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1.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204583

RESUMO

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.


Assuntos
Doença de Fabry/genética , Predisposição Genética para Doença , Mutação , alfa-Galactosidase/genética , Alelos , Animais , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Gerenciamento Clínico , Ativação Enzimática , Doença de Fabry/diagnóstico , Doença de Fabry/metabolismo , Doença de Fabry/terapia , Humanos , Relação Estrutura-Atividade , Resultado do Tratamento , alfa-Galactosidase/química , alfa-Galactosidase/metabolismo
2.
FASEB J ; 35(8): e21818, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34320241

RESUMO

Fabry disease results from a deficiency of the lysosomal enzyme ⍺-Galactosidase-A (⍺-Gal A) and is estimated to occur in approximately 1:4100 live births. Characteristic of the disease is the accumulation of α-Gal-A substrates, primarily the glycosphingolipids (GSLs) globotriaosylceramide and globotriaosylsphingosine. Thrombotic events are a significant concern for Fabry patients, with strokes contributing to a significant decrease in overall lifespan. Currently, the mechanisms underlying the increased risk of thrombotic events experienced by Fabry patients are incompletely defined. Using a rat model of Fabry disease, we provide an improved understanding of the mechanisms linking GSL accumulation to thrombotic risk. We found that ⍺-Gal A-deficient rats accumulate myeloid-derived leukocytes at sites of GSL accumulation, including in the bone marrow and circulation, and that myeloid-derived leukocyte and megakaryocyte populations were prominent among cell types that accumulated GSLs. In the circulation, ⍺-Gal A-deficient rats had increases in cytokine-producing cell types and a corresponding elevation of pro-inflammatory cytokines. Lastly, circulating platelets from ⍺-Gal A-deficient rats accumulated a similar set of ⍺-Galactosidase-A substrates as was observed in megakaryocytes in the bone marrow, and exhibited increased platelet binding to fibrinogen in microfluidic and flow cytometric assays.


Assuntos
Plaquetas/citologia , Doença de Fabry/metabolismo , Células Mieloides/classificação , Células Mieloides/fisiologia , alfa-Galactosidase/metabolismo , Animais , Medula Óssea/enzimologia , Sistemas CRISPR-Cas , Feminino , Leucócitos/fisiologia , Masculino , Megacariócitos/fisiologia , Ativação Plaquetária , Agregação Plaquetária , Ratos , alfa-Galactosidase/genética
3.
Int J Mol Sci ; 22(9)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922740

RESUMO

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene that result in a deficiency of the enzymatic activity of α-galactosidase A and consequent accumulation of glycosphingolipids in body fluids and lysosomes of the cells throughout the body. GB3 accumulation occurs in virtually all cardiac cells (cardiomyocytes, conduction system cells, fibroblasts, and endothelial and smooth muscle vascular cells), ultimately leading to ventricular hypertrophy and fibrosis, heart failure, valve disease, angina, dysrhythmias, cardiac conduction abnormalities, and sudden death. Despite available therapies and supportive treatment, cardiac involvement carries a major prognostic impact, representing the main cause of death in FD. In the last years, knowledge has substantially evolved on the pathophysiological mechanisms leading to cardiac damage, the natural history of cardiac manifestations, the late-onset phenotypes with predominant cardiac involvement, the early markers of cardiac damage, the role of multimodality cardiac imaging on the diagnosis, management and follow-up of Fabry patients, and the cardiac efficacy of available therapies. Herein, we provide a comprehensive and integrated review on the cardiac involvement of FD, at the pathophysiological, anatomopathological, laboratory, imaging, and clinical levels, as well as on the diagnosis and management of cardiac manifestations, their supportive treatment, and the cardiac efficacy of specific therapies, such as enzyme replacement therapy and migalastat.


Assuntos
Arritmias Cardíacas/terapia , Terapia de Reposição de Enzimas , Doença de Fabry/terapia , alfa-Galactosidase/administração & dosagem , alfa-Galactosidase/metabolismo , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/etiologia , Doença de Fabry/complicações , Doença de Fabry/enzimologia , Humanos
4.
Microb Cell Fact ; 20(1): 71, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33736637

RESUMO

BACKGROUND: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and ß-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. RESULTS: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on ß-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as ß-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant ß-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. CONCLUSIONS: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and ß-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases.


Assuntos
Bacillus coagulans/enzimologia , Galactosídeos/metabolismo , Oligossacarídeos/biossíntese , Prebióticos , beta-Galactosidase/metabolismo , Bacillus coagulans/crescimento & desenvolvimento , Bacillus coagulans/metabolismo , Biocatálise , Clonagem Molecular , Estabilidade Enzimática , Galactose/análise , Galactose/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Oligossacarídeos/química , Análise de Sequência de DNA , Especificidade por Substrato , alfa-Galactosidase/metabolismo , beta-Galactosidase/química , beta-Galactosidase/genética
5.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669157

RESUMO

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.


Assuntos
Galactose/biossíntese , Oligossacarídeos/biossíntese , Prebióticos/análise , alfa-Galactosidase/metabolismo , Aspergillus niger/enzimologia , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactose/química , Oligossacarídeos/química , alfa-Galactosidase/química
6.
Biomolecules ; 11(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673160

RESUMO

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.


Assuntos
Doença de Fabry , Animais , Inibidores Enzimáticos/farmacologia , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico , Doença de Fabry/genética , Doença de Fabry/fisiopatologia , Feminino , Humanos , Masculino , Sondas Moleculares/metabolismo , Mutação , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
7.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573215

RESUMO

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA-) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA- cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA- hFUT2×hGLA cells as compared to the TSA- nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.


Assuntos
Epigênese Genética/efeitos dos fármacos , Epitopos/metabolismo , Rejeição de Enxerto/prevenção & controle , Ácidos Hidroxâmicos/farmacologia , Transplante Heterólogo/efeitos adversos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Clonagem de Organismos/métodos , Criopreservação , Embrião de Mamíferos , Epitopos/genética , Epitopos/imunologia , Fibroblastos , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Fucosiltransferases/metabolismo , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Suínos , Transplante Heterólogo/métodos , alfa-Galactosidase/genética , alfa-Galactosidase/imunologia , alfa-Galactosidase/metabolismo
8.
Eur Rev Med Pharmacol Sci ; 25(2): 845-855, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33577039

RESUMO

Anderson-Fabry disease (FD) is a rare genetic, progressive, and multi-systemic condition, with X-linked inheritance. This is caused by pathogenic variants in the GLA gene, coding for the lysosomal enzyme called alpha-galactosidase A (aGLA), responsible for the cleavage of globotriaosylceramide (Gb3). The reduced or absent activity of aGLA causes the intracellular accumulation of Gb3, particularly in smooth and endothelial muscle cells, which causes cellular dysfunction. The main organs involved are the central nervous system, heart, and kidneys. However, being a ubiquitous enzyme, FD disease must be considered a systemic disease involving the peripheral nervous system, ocular and audio-vestibular systems. Also, the vascular district is damaged but the pathophysiology of vasculopathy in FD is not yet entirely understood. In literature, many vascular diagnostic tests were used to evaluate this specific involvement in FD, i.e., carotid intima media thickness (cIMT), arterial stiffness (AS), flow-mediated dilation (FMD) and atherosclerotic plaques; evaluation of vascular calcifications in FD patients is not presently available. In this review, we examined the current available literature on vascular aspects in FD. Moreover, we presented our global vascular evaluation, based on Radio Frequency Duplex Ultrasound (RF-DU), plaques, and vascular calcifications, to apply to FD patients.


Assuntos
Artérias/patologia , Doença de Fabry/diagnóstico , Artérias/metabolismo , Doença de Fabry/metabolismo , Humanos , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
9.
ACS Appl Mater Interfaces ; 13(7): 7825-7838, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33583172

RESUMO

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.


Assuntos
Terapia de Reposição de Enzimas , Doença de Fabry/terapia , Nanoestruturas/química , alfa-Galactosidase/metabolismo , Doença de Fabry/enzimologia , Humanos , Oligopeptídeos/química , Tamanho da Partícula , Propriedades de Superfície , Tensoativos/química
10.
J Agric Food Chem ; 69(3): 955-965, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33434031

RESUMO

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with ß-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.


Assuntos
Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Lactobacillus plantarum/metabolismo , Oligossacarídeos/biossíntese , alfa-Galactosidase/metabolismo , Proteínas de Bactérias/genética , Galactose/análise , Lactobacillus plantarum/enzimologia , Lactobacillus plantarum/genética , Lactulose/metabolismo , Oligossacarídeos/química , alfa-Galactosidase/genética
11.
Protein Expr Purif ; 177: 105752, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32949707

RESUMO

Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha-d-galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies.


Assuntos
Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Galactose/química , Imino Piranoses/química , Animais , Células CHO , Cricetulus , Estabilidade Enzimática , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
12.
Molecules ; 25(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114310

RESUMO

Pleurotus ostreatus is one of the widely cultivated edible fungi across the world. Mycelial subculture is an indispensable part in the process of cultivation and production for all kinds of edible fungi. However, successive subcultures usually lead to strain degeneration. The degenerated strains usually have a decrease in stress resistance, yield, and an alteration in fruiting time, which will subsequently result in tremendous economic loss. Through proteomic analysis, we identified the differentially expressed proteins (DEPs) in the mycelium of Pleurotus ostreatus from different subcultured generations. We found that the DNA damage repair system, especially the double-strand breaks (DSBs), repairs via homologous recombination, was impaired in the subcultured mycelium, and gradual accumulation of the DSBs would lead to the strain degeneration after successive subculture. The TUNEL assay further confirmed our finding about the DNA breaks in the subcultured mycelium. Interestingly, the enzyme activity of laccase, carboxylic ester hydrolase, α-galactosidase, and catalase directly related to passage number could be used as the characteristic index for strain degeneration determination. Our results not only reveal for the first time at the molecular level that genomic instability is the cause of degeneration, but also provide an applicable approach for monitoring strain degeneration in process of edible fungi cultivation and production.


Assuntos
Extratos Celulares/química , Carpóforos/metabolismo , Proteínas Fúngicas/genética , Micélio/enzimologia , Pleurotus/química , Proteômica/métodos , Carboxilesterase/genética , Carboxilesterase/metabolismo , Catalase/genética , Catalase/metabolismo , Células Cultivadas , Estabilidade Enzimática , Indústria Alimentícia , Proteínas Fúngicas/metabolismo , Lacase/genética , Lacase/metabolismo , Espectrometria de Massas em Tandem , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
13.
Int J Mol Sci ; 21(20)2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33081309

RESUMO

The biofilm-producing strains of P. aeruginosa colonize various surfaces, including food products and industry equipment that can cause serious human and animal health problems. The biofilms enable microorganisms to evolve the resistance to antibiotics and disinfectants. Analysis of the P. aeruginosa strain (serotype O6, sequence type 2502), isolated from an environment of meat processing (PAEM) during a ready-to-cook product storage (-20 °C), showed both the mosaic similarity and differences between free-living and clinical strains by their coding DNA sequences. Therefore, a cold shock protein (CspA) has been suggested for consideration of the evolution probability of the cold-adapted P. aeruginosa strains. In addition, the study of the action of cold-active enzymes from marine bacteria against the food-derived pathogen could contribute to the methods for controlling P. aeruginosa biofilms. The genes responsible for bacterial biofilm regulation are predominantly controlled by quorum sensing, and they directly or indirectly participate in the synthesis of extracellular polysaccharides, which are the main element of the intercellular matrix. The levels of expression for 14 biofilm-associated genes of the food-derived P. aeruginosa strain PAEM in the presence of different concentrations of the glycoside hydrolase of family 36, α-galactosidase α-PsGal, from the marine bacterium Pseudoalteromonas sp. KMM 701 were determined. The real-time PCR data clustered these genes into five groups according to the pattern of positive or negative regulation of their expression in response to the action of α-galactosidase. The results revealed a dose-dependent mechanism of the enzymatic effect on the PAEM biofilm synthesis and dispersal genes.


Assuntos
Biofilmes , Microbiologia de Alimentos , Genes Bacterianos , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas e Peptídeos de Choque Frio/genética , Proteínas e Peptídeos de Choque Frio/metabolismo , Produtos da Carne/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
14.
Nat Cell Biol ; 22(11): 1371-1381, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33077910

RESUMO

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.


Assuntos
Neoplasias da Mama/enzimologia , Calnexina/metabolismo , Movimento Celular , Retículo Endoplasmático/enzimologia , Matriz Extracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Pulmonares/enzimologia , Podossomos/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Calnexina/antagonistas & inibidores , Linhagem Celular Tumoral , Retículo Endoplasmático/patologia , Matriz Extracelular/patologia , Feminino , Glicosilação , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Podossomos/patologia , Transporte Proteico , Proteólise , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Galactosidase/metabolismo
15.
J Agric Food Chem ; 68(39): 10560-10573, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32829638

RESUMO

α-Galactosidase has potential applications, and attempts to improve proteolytic resistance of enzymes have important values. We use a novel strategy for genetic manipulation of a pepsin-sensitive region specific for a pepsin-sensitive but trypsin-resistant high-temperature-active Gal27B from Neosartorya fischeri to screen mutants with enhanced pepsin resistance. All enzymes were produced in Pichia pastoris to identify the roles of loop 4 (Gal27B-A23) and its key residue at position 156 (Gly156Arg/Pro/His) in pepsin resistance. Gal27B-A23 and Gly156Arg/Pro/His elevated pepsin resistance, thermostability, stability at low pH, activity toward raffinose (5.3-6.9-fold) and stachyose (about 1.3-fold), and catalytic efficiencies (up to 4.9-fold). Replacing the pepsin cleavage site Glu155 with Gly improved pepsin resistance but had no effect on pepsin resistance when Arg/Pro/His was at position 156. Thus, pepsin resistance could appear to occur through steric hindrance between the residue at the altered site and neighboring pepsin active site. In the presence of pepsin or trypsin, all mutations increased the ability of Gal27B to hydrolyze galactosaccharides in soybean flour (up to 9.6- and 4.3-fold, respectively) and promoted apparent metabolizable energy and nutrient digestibility in soybean meal for broilers (1.3-1.8-fold). The high activity and tolerance to heat, low pH, and protease benefit food and feed industry in a cost-effective way.


Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pepsina A/química , Tripsina/química , alfa-Galactosidase/química , alfa-Galactosidase/genética , Ácidos/química , Motivos de Aminoácidos , Ração Animal/análise , Animais , Aspergillus/química , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Engenharia de Proteínas , Especificidade por Substrato , alfa-Galactosidase/metabolismo
16.
Int J Mol Sci ; 21(14)2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32650402

RESUMO

Fruit ripening and abscission are the results of the cell wall modification concerning different components of the signaling network. However, molecular-genetic information on the cross-talk between ripe fruit and their abscission zone (AZ) remains limited. In this study, we investigated transcriptional and hormonal changes in olive (Olea europaea L. cv Picual) pericarp and AZ tissues of fruit at the last stage of ripening, when fruit abscission occurs, to establish distinct tissue-specific expression patterns related to cell-wall modification, plant-hormone, and vesicle trafficking in combination with data on hormonal content. In this case, transcriptome profiling reveals that gene encoding members of the α-galactosidase and ß-hexosaminidase families associated with up-regulation of RabB, RabD, and RabH classes of Rab-GTPases were exclusively transcribed in ripe fruit enriched in ABA, whereas genes of the arabinogalactan protein, laccase, lyase, endo-ß-mannanase, ramnose synthase, and xyloglucan endotransglucosylase/hydrolase families associated with up-regulation of RabC, RabE, and RabG classes of Rab-GTPases were exclusively transcribed in AZ-enriched mainly in JA, which provide the first insights into the functional divergences among these protein families. The enrichment of these protein families in different tissues in combination with data on transcript abundance offer a tenable set of key genes of the regulatory network between olive fruit tissues in late development.


Assuntos
Frutas/genética , Frutas/metabolismo , Olea/genética , Olea/metabolismo , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Transcriptoma/genética , Parede Celular/genética , Parede Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes/genética , Transdução de Sinais/genética , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo
17.
PLoS One ; 15(7): e0235687, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678825

RESUMO

Lactobacillus amylolyticus L6, a gram-positive amylolytic bacterium isolated from naturally fermented tofu whey (NFTW), was able to hydrolyze raffinose and stachyose for the production of α-galactosidase. The cell-free extract of L. amylolyticus L6 was found to exhibit glycosyltransferase activity to synthesize α-galacto-oligosaccharides (GOS) with melibiose as substrate. The coding genes of α-galactosidase were identified in the genome of L. amylolyticus L6. The α-galactosidase (AglB) was placed into GH36 family by amino acid sequence alignments with other α-galactosidases from lactobacilli. The optimal reaction conditions of pH and temperature for AglB were pH 6.0 and 37°C, respectively. Besides, potassium ion was found to improve the activity of AglB while divalent mercury ion, copper ion and zinc ion displayed different degrees of inhibition effect. Under the optimum reaction condition, AglB could catalyze the synthesis of GOS with degree of polymerization (DP) ≥5 by using 300 mM melibiose concentration as substrate. The maximum yield of GOS with (DP) ≥3 could reach 31.56% (w/w). Transgalactosyl properties made AglB a potential candidate for application in the production of GOS.


Assuntos
Proteínas de Bactérias/metabolismo , Clonagem Molecular , Lactobacillus/enzimologia , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura , alfa-Galactosidase/química , alfa-Galactosidase/genética
18.
Drug Des Devel Ther ; 14: 2149-2158, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32581513

RESUMO

Purpose: To determine the impact of initiating enzyme replacement therapy (ERT) with agalsidase alfa early in the course of Fabry disease, we evaluated renal and cardiac outcomes for ≤10 years after ERT initiation in males from the Fabry Outcome Survey (FOS). Patients and Methods: Male patients from FOS were stratified into three cohorts by age at ERT initiation: ≤18 years (cohort 1), >18 and ≤30 years (cohort 2), and >30 years (cohort 3). Analysis included age at symptom onset, diagnosis, and ERT initiation; ERT duration; FOS-Mainz Severity Score Index (FOS-MSSI); estimated glomerular filtration rate (eGFR); proteinuria level; and left ventricular mass indexed to height (LVMI). Mixed-effect models estimated renal and cardiac outcomes during follow-up between and within cohorts. Findings: The analysis included 560 male patients: 151 (27.0%) in cohort 1, 155 (27.7%) in cohort 2, and 254 (45.4%) in cohort 3. Mean±SD duration of ERT for cohorts 1, 2, and 3 was 6.3±4.3, 8.6±4.9, and 7.9±4.9 years, respectively. Mean±SD baseline FOS-MSSI scores increased with age from 9.8±7.2 in cohort 1 to 24.7±11.4 in cohort 3. Cohort 3 showed the lowest baseline mean±SD value for eGFR (87.1±29.0 mL/min/1.73m2) and highest baseline mean±SD values for proteinuria (801.9±952.6 mg/day) and LVMI (56.7±16.0 g/m2.7) among the three cohorts. Evaluation of mean annual rates of change in eGFR, proteinuria, and LVMI revealed no significant differences in any parameter for cohort 1. For cohort 2, proteinuria and LVMI remained stable, whereas eGFR significantly deteriorated annually (-1.12 mL/min/1.73m2; P<0.001). Cohort 3 demonstrated significant annual deteriorations in eGFR (-2.60 mL/min/1.73m2; P<0.001), proteinuria (+34.10 mg/day; P<0.001), and LVMI (+0.59 g/m2.7; P=0.001). Implications: Renal and/or cardiac disease progression appears attenuated in patients starting ERT in childhood or early adulthood versus patients starting ERT in later adulthood. These findings support early ERT initiation in Fabry disease. ClinicalTrials.gov identifier: NCT03289065.


Assuntos
Terapia de Reposição de Enzimas , Doença de Fabry/terapia , Isoenzimas/metabolismo , Proteínas Recombinantes/metabolismo , alfa-Galactosidase/metabolismo , Adolescente , Adulto , Criança , Doença de Fabry/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
19.
Biomolecules ; 10(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486191

RESUMO

Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A (rh-α-Gal A) is the standard treatment for Fabry disease (FD). ERT has shown a significant impact on patients; however, there is still morbidity and mortality in FD, resulting in progressive cardiac, renal, and cerebrovascular pathology. The main pathway for delivery of rh-α-Gal A to lysosome is cation-independent mannose-6-phosphate receptor (CI-M6PR) endocytosis, also known as insulin-like growth factor 2 receptor (IGF2R) endocytosis. This study aims to investigate the mechanisms of uptake of rh-α-Gal-A in different cell types, with the exploration of clathrin-dependent and caveolin assisted receptor-mediated endocytosis and the dynamics of autophagy-lysosomal functions. rh-α-Gal-A uptake was evaluated in primary fibroblasts, urine originated kidney epithelial cells, and peripheral blood mononuclear cells derived from Fabry patients and healthy controls, and in cell lines HEK293, HTP1, and HUVEC. Uptake of rh-α-Gal-A was more efficient in the cells with the lowest endogenous enzyme activity. Chloroquine and monensin significantly blocked the uptake of rh-α-Gal-A, indicating that the clathrin-mediated endocytosis is involved in recombinant enzyme delivery. Alternative caveolae-mediated endocytosis coexists with clathrin-mediated endocytosis. However, clathrin-dependent endocytosis is a dominant mechanism for enzyme uptake in all cell lines. These results show that the uptake of rh-α-Gal-A occurs rapidly and activates the autophagy-lysosomal pathway.


Assuntos
Autofagia , Doença de Fabry/terapia , alfa-Galactosidase/metabolismo , Linhagem Celular , Clatrina/metabolismo , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico , Humanos , Lisossomos/metabolismo , Proteínas Recombinantes/metabolismo
20.
J Agric Food Chem ; 68(22): 6161-6169, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32390413

RESUMO

α-Galactosidase catalyzes the hydrolysis of a terminal α-galactose residue in galacto-oligosaccharides and has potential in various industrial applications and food processing. We determined the crystal structures of α-galactosidase from the thermophilic microorganism Thermus thermophilus (TtGalA) and its complexes with pNPGal and stachyose. The monomer folds into an N-terminal domain, a catalytic (ß/α)8 barrel domain, and a C-terminal domain. The domain organization is similar to that of the other family of 36 α-galactosidases, but TtGalA presents a cagelike hexamer. Structural analysis shows that oligomerization may be a key factor for the thermal adaption of TtGalA. The structure of TtGalA complexed with stachyose reveals only the existence of one -1 subsite and one +1 subsite in the active site. Structural comparison of the stachyose-bound complexes of TtGalA and GsAgaA, a tetrameric enzyme with four subsites, suggests evolutionary divergence of substrate specificity within the GH36 family of α-galactosidases. To the best of our knowledge, the crystal structure of TtGalA is the first report of a quaternary structure as a hexameric assembly in the α-galactosidase family.


Assuntos
Proteínas de Bactérias/química , Thermus thermophilus/enzimologia , alfa-Galactosidase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Galactose/metabolismo , Domínios Proteicos , Multimerização Proteica , Especificidade por Substrato , Thermus thermophilus/química , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
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