Β-linked Galactose Research Articles

Characterization of glycan substrates accumulating in GM1 Gangliosidosis

IntroductionGM1 gangliosidosis is a rare autosomal recessive genetic disorder caused by the disruption of the GLB1 gene that encodes β-galactosidase, a lysosomal hydrolase that removes β-linked galactose from the non-reducing end of glycans. Deficiency of this catabolic enzyme leads to the lysosomal accumulation of GM1 and its asialo derivative GA1 in β-galactosidase deficient patients and animal models. In addition to GM1 and GA1, there are other glycoconjugates that contain β-linked galactose whose metabolites are substrates for β-galactosidase. For example, a number of N-linked glycan structures that have galactose at their non-reducing end have been shown to accumulate in GM1 gangliosidosis patient tissues and biological fluids. ObjectiveIn this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover. ResultsUsing tandem mass spectrometry and glycan reductive isotope labeling with data-dependent mass spectrometry, we have confirmed the accumulation of glycolipids (GM1 and GA1) and N-linked glycans with terminal beta-linked galactose. We have also discovered a novel set of core 1 and 2 O-linked glycan metabolites, many of which are part of structurally-related isobaric series that accumulate in disease. In the brain of GLB1 null mice, the levels of these glycan metabolites increased along with those of both GM1 and GA1 as a function of age. In addition to brain tissue, we found elevated levels of both N-linked and O-linked glycan metabolites in a number of peripheral tissues and in urine. Both brain and urine samples from human GM1 gangliosidosis patients exhibited large increases in steady state levels for the same glycan metabolites, demonstrating their correlation with this disease in humans as well. ConclusionsOur studies illustrate that GLB1 deficiency is not purely a ganglioside accumulation disorder, but instead a broad oligosaccharidosis that include representatives of many β-linked galactose containing glycans and glycoconjugates including glycolipids, N-linked glycans, and various O-linked glycans. Accounting for all β-galactosidase substrates that accumulate when this enzyme is deficient increases our understanding of this severe disorder by identifying metabolites that may drive certain aspects of the disease and may also serve as informative disease biomarkers to fully evaluate the efficacy of future therapies.

Lectin activity of Pseudomonas aeruginosa vaccine candidates PSE17-1, PSE41-5 and PSE54

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal β-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal β-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile.

Clostridium difficile is a major cause of hospital-acquired antibiotic-associated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and β-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and β-linked galactose, including blood groups, Lewis antigens, N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues in vivo.

Identification and Characterization of Glycopeptides from Egg Protein Ovomucin with Anti-Agglutinating Activity against Porcine K88 Enterotoxigenic Escherichia coli Strains.

Ovomucin is a glycoprotein from egg white with potential to act as an anti-adhesive agent against infectious diseases. This study aimed to determine whether ovomucin or ovomucin hydrolysates could prevent adhesion of two porcine K88 enterotoxigenic Escherichia coli (ETEC) strains. Adhesion was assessed in vitro using a hemagglutination assay. Ovomucin hydrolysates, but not intact ovomucin, prevented adhesion of ETEC to porcine erythrocytes. The ovomucin hydrolysate prepared by acid protease II exhibited the best anti-agglutinating activity against both strains; this hydrolysate was fractionated by cation exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC). The most active fractions, F3(9) and F7(1), with minimal inhibitory concentrations of 0.03 and 0.25 g/L against strains ECL13795 and ECL13998, respectively, were subjected to structural characterization. Six identified glycopeptides were all derived from α-ovomucin, composed of a pentasaccharide core of two N-acetylglucosamine and three mannose residues (GlcNAc2Man3) and a bisecting N-acetylglucosamine (GlcNAc). The terminal β-linked galactose from these glycopeptides could be one of the binding sites for K88ac fimbriae.

Molecular phylogeny, 3D-structural insights, docking and mechanisms of action of plant beta-galactosidases

Beta-galactosidase BGAL is an exoglycosidase that catalyses the hydrolysis of terminal β-linked galactose residues. To better understand the molecular characteristics and structural insights of mango BGAL MiBGAL, we performed the sequence analyses, reconstruction of the evolutionary tree, homology modelling and molecular docking. BGALs are widely distributed enzymes that evolved from a common bacterial ancestor. Plant BGALs pBGALs belong to glycosyl hydrolase-35GH35 family and had close similarities with fungi BGALs. Three conserved motifs and GH35 putative active site with a consensus sequence G-G-P-[LIVM]2-x2-Q-x-E-N-E-[FY] were identified in 67 BGAL sequences. Modelled 3D structure of MiBGAL is composed of a catalytic TIM barrel domain domain-I and three other β-domains, II, III & IV. Structural studies identified the residues Glu182 and Glu251 as the proton donor and nucleophile, respectively in pBGALs that could function through retaining mechanism. p-nitrophenyl-β-D-galactopyranoside and 2-[4-2-hydroxyethylpiperazin-1-yl]ethanesulfonic acid could be potential substrate and inhibitor, respectively among the docked-ligands for both tomato BGAL4 and MiBGAL.

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

BackgroundProkaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs. MethodSpecific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties. ResultsThis study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated. ConclusionsThe carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties. General significanceThe novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.

The Yariv reagent: Behaviour in different solvents and interaction with a gum arabic arabinogalactanprotein

The β-d-Glc Yariv reagent is frequently used to isolate and to study the structure of arabinogalactan-proteins with the arabinogalactan type II structure. The present paper describes the aggregation features of the Yariv reagent in water, salt solutions and in organic solvents as determined by NMR, absorption spectroscopy and light scattering experiments. The results indicate that in water the Yariv reagent forms aggregates of up to 300 units and in 1% aqueous NaCl the degree of aggregation is approx. 150. The aggregates are formed both by H-bonds and hydrophobic interactions, the former appearing to be of most importance in water.The interaction between the Yariv reagent and an AGP fraction from gum arabic, showed a degree of aggregation of the Yariv reagent when using 1% NaCl to be of approx. 150 units, whereas disruption of the aggregate took place in 10% NaCl with an aggregation number of approx. 100.Partial acid hydrolysis of an AGP from gum Arabic (Acacia Senegal) and analyses of the linkage types remaining indicated that a certain length of (1→3)-β-linked galactose units was necessary for binding between the Yariv reagent and the AGP. This is in accordance to what also was recently observed by Kitazawa et al. (2013).

A HPLC-based glycoanalytical protocol allows the use of natural O-glycans derived from glycoproteins as substrates for glycosidase discovery from microbial culture

Many disorders are characterised by changes in O-glycosylation, but analysis of O-glycosylation has been limited by the availability of specific endo- and exo-glycosidases. As a result chemical methods are employed. However, these may give rise to glycan degradation, so therefore novel O-glycosidases are needed. Artificial substrates do not always identify every glycosidase activity present in an extract. To overcome this, an HPLC-based protocol for glycosidase identification from microbial culture was developed using natural O-glycans and O-glycosylated glycoproteins (porcine stomach mucin and fetuin) as substrates. O-glycans were released by ammonia-based β-elimination for use as substrates, and the bacterial culture supernatants were subjected to ultrafiltration to separate the proteins from glycans and low molecular size molecules. Two bacterial cultures, the psychrotroph Arthrobacter C1-1 and a Corynebacterium isolate, were examined as potential sources of novel glycosidases. Arthrobacter C1-1 culture contained a β-galactosidase and N-acetyl-β-glucosaminidase when assayed using 4-methylumbelliferyl substrates, but when defucosylated O-glycans from porcine stomach mucin were used as substrate, the extract did not cleave β-linked galactose or N-acetylglucosamine. Sialidase activity was identified in Corynebacterium culture supernatant, which hydrolysed sialic acid from fetuin glycans. When both culture supernatants were assayed using the glycoproteins as substrate, neither contained endoglycosidase activity. This method may be applied to investigate a microbial or other extract for glycosidase activity, and has potential for scale-up on high-throughput platforms.

Structural Basis for β-Galactosidase Associated with Lysosomal Disease

G(M1)-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-D-galactosidase (h-β-GAL), which lead to accumulations of the h-β-GAL substrates, G(M1) ganglioside and keratan sulfate due to mutations in the h-β-GAL gene. H-β-GAL is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. Here, we present the crystal structures of h-β-GAL in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. H-β-GAL showed a novel homodimer structure; each monomer was comprised of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. The long loop region connecting the TIM barrel domain with β-domain 1 was responsible for the dimerization. To gain structural insight into the molecular defects of h-β-GAL in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

The fission yeast Pvg1p has galactose-specific pyruvyltransferase activity

N-Glycan from the fission yeast Schizosaccharomyces pombe contains outer-chain pyruvic acid 4,6-ketal-linked galactose (PvGal). Here, we characterized a putative S. pombe pyruvyltransferase, Pvg1p, reported to be essential for biosynthesis of PvGal. When p-nitrophenyl-β-Gal (pNP-β-Gal) was used as a substrate, the structure of the recombinant Pvg1p product was determined to be pNP-PvGal by one- and two-dimensional NMR spectroscopy. The recombinant Pvg1p transferred pyruvyl residues from phosphoenolpyruvate specifically to β-linked galactose.

Crystal Structure of Human β-Galactosidase: STRUCTURAL BASIS OF GM1 GANGLIOSIDOSIS AND MORQUIO B DISEASES

G(M1) gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, G(M1) ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.

The fine specificity of mannose-binding and galactose-binding lectins revealed using outlier motif analysis of glycan array data

Glycan-binding proteins are commonly used as analytical reagents to detect the levels of specific glycan structures in biological samples. A detailed knowledge of the specificities of glycan-binding proteins is required for properly interpreting their binding data. A powerful technology for characterizing glycan-binding specificity is the glycan array. However, the interpretation of glycan-array data can be difficult due to the complex fine specificities of certain glycan-binding proteins. We developed a systematic approach, called outlier-motif analysis, for extracting fine-specificity information from glycan-array data, and we applied the method to the study of four commonly used lectins: two mannose binders (concanavalin A and Lens culinaris) and two galactose binders (Bauhinia purpurea and peanut agglutinin). The study confirmed the known, primary specificity of each lectin and also revealed new insights into their binding preferences. Lens culinaris's main specificity may be non-terminal, α-linked mannose with a single linkage at its 2' carbon, which is more restricted than previous definitions. We found broader specificity for bauhinea purpurea (BPL) than previously reported, showing that BPL can bind terminal N-acetylgalactosamine (GalNAc) and penultimate β-linked galactose under certain limitations. Peanut agglutinin may bind terminal Galβ1,3Gal, a glycolipid motif, in addition to terminal Galβ1,3GalNAc, a common O-linked glycoprotein motif. These results could be used to more accurately interpret data obtained using these well-studied lectins. Furthermore, this study demonstrates a systematic and general approach for extracting fine-specificity information from glycan-array data.

Exploring the Structural Diversity of Mammalian Carbohydrates (“Glycospace”) by Statistical Databank Analysis

The diversity of three major classes of mammalian carbohydrates, mainly glycolipids and O- and N-linked glycans, deposited in the databank GLYCOSCIENCES.de was subjected to statistical analyses. Size, chain length, and branching complexity were accessed and revealed that the average oligosaccharide is composed of about eight monosaccharide units. About a quarter of all oligosaccharides are strictly linear, and the remainder are branched at least once. Glucosamine, galactose, and mannose are dominating and comprise ~75% of the monosaccharides within mammalian oligosaccharide frameworks. alpha-Linked sialic acid, alpha-linked fucose, and beta-linked galactose decorate the majority of reducing termini. Glucose as the most abundant carbohydrate in mammals plays only a very minor role within these structures. Particular emphasis was placed on analyzing the way the monosaccharide units are linked within the oligomeric framework. Just 11 monosaccharide connections account for >75% of all linkages. Thus, the number of structural combinations found in nature, the part of the occupied mammalian glycospace, is much smaller than expected. As a result, a potential set of building blocks for oligosaccharide assembly is presented. This potential building block set was correlated with the accessible 3299 mammalian carbohydrate structures in the GLYCOSCIENCES.de databank. Only 36 building blocks are required to construct 75% of the 3299 mammalian oligosaccharides.

Quantitation of Binding of Ricinus communis Agglutinin I to Von Willebrand Factor (VWF): Investigation of Relationship with Plasma Clearance of VWF in Type 1 Von Willebrand Disease.

Type 1 von Willebrand disease (VWD) is a heterogeneous bleeding disorder manifest by a partial quantitative deficiency of the sialoglycoprotein von Willebrand factor (VWF). Glycosylation of VWF results in the addition of 12 N-linked and 10 O-linked oligosaccharide chains and modification of these structures has been shown to result in increased plasma clearance of VWF. Exposed galactose residues are required for the binding of VWF to the hepatic asialoglycoprotein receptor. The lectin Ricinus communis agglutinin I (RCA I) has a high affinity for the terminal β-linked galactose residues on VWF. Recent findings have reported increased binding of RCA-I to VWF in a proportion of patients with a partial quantitative deficiency of VWF. We have evaluated the binding of RCA-I to VWF in patients with type 1 VWD and normal controls and investigated its relationship with the plasma clearance of VWF antigen (VWF:Ag) in patients with type 1 VWD. An ELISA was developed to assess binding of biotinylated RCA-I to VWF in 30 patients with type 1 VWD and 17 normal controls. Plasma samples were analysed at baseline in both groups and following desmopressin (DDAVP) infusion in VWD group. VWF:Ag levels were assessed in parallel and RCA-I binding to VWF was expressed as a ratio of RCA-I/VWF:Ag. In the VWD patients VWF:Ag levels were quantitated over 6 hours following the administration of intravenous DDAVP. The half-life of VWF (VWF:Ag t1/2) was calculated by determining the first-order rate constant for the elimination phase of VWF:Ag.1 Median VWF:Ag levels in the VWD patients and normal controls were 36 IU/dL (range, 4–50), and 103 IU/dL (64–197) respectively. At baseline, RCA-I lectin was found to bind to VWF with a median RCA-I/VWF:Ag ratio of 0.98 (0.48–1.74) in VWD group as compared to 0.85 (0.69–1.28) in the controls. This represents a significant difference in RCA-I/VWF:Ag ratios between the two groups, p<0.05. Quantitation of RCA-I binding to VWF secreted post DDAVP in VWD group is pending. Median VWF:Ag t1/2 in the type 1 VWD patients was 4.1 hours (0.92–8.7) representing significantly increased plasma clearance of VWF:Ag in these patients.2 There was significant correlation between baseline RCA/VWF:Ag ratio and VWF:Ag t1/2 in the type 1 VWD patients, r=0.48, p<0.01. This represents significant inverse correlation between RCA-I/VWF binding and plasma clearance of VWF:Ag. There was no correlation between baseline RCA/VWF:Ag ratio and VWF:Ag in either the patient or control groups, p>0.1. The finding of increased binding of RCA-I to VWF in type 1 VWD patients at baseline is suggestive of variation in glycosylation in a proportion of these patients. This could be causative of or contributory towards increased VWF clearance, be otherwise related to the type 1 VWD disease phenotype or may represent normal variability. The inverse correlation found between baseline RCA-I/VWF binding and plasma clearance of VWF:Ag may reflect differences between VWF secreted post-DDAVP and baseline VWF: investigation is ongoing.

Spermadhesin AQN1 Is a Candidate Receptor Molecule Involved in the Formation of the Oviductal Sperm Reservoir in the Pig1

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.

One-pot synthesis of glycosyl poly(arbutin) by enzymatic glycosylation followed by polymerization with peroxidase

Polyaromatic compounds that carry several side residues of d-glucose, d-galactose, and d-mannose were synthesized from hydroquinone β-glucoside (arbutin) through two successive enzymatic reactions: glycosylation of the glucose residue in arbutin, followed by oxidative polymerization of the hydroquinone moieties. In the first step, Bacillus macerans cyclodextrin glucanotransferase, B. circulans β-galactosidase, and jack bean α-mannosidase were used to obtain glycosylation products in yields of 56.0, 29.5, and 25.2%, respectively, where the main products were a series of α-1,4-linked glucosyl/maltooligosyl arbutin, β-1,4-linked galactosyl arbutin, and α-1,6-linked mannosyl arbutin, respectively. In the second step, horseradish peroxidase was used and the enzyme efficiently polymerized glycosyl arbutin(s) together with the remaining arbutin using hydrogen peroxide as an oxidant. The water-soluble products, glycosyl poly(arbutin)s, showed approximate molecular weight distribution of 0.5–25 kDa which was estimated by gel permeation chromatography calibrated with maltooligosaccharides and pullulan markers. There were no differences in the phenolic main chain structures of glycosyl poly(arbutin)s and poly(arbutin), where the binding of neighboring hydroquinone moieties occurred at ortho-positions. Glycosyl poly(arbutin)s containing α-linked mannose and β-linked galactose residues exhibited increased absorbability to immobilized concanavalin A and castor bean lectin (RCA 120), respectively.

The Drosophila Gene brainiac Encodes a Glycosyltransferase Putatively Involved in Glycosphingolipid Synthesis

The Drosophila genes fringe and brainiac exhibit sequence similarities to glycosyltransferases. Drosophila and mammalian fringe homologs encode UDP-N-acetylglucosamine:fucose-O-Ser beta1,3-N-acetylglucosaminyltransferases that modulate the function of Notch family receptors. The biological function of brainiac is less well understood. brainiac is a member of a large homologous mammalian beta3-glycosyltransferase family with diverse functions. Eleven distinct mammalian homologs have been demonstrated to encode functional enzymes forming beta1-3 glycosidic linkages with different UDP donor sugars and acceptor sugars. The putative mammalian homologs with highest sequence similarity to brainiac encode UDP-N-acetylglucosamine:beta1,3-N-acetylglucosaminyltransferases (beta3GlcNAc-transferases), and in the present study we show that brainiac also encodes a beta3GlcNAc-transferase that uses beta-linked mannose as well as beta-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galbeta1-4Glcbeta1-Cer) and insects (Manbeta1-4Glcbeta1-Cer) are different. Both disaccharide glycolipids served as substrates for brainiac, but glycolipids of insect cells have so far only been found to be based on the GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer core structure. Infection of High Five(TM) cells with baculovirus containing full coding brainiac cDNA markedly increased the ratio of GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer glycolipids compared with Galbeta1-4Manbeta1-4Glcbeta1-Cer found in wild type cells. We suggest that brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids.

Characterization of a thermostable extracellular β-galactosidase from a thermophilic fungus Rhizomucor sp.

An extracellular β-galactosidase from a thermophilic fungus Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250 000 and it is composed of two identical subunits with molecular mass of 120 000. It is an acidic protein with a p I of 4.2. Purified β-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60°C, respectively. The enzyme is stable at 60°C for 4 h, and has a t 1/2 of 150 min at 70°C which is one of the highest reported for fungal β-galactosidases. Substrate specificity studies indicated that the enzyme is specific for β-linked galactose residues with a preference for p-nitrophenyl-β- D-galactopyranoside ( pNPG). The K m and V max values for the synthetic substrates pNPG and o-nitrophenyl-β- D-galactopyranoside ( oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min −1 mg −1 and 4.45 mmol min −1 mg −1, respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min −1 mg −1. The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with K i of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using pNPG and oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.