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1.
Biomolecules ; 12(11)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36358928

RESUMO

ß-Galactosidases (ß-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing ß-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a ß-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative ß-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose ß1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both ß-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.


Assuntos
Galactose , Galactosidases , Animais , beta-Galactosidase/genética , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Galactose/metabolismo , Oligossacarídeos , Moluscos/metabolismo
2.
PLoS One ; 17(10): e0275580, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36251631

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by a trinucleotide CAG repeat. SCA7 predominantly causes a loss of photoreceptors in the retina and Purkinje cells of the cerebellum. Severe infantile-onset SCA7 also causes renal and cardiac irregularities. Previous reports have shown that SCA7 results in increased susceptibility to DNA damage. Since DNA damage can lead to accumulation of senescent cells, we hypothesized that SCA7 causes an accumulation of senescent cells over the course of disease. A 140-CAG repeat SCA7 mouse model was evaluated for signs of disease-specific involvement in the kidney, heart, and cerebellum, tissues that are commonly affected in the infantile form. We found evidence of significant renal abnormality that coincided with an accumulation of senescent cells in the kidneys of SCA7140Q/5Q mice, based on histology findings in addition to RT-qPCR for the cell cycle inhibitors p16Ink4a and p21Cip1 and senescence-associated ß-galactosidase (SA-ßgal) staining, respectively. The Purkinje layer in the cerebellum of SCA7140Q/5Q mice also displayed SA-ßgal+ cells. These novel findings offer evidence that senescent cells accumulate in affected tissues and may possibly contribute to SCA7's specific phenotype.


Assuntos
Proteínas do Tecido Nervoso , Ataxias Espinocerebelares , Animais , Ataxina-7/genética , Modelos Animais de Doenças , Galactosidases , Camundongos , Proteínas do Tecido Nervoso/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Repetições de Trinucleotídeos
3.
Int J Mol Sci ; 23(19)2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36232994

RESUMO

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.


Assuntos
Paracoccus , Saccharomycetales , beta-Galactosidase , Fermentação , Galactosidases , Paracoccus/enzimologia , Saccharomycetales/metabolismo , beta-Galactosidase/biossíntese
4.
Sci Rep ; 12(1): 16847, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207369

RESUMO

Minimally invasive techniques and biological autograft alternatives such as the bone morphogenetic proteins (BMPs) can reduce morbidity associated with spinal fusions. This study was a proof-of-concept for gene-therapy-mediated anterior spine fusion that could be adapted to percutaneous technique for clinical use. Isogeneic bone marrow stromal cells genetically programmed to express b-galactosidase (LACZ, a marker gene), BMP2, BMP7, a mixture of BMP2 and BMP7 infected cells (homodimers, HM), or BMP2/7 heterodimers (HT) were implanted into the discs between lumbar vertebrae 4 and 5 (L4/5) and L5/6 of male Lewis rats. Spine stiffening was monitored at 4, 8 and 12 weeks using noninvasive-induced angular displacement (NIAD) testing. At 12 weeks isolated spines were assessed for fusion and bone formation by palpation, biomechanical testing [four-point bending stiffness, moment to failure in extension, and in vitro angular displacement (IVAD)], faxitron x-rays, microCT, and histology. Progressive loss of NIAD occurred in only the HT group (p < 0.001), and biomechanical tests correlated with the NIAD results. Significant fusion occurred only in the HT group (94% of animals with one or both levels) as assessed by palpation (p < 0.001), which predicted HT bone production assessed by faxitron (p ≤ 0.001) or microCT (p < 0.023). Intervertebral bridging bone was consistently observed only in HT-treated specimens. Induced bone was located anterior and lateral to the disc space, with no bone formation noted within the disc. Percutaneous anterior spine fusions may be possible clinically, but induction of bone inside the disc space remains a challenge.


Assuntos
Disco Intervertebral , Fusão Vertebral , Animais , Fenômenos Biomecânicos , Proteínas Morfogenéticas Ósseas/genética , Galactosidases , Terapia Genética/métodos , Vértebras Lombares/cirurgia , Masculino , Ratos , Ratos Endogâmicos Lew , Fusão Vertebral/métodos
5.
Metab Brain Dis ; 37(8): 3023-3026, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36178639

RESUMO

Fabry disease is a rare X-linked lysosomal storage disorder due to pathogenic variants of the galactosidase alpha (GLA) gene, leading to a deficiency of alpha-galactosidase A. The inadequate enzymatic activity leads to progressive glycosphingolipids accumulation within tissues and subsequent multi-systemic dysfunction, with predominant involvement of heart, kidney, and nervous system. Two subtypes are recognized: the classic type and the late-onset type. We here describe the clinical characteristics of a patient with late-onset Fabry disease carrying a not previously identified GLA gene variant. This 50-year-old man came to hospital because of an acute ischemic stroke. He also complained of acroparesthesia and had angiokeratomas in the nape and the back. Blood alpha-galactosidase A activity was low, plasmatic lyso-Gb3 level was borderline, cardiac MRI showed cardiac fibrosis, brain MRI documented cerebrovascular disease, and skin biopsy revealed small fiber neuropathy without globotriaosylceramide-3 skin deposits. Genetic study by means of targeted next-generation sequencing analysis disclosed a missense substitution c.1139C>T (p.Pro380Leu) in the GLA gene. We suggest that this novel variant should be considered as pathogenic and associated with a late-onset variant of Fabry disease with a predominant neurological phenotype.


Assuntos
Doença de Fabry , AVC Isquêmico , Masculino , Humanos , alfa-Galactosidase/genética , Doença de Fabry/genética , Galactosidases/genética , Fenótipo , Mutação
6.
Appl Environ Microbiol ; 88(18): e0110022, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36036580

RESUMO

Carbohydrate-active enzymes are important components of the polysaccharide metabolism system in marine bacteria. Carrageenase is indispensable for forming carrageenan catalytic pathways. Here, two GH16_13 carrageenases showed likely hydrolysis activities toward different types of carrageenans (e.g., κ-, hybrid ß/κ, hybrid α/ι, and hybrid λ), which indicates that a novel pathway is present in the marine bacterium Flavobacterium algicola to use κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). A comparative study described the different features with another reported pathway based on the specific carrageenans (κ, ι, and λ) and expanded the carrageenan metabolic versatility in F. algicola. A further comparative genomic analysis of carrageenan-degrading bacteria indicated different distributions of carrageenan metabolism-related genes in marine bacteria. The crucial core genes encoding the GH127 α-3,6-anhydro-d-galactosidase (ADAG) and 3,6-anhydro-d-galactose (d-AHG)-utilized cluster have been conserved during evolution. This analysis further revealed the horizontal gene transfer (HGT) phenomenon of the carrageenan polysaccharide utilization loci (CarPUL) from Bacteroidetes to other bacterial phyla, as well as the versatility of carrageenan catalytic activities in marine bacteria through different metabolic pathways. IMPORTANCE Based on the premise that the specific carrageenan-based pathway involved in carrageenan use by Flavobacterium algicola has been identified, another pathway was further analyzed, and it involved two GH16_13 carrageenases. Among all the characterized carrageenases, the members of GH16_13 accounted for only a small portion. Here, the functional analysis of two GH16_13 carrageenases suggested their hydrolysis effects on different types of carrageenans (e.g., κ, hybrid ß/κ, hybrid α/ι-, and hybrid λ-), which led to the identification of another pathway. Further exploration enabled us to elucidate the novel pathway that metabolizes KC and IC in F. algicola successfully. The coexistence of these two pathways may provide improved survivability by F. algicola in the marine environment.


Assuntos
Galactose , Glicosídeo Hidrolases , Carragenina/metabolismo , Flavobacterium/genética , Flavobacterium/metabolismo , Galactosidases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Redes e Vias Metabólicas/genética , Polissacarídeos
7.
Stem Cell Res ; 61: 102747, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35325818

RESUMO

Human dermal fibroblasts (HDF) were obtained by skin punch biopsy from a 51-year old man with suspected Fabry disease (FD), carrying the hemizygous c.376A > G variant in the α-galactosidase A gene (GLA). Cultured HDF were reprogrammed to induced pluripotent stem cells (iPSC) using a non-modified RNA-based transfection protocol. GLA-S126G-iPSC exhibit typical embryonic stem cell-like morphology, normal karyotype, expression of all tested pluripotency markers, and three germ layer differentiation potential. We provide a novel patient-specific cell line that can be used to investigate a genetic variation of yet unknown significance.


Assuntos
Doença de Fabry , Células-Tronco Pluripotentes Induzidas , Doença de Fabry/genética , Doença de Fabry/patologia , Galactosidases/genética , Galactosidases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Virulência
8.
J Med Genet ; 59(3): 279-286, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33547137

RESUMO

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesised that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry disease. METHODS: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry disease (n=58), age-matched and sex-matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time PCR. Circular RNA biomarker sensitivity, specificity, predictive values and area under the curve (AUC) were determined. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics. RESULTS: A distinct circular RNA transcriptome signature identified patients with Fabry disease. Level of circular RNAs hsa_circ_0006853 (AUC=0.73), hsa_circ_0083766 (AUC=0.8) and hsa_circ_0002397 (AUC=0.8) distinguished patients with Fabry disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 was, furthermore, female-specifically expressed. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry disease. CONCLUSION: The discovery of circular RNA-based and Fabry disease-specific biomarkers may advance future diagnosis of Fabry disease and help to distinguish related phenotypes.


Assuntos
Injúria Renal Aguda , Doença de Fabry , Biomarcadores/metabolismo , Biomarcadores Tumorais , Doença de Fabry/diagnóstico , Doença de Fabry/genética , Feminino , Galactosidases/genética , Humanos , Masculino , Fenótipo , RNA/genética , RNA/metabolismo , RNA Circular/genética
9.
Sci Rep ; 11(1): 23328, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857830

RESUMO

Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two ß-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6'-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut.


Assuntos
Bifidobacterium/fisiologia , Fezes/microbiologia , Galactose/metabolismo , Trato Gastrointestinal/microbiologia , Leite Humano/metabolismo , Oligossacarídeos/metabolismo , Galactosidases/metabolismo , Humanos , Lactente , Leite Humano/microbiologia , Trissacarídeos/metabolismo
10.
Front Cell Infect Microbiol ; 11: 775371, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34926322

RESUMO

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick's specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and ß-1,4 galactosyltransferases (ß-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas ß-1,4GalT transfers α-galactose to a ß1,4 terminal linkage acceptor sugars-GlcNAc, Glc, and Xyl-in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and ß-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in ß-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and ß-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS.


Assuntos
Amblyomma , Imunoglobulina E , Animais , Hipersensibilidade Alimentar , Galactosidases , Galactosiltransferases/genética , Humanos , alfa-Galactosidase/genética
11.
mBio ; 12(6): e0276621, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34809461

RESUMO

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable ß-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different ß-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.


Assuntos
Expressão Gênica , Vetores Genéticos/genética , Geobacillus/enzimologia , Methanobacteriaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosidases/genética , Galactosidases/metabolismo , Vetores Genéticos/metabolismo , Geobacillus/genética , Metano/metabolismo , Methanobacteriaceae/crescimento & desenvolvimento , Methanobacteriaceae/metabolismo
12.
J Chem Inf Model ; 61(9): 4554-4570, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34423980

RESUMO

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-ß-galactosidase/6-phospho-ß-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands p-nitrophenyl-ß-d-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-ß-d-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BlBglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BlBglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BlBglC complex. Additionally, the BlBglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BlBglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.


Assuntos
Bacillus licheniformis , Glucosidases , Bacillus licheniformis/metabolismo , Galactosidases , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato
13.
Adv Mater ; 33(34): e2101707, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34278613

RESUMO

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.


Assuntos
Vacinas contra COVID-19/genética , Galactosidases/química , Nanopartículas/química , Fosfatidiletanolaminas/química , RNA Mensageiro/química , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/prevenção & controle , Vacinas contra COVID-19/química , Vacinas contra COVID-19/metabolismo , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Galactosidases/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , RNA Mensageiro/genética
14.
J Dairy Sci ; 104(9): 9465-9477, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34127264

RESUMO

A novel galactosidase gene (gal3149) was identified from Bacillus velezensis SW5 and heterologously expressed in Escherichia coli BL21 (DE3). The novel galactosidase, Gal3149, encoded by gal3149 in an open reading frame of 1,299 bp, was 433 amino acids in length. Protein sequence analysis showed that Gal3149 belonged to family 4 of glycoside hydrolases (GH4). Gal3149 displayed higher enzyme activity for the substrate 2-nitrophenyl-ß-d-galactopyranoside (oNPG) than for 4-nitrophenyl-α-d-galactopyranoside (pNPαG). This is the first time that an enzyme belonging to GH4 has been shown to exhibit ß-galactosidase activity. Gal3149 showed optimal activity at pH 8.0 and 50°C, and exhibited excellent thermal stability, with retention of 50% relative activity after incubation at a temperature range of 0 to 50°C for 48 h. Gal3149 activity was significantly improved by K+ and Na+, and was strongly or completely inhibited by Ag+, Zn2+, Tween-80, Cu2+, carboxymethyl cellulose, and oleic acid. The rate of hydrolyzed lactose in 1 mL of milk by 1 U of Gal3149 reached about 50% after incubation for 4 h. These properties lay a solid foundation for Gal3149 in application of the lactose-reduced dairy industry.


Assuntos
Bacillus , Galactosidases , Animais , Bacillus/genética , Bacillus/metabolismo , Clonagem Molecular , Concentração de Íons de Hidrogênio , Cinética , Lactose , Temperatura , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
15.
Food Funct ; 12(11): 4960-4971, 2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34100482

RESUMO

The composition and structure of gut microbiota plays an important role in obesity induced by a high-fat diet (HFD) and related metabolic syndrome (MetS). Previous studies have shown that galacto-oligosaccharides (GOSs) have an effective anti-obesity effect. In this study, we aimed to investigate the effect of enzymatically synthesized α-galacto-oligosaccharides (ES-α-GOSs) on MetS and gut microbiota dysbiosis in HFD-fed mice, and to further investigate whether the attenuation of MetS is associated with the modulation of gut microbiota. Our results indicated that ES-α-GOS could notably ameliorate obesity-related MetS, including hyperlipidemia, insulin resistance and mild inflammation. The subsequent analysis of gut microbiota further showed that ES-α-GOS supplements can significantly modulate the overall composition of the gut microbiota and reverse the gut microbiota disorder caused by HFD feeding. Moreover, Spearman correlation analysis showed that 40 key bacteria reversed by ES-α-GOS were highly associated with metabolic parameters. These results suggested that ES-α-GOSs could serve as a potential candidate for preventing obesity-induced MetS in association with the modulation of gut microbiota.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Síndrome Metabólica/microbiologia , Oligossacarídeos/farmacologia , Tecido Adiposo/patologia , Animais , Aspergillus niger/metabolismo , Bactérias , Glicemia , Suplementos Nutricionais , Disbiose , Dislipidemias , Galactosidases/metabolismo , Hiperlipidemias , Inflamação , Resistência à Insulina , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo
16.
Appl Microbiol Biotechnol ; 105(11): 4621-4634, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34057561

RESUMO

Two GH117 family α-neoagarobiose hydrolases (GH117A α-NABH and GH117B α-NABH) from the freshwater agar-degrading Cellvibrio sp. KY-GH-1 were expressed and purified as recombinant His-tagged proteins using an Escherichia coli expression system to compare activities. The amino acid sequence of GH117A α-NABH (364 amino acids, 40.9 kDa) showed 35% identity with that of GH117B α-NABH (392 amino acids, 44.2 kDa). GH117A α-NABH, but not GH117B α-NABH, could hydrolyze neoagarobiose (NA2) into monosaccharides 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The presence of GH117A α-NABH homologues in all of the agar-degrading bacteria aligned suggests that GH117A α-NABH hydrolyzing NA2 into L-AHG and D-galactose is an essential component of the agar-degrading enzyme machinery. For GH117A α-NABH-catalyzed hydrolysis, NA2 was the sole substrate among various neoagaro-oligosaccharides (NA2~NA18). GH117A α-NABH appeared to exist as a dimer, and optimal enzymatic temperature and pH were 35 °C and 7.5, respectively. GH117A α-NABH was stable up to 35 °C and at pH 7.5 and unstable beyond 35 °C and outside pH 7.0~7.5. The kinetic parameters Km, Vmax, kcat, and kcat/Km for NA2 were 16.0 mM, 20.8 U/mg, 14.2 s-1, and 8.9 × 102 s-1 M-1, respectively. Combined addition of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine enhanced the enzyme activity by 2.4-fold. The enzyme-mediated hydrolysis of 5.0% NA2 into monosaccharide and purification of L-AHG from hydrolysis products by Sephadex G-10 column chromatography recovered ~ 192 mg L-AHG from 400 mg NA2 (~ 92% of the theoretical maximum yield). These results indicate that the recombinant GH117A α-NABH is NA2-specific and useful to produce L-AHG from NA2. KEY POINTS: • Recombinant GH117A α-NABH (364 aa, 40.9 kDa) purified from E. coli forms a dimer. • The enzyme hydrolyzes only NA2 among various neoagaro-oligosaccharides (NA2~NA18). • The enzyme completely hydrolyzes up to 5% NA2 into monomers under optimal conditions.


Assuntos
Galactosidases , Monossacarídeos , Dissacarídeos , Escherichia coli/genética , Glicosídeo Hidrolases , Hidrólise
17.
Genetics ; 217(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33724406

RESUMO

Dollo's law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.


Assuntos
Evolução Molecular , Proteínas Fúngicas/genética , Fungos/genética , Galactosidases/genética , Transferência Genética Horizontal , Fungos/classificação , Galactose/genética , Galactose/metabolismo , Filogenia
18.
J Dairy Sci ; 104(3): 2735-2747, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33455743

RESUMO

The activities of ß-galactosidases from bacteria and molds are affected by temperature, pH, and other factors in the processing of dairy products, limiting their application, so it is necessary to find alternative lactases. In this study, the ß-galactosidase gene from Bacillus coagulans T242 was cloned, co-expressed with a molecular chaperone in Escherichia coli BL21, and subjected to bioinformatic and kinetic analyses and lactase characterization. The results show that the enzyme is a novel thermostable neutral lactase with optimum hydrolytic activity at pH 6.8 and 50°C. The thermal stability and increased lactose hydrolysis activity of ß-galactosidase in the presence of Ca2+ indicated its potential application in the dairy industry.


Assuntos
Bacillus coagulans , Galactosidases , Animais , Clonagem Molecular , Biologia Computacional , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Lactose , Temperatura , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
19.
Ann Palliat Med ; 10(4): 4926-4931, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33040545

RESUMO

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA). Male patients of FD develop early sign and symptoms in childhood or adolescence. However, "de novo somatic mosaicism" is rare and might be developed a relatively mild phenotype despite carrying a classic type. A 34-year-old male patient visited with foamy urine. Renal biopsy findings were consistent with FD. Leukocyte α-galactosidase activity was markedly reduced at 5.3 nmol/hr/mg (normal range, 25-126). Sequence analysis of the patient's GLA gene identified mosaicism for the mutation GLA[NM_000169.2] c.820=/G>C. This mutation results in a substitution of the amino acid in position 274 from glycine to arginine. However, no family members showed FD-related symptoms, and the daughter of the patient was also tested for paternity and was identified as a real biological daughter, but DNA sequence analysis for FD showed no mutations. Based on these results, we diagnosed the patients as de novo mutation with somatic mosaicism. Next generation sequencing turned out that 58% of the readings had the mutated allele in buccal cells, 84% in blood, and 85% in urine, when 100% should be expected in a hemizygous affected male confirming the presence of somatic mosaicisms. The patient has been on treatment for enzyme replacement therapy (agalsidase-ß, 1.0 mg/kg biweekly) for past 9 years and has maintained normal renal function (serum creatinine 1.0 mg/dL) with mild albuminuria (123 mg/g Cr). Therefore, this case suggests somatic mosaicism is one of important phenotype modifiers.


Assuntos
Doença de Fabry , Mosaicismo , Adulto , Doença de Fabry/genética , Galactosidases/genética , Humanos , Masculino , Mutação
20.
Pharmacology ; 106(1-2): 3-8, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32739918

RESUMO

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.


Assuntos
Equinococose/terapia , Echinococcus multilocularis/química , Echinococcus multilocularis/efeitos dos fármacos , Galactosidases/farmacologia , Açúcares/química , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Meios de Cultura , Equinococose/parasitologia , Echinococcus multilocularis/imunologia , Echinococcus multilocularis/ultraestrutura , Feminino , Citometria de Fluxo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Polissacarídeos/química , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo
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