence identity and are type II transmembrane proteins. The extracellular domain of each comprises seven 23-residue tandem repeats and a C-terminal C-type carbohydrate-recognition domain (CRD). Cross-linking, equilibrium ultracentrifugation, and circular dichroism studies of soluble recombinant fragments of DC-SIGN and DC-SIGNR have been used to show that the extracellular domain of each receptor is a tetramer stabilized by an alpha-helical stalk. Both DC-SIGN and DC-SIGNR bind ligands bearing mannose and related sugars through the CRDs. The CRDs of DC-SIGN and DC-SIGNR bind Man(9)GlcNAc(2) oligosaccharide 130- and 17-fold more tightly than mannose, and affinity for a glycopeptide bearing two such oligosaccharides is increased by a further factor of 5- to 25-fold. These results indicate that the CRDs contain extended or secondary oligosaccharide binding sites that accommodate mammalian-type glycan structures. When the CRDs are clustered in the tetrameric extracellular domain, their arrangement provides a means of amplifying specificity for multiple glycans on host molecules targeted by DC-SIGN and DC-SIGNR. Binding to clustered oligosaccharides may also explain the interaction of these receptors with the gp120 envelope protein of human immunodeficiency virus-1, which contributes to virus infection.

ChemInform Abstract: Characterization of the Subsite Structure of the β‐Glucosidase from Aspergillus niger, an Aspect of the Mechanism of Carbohydrate Recognition.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Characterization of the subsite structure of the β-glucosidase from Aspergillus niger, an aspect of the mechanism of carbohydrate recognition

Steady-state kinetics on the reaction catalyzed by the β-glucosidase of Aspergillus niger were carried out to evaluate the kinetic parameters, K m and k o , for phenyl β- d-glucosides. The k o/K m values, which may relate to productive binding at subsites, were found to correlate with the substituent constant π (hydrophobicity), suggesting that subsite 2 has a hydrophobic character. A “hydrophobic-driven” mechanism is considered to contribute to the productive E–S complex for recognition of the substrate.

Secondary structure and NMR assignments ofbacillus circulansxylanase

Bacillus circulans xylanase (BCX) is a member of the family of low molecular weight endo-beta-(1,4)-xylanases. The main-chain 1H, 13C, and 15N resonances of this 20.4-kDa enzyme were assigned using heteronuclear NMR experiments recorded on a combination of selectively and uniformly labeled protein samples. Using chemical shift, NOE, J coupling, and amide hydrogen exchange information, 14 beta-strands, arranged in a network of three beta-sheets, and a single alpha-helix were identified in BCX. The NMR-derived secondary structure and beta-sheet topology agree closely with that observed in the crystal structure of this protein. The HN of Ile 118 has a strongly upfield-shifted resonance at 4.03 ppm, indicative of a potential amide-aromatic hydrogen bond to the indole ring of Trp 71. This interaction, which is conserved in all low molecular weight xylanases of known structure, may play an important role in establishing the active site conformation of these enzymes. Following hen egg white and bacteriophage T4 lysozymes, B. circulans xylanase represents the third family of beta-glycanases for which extensive NMR assignments have been reported. These assignments provide the background for detailed studies of the mechanism of carbohydrate recognition and hydrolysis by this bacterial xylanase.

Pharmacokinetics in design of polymeric drug delivery systems

Abstract In this paper, the utilities of the clearance concept-based pharmacokinetic analysis to study systematic disposition characteristics and targeting efficacy of various macromolecular drug carriers are demonstrated. The relationships between physicochemical and biological properties and uptake clearance values of macromolecules in various organs are discussed for designing a targeting system employing them. Based on the information about the effects of molecular size and electric charges on in vivo disposition of model macromolecules, the passive and active targeting system for an anticancer agent to a tumor is developed. Targeting efficacy of carrier systems utilizing carbohydrate-recognition mechanism were also quantitatively characterized in the same way. Thus, pharmacokinetic analysis was demonstrated to make a significant contribution in the rational design of a targeting system.

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.

Cell adhesive activity of two animal lectins through different recognition mechanisms

Cell adhesive activity of two animal lectins. frog ( Rama catesbeiana) S-type 14K lectin and echinoidin (a C-type lectin from sea urchin plasma), was studied with human rhabdomyosarcoma (RD) cells. RD cells attached to and spread on plastic plates coatce with each lectin. Cell adhesion by the frog lectin was completely inhibited by the addition of lactose or asialofetuin glycopeptide. Echinoidin-induced cell adhesion was only inhibited by peptide GRGDS. Since echinoidin is known to contain an RGD-sequence, our results clearly indicate that this sequence is active as the cell adhesive signal. These results suggest that some of the animal lectins may function as a cell adhesive molecule rather than using the carbohydrate-recognition mechanism.

糖鎖認識機構を利用した肝臓への蛋白質のターゲティング

Effectiveness of a hepatic targeting system utilizing carbohydrate recognition mechanism was evaluated. Three types of bovine serum albumin (BSA)derivatives with 1-thiogalactoside(Gal-BSA), 1-thioglucoside(Glc-BSA), and 1-thiomannoside (Man-BSA) were synthesized by the amidation method and their disposition characteristics in mice were studied. Gal-BSA and Glc-BSA were rapidly taken up by the liver parenchymal cells, while Man-BSA was accumulated in the non-parenchymal cells due to receptor-mediated endocytosis. Similar results were observed in glycosylated superoxide dismutase and carboxymethyldextran, suggesting wide applicability of this methodology.