Partial purification of goat kidney beta-mannosidase.
Ontology highlight
ABSTRACT:
SUBMITTER: Frei JI
PROVIDER: S-EPMC1148787 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.
| S-EPMC1132173 | biostudies-other
Project description:beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.
| S-EPMC1147897 | biostudies-other
Project description:Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1----4)GlcNAc beta (1----4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1----4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.
| S-EPMC1146542 | biostudies-other
Project description:More than 800 million people in the world suffer from chronic kidney disease (CKD). Genome-wide association studies (GWAS) have identified hundreds of loci where genetic variants are associated with kidney function; however, causal genes and pathways for CKD remain unknown. Here, we performed integration of kidney function GWAS and human kidney-specific expression quantitative trait analysis and identified that the expression of beta-mannosidase (MANBA) was lower in kidneys of subjects with CKD risk genotype. We also show an increased incidence of renal failure in subjects with rare heterozygous loss-of-function coding variants in MANBA using phenome-wide association analysis of 40,963 subjects with exome sequencing data. MANBA is a lysosomal gene highly expressed in kidney tubule cells. Deep phenotyping revealed structural and functional lysosomal alterations in human kidneys from subjects with CKD risk alleles and mice with genetic deletion of Manba Manba heterozygous and knockout mice developed more severe kidney fibrosis when subjected to toxic injury induced by cisplatin or folic acid. Manba loss altered multiple pathways, including endocytosis and autophagy. In the absence of Manba, toxic acute tubule injury induced inflammasome activation and fibrosis. Together, these results illustrate the convergence of common noncoding and rare coding variants in MANBA in kidney disease development and demonstrate the role of the endolysosomal system in kidney disease development.
| S-EPMC8627675 | biostudies-literature
Project description:The lysosomal α-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant α-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5 Å.
| S-EPMC3412785 | biostudies-literature
Project description:Neurological dysfunction is common in humans and animals with lysosomal storage diseases. β-Mannosidosis, an autosomal recessive inherited disorder of glycoprotein catabolism caused by deficiency of the lysosomal enzyme β-mannosidase, is characterized by intracellular accumulation of small oligosaccharides in selected cell types. In ruminants, clinical manifestation is severe, and neuropathology includes extensive intracellular vacuolation and dysmyelination. In human cases of β-mannosidosis, the clinical symptoms, including intellectual disability, are variable and can be relatively mild. A β-mannosidosis knockout mouse was previously characterized and showed normal growth, appearance, and lifespan. Neuropathology between 1 and 9 months of age included selective, variable neuronal vacuolation with no hypomyelination. This study characterized distribution of brain pathology in older mutant mice, investigating the effects of two strain backgrounds. Morphological analysis indicated a severe consistent pattern of neuronal vacuolation and disintegrative degeneration in all five 129X1/SvJ mice. However, the mice with a mixed genetic background showed substantial variability in the severity of pathology. In the severely affected animals, neuronal vacuolation was prominent in specific layers of piriform area, retrosplenial area, anterior cingulate area, selected regions of isocortex, and in hippocampus CA3. Silver degeneration reaction product was prominent in regions including specific cortical layers and cerebellar molecular layer. The very consistent pattern of neuropathology suggests metabolic differences among neuronal populations that are not yet understood and will serve as a basis for future comparison with human neuropathological analysis. The variation in severity of pathology in different mouse strains implicates genetic modifiers in the variable phenotypic expression in humans.
| S-EPMC4110335 | biostudies-literature
Project description:We purified three proline-rich antimicrobial peptides from elastase-treated extracts of sheep and goat leukocytes and subjected two of them, OaBac5alpha and ChBac5, to detailed analysis. OaBac5alpha and ChBac5 were homologous to each other and to bovine Bac5. Both exhibited potent, broad-spectrum antimicrobial activity under low-concentration salt conditions. While the peptides remained active against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Listeria monocytogenes in 100 mM NaCl, they lost activity against Staphylococcus aureus and Candida albicans under these conditions. ChBac5 was shown to bind lipopolysaccharide, a property that could enhance its ability to kill gram-negative bacteria. Proline-rich Bac5 peptides are highly conserved in ruminants and may contribute significantly to their innate host defense mechanisms.
| S-EPMC96712 | biostudies-literature
Project description:BackgroundMannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source.ResultsParental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate.ConclusionsWe successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.
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Project description:?-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this ?-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. ?-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other ?-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus ?-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of ?-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred ?g/mL ?-D-glucose inhibited ?-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.
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Project description:Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested-actin, myosin heavy and light chain, and IQGAP-as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis.
| S-EPMC2825282 | biostudies-literature