Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Muller.
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ABSTRACT:
SUBMITTER: Calvo P
PROVIDER: S-EPMC1186125 | biostudies-other | 1978 Nov
REPOSITORIES: biostudies-other
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Project description:A beta-N-acetylhexosaminidase was purified 800-fold from extracts of Trichomonas foetus by affinity chromatography on a column of N-(epsilon-aminohexanoyl)-2-acetamido-2-deoxy-beta-D-glucopyranosylamine bound to CNBr-activated Sepharose. The enzyme has a dual specificity for the p-nitrophenyl beta-D-glycosides of N-acetylglucosamine and N-acetyl-galactosamine. The parent sugars are both competitive inhibitors. The enzyme has a mol. wt. approx. 150000 and a pH optimum of 6.2. It is suggested that the same active site catalyses both activities and that no part is played by the 4-hydroxyl group in substrate binding, but it is involved in determining the catalytic rate.
| S-EPMC1172336 | biostudies-other
Project description:beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.
| S-EPMC1146539 | biostudies-other
Project description:Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.
| S-EPMC6940953 | biostudies-literature
Project description:beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α)8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and Glu(331) could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom.
| S-EPMC4129151 | biostudies-literature
Project description:The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a β-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a β-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-β-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-β-d-galactopyranoside, and 4-Nitrophenyl β-d-glucopyranoside, presenting β-N-acetylglucosaminidase, β-glucosidase, and β-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel β-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.
| S-EPMC5615308 | biostudies-literature
Project description:N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.
| S-EPMC6384963 | biostudies-literature
Project description:Backgroundβ-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.ResultsComputational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.ConclusionsThe main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.
| S-EPMC4384365 | biostudies-literature
Project description:1. Hyaluronate extracted from rooster comb was digested by a mixture of beta-N-acetylhexosaminidase and beta-glucuronidase with simultaneous dialysis for 96 h. 2. The produjct, yielding 99.6% of a mixture of mono- and oligo-saccharides, was purified by gel chromatography and analysed for glucuronic acid, N-acetylglucosamine and other sugars. 3. The oligosaccharide portion was chromatographed on DEAE-cellulose, and the effluent fractions were analysed for glucuronic acid and N-acetylglucosamine, reduced with NaBH4, digested by beta-N-acetylhexosaminidase and subjected to acid hydrolysis and glucosamine determination. 4. GlcNAc-GlcA-GlcNAc, GlcA-GlcA-GlcNAc and GlcA-GlcA-GlcA-GlcNAc were the oligosaccharides obtained, which resulted from the transferase activity of the enzymes and represented 57% of the digestion products. The results demonstrate that this hyaluronate is an unbranched polymer of approximatey equal amounts of glucuronic acid and N-acetylglucosamine. The data also indicate that if this glycosaminoglycan contains any of the neutral sugars for which it was analysed, their concentration must be less than 0.020% of the sum of the known components.
| S-EPMC1163124 | biostudies-other
Project description:GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in β-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or β-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and β-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/β developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/β, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/β intracranial injection among different species, despite encoding for self-proteins.
| S-EPMC5488349 | biostudies-literature
Project description:Emerging therapies have begun to evaluate the abilities of Müller glial cells (MGCs) to protect and/or regenerate neurons following retina injury. The migration of donor cells is central to many reparative strategies, where cells must achieve appropriate positioning to facilitate localized repair. Although chemical cues have been implicated in the MGC migratory responses of numerous retinopathies, MGC-based therapies have yet to explore the extent to which external biochemical stimuli can direct MGC behavior. The current study uses a microfluidics-based assay to evaluate the migration of cultured rMC-1 cells (as model MGC) in response to quantitatively-controlled microenvironments of signaling factors implicated in retinal regeneration: basic Fibroblast Growth factor (bFGF or FGF2); Fibroblast Growth factor 8 (FGF8); Vascular Endothelial Growth Factor (VEGF); and Epidermal Growth Factor (EGF). Findings indicate that rMC-1 cells exhibited minimal motility in response to FGF2, FGF8 and VEGF, but highly-directional migration in response to EGF. Further, the responses were blocked by inhibitors of EGF-R and of the MAPK signaling pathway. Significantly, microfluidics data demonstrate that changes in the EGF gradient (i.e. change in EGF concentration over distance) resulted in the directional chemotactic migration of the cells. By contrast, small increases in EGF concentration, alone, resulted in non-directional cell motility, or chemokinesis. This microfluidics-enhanced approach, incorporating the ability both to modulate and asses the responses of motile donor cells to a range of potential chemotactic stimuli, can be applied to potential donor cell populations obtained directly from human specimens, and readily expanded to incorporate drug-eluting biomaterials and combinations of desired ligands.
| S-EPMC6054825 | biostudies-literature