RESUMO
Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.
Assuntos
Acetilglucosaminidase , Mucopolissacaridose III , Humanos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Acetilglucosaminidase/metabolismo , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/química , Acetilglucosaminidase/genética , Sítio Alostérico/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacosRESUMO
The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.
Assuntos
Acetilglucosaminidase/química , Acetilglucosaminidase/genética , Clonagem Molecular/métodos , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Acetilglucosaminidase/farmacologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Queijo/microbiologia , Cromatografia Líquida , Enterococcus/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Indústria Alimentícia , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Listeria monocytogenes/efeitos dos fármacos , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Staphylococcus aureus/efeitos dos fármacos , Especificidade por Substrato , Espectrometria de Massas em Tandem , TemperaturaRESUMO
A phosphorolytic activity has been reported for beta-N-acetylglucosaminidases from glycoside hydrolase family 3 (GH3) giving an interesting explanation for an unusual histidine as catalytic acid/base residue and suggesting that members from this family may be phosphorylases [J. Biol. Chem. 2015, 290, 4887]. Here, we describe the characterization of Hsero1941, a GH3 beta-N-acetylglucosaminidase from the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1. The enzyme has significantly higher activity against pNP-beta-D-GlcNAcp (Km = 0.24 mM, kcat = 1.2 s-1, kcat/Km = 5.0 mM-1s-1) than pNP-beta-D-Glcp (Km = 33 mM, kcat = 3.3 × 10-3 s-1, kcat/Km = 9 × 10-4 mM-1s-1). The presence of phosphate failed to significantly modify the kinetic parameters of the reaction. The enzyme showed a broad aglycone site specificity, being able to hydrolyze sugar phosphates beta-D-GlcNAc 1P and beta-D-Glc 1P, albeit at a fraction of the rate of hydrolysis of aryl glycosides. GH3 beta-glucosidase EryBI, that does not have a histidine as the general acid/base residue, also hydrolyzed beta-D-Glc 1P, at comparable rates to Hsero1941. These data indicate that Hsero1941 functions primarily as a hydrolase and that phosphorolytic activity is likely adventitious. The prevalence of histidine as a general acid/base residue is not predictive, nor correlative, with GH3 beta-N-acetylglucosaminidases having phosphorolytic activity.
Assuntos
Acetilglucosaminidase/metabolismo , Glucosidases/metabolismo , Herbaspirillum/enzimologia , Saccharopolyspora/enzimologia , Acetilglucosaminidase/química , Acetilglucosaminidase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Glucosidases/química , Glucosidases/genética , Herbaspirillum/genética , Hidrólise , Fosforilação , Saccharopolyspora/genética , Especificidade por SubstratoRESUMO
The use of cell wall degrading enzymes from Trichoderma asperellum immobilized on biodegradable support is an alternative for food packaging. In this study, hydrolytic enzymes produced by T. asperellum were tested as a fungal growth inhibitor, in free form or immobilized on a biodegradable film composed of cassava starch and poly(butylene adipate-co-terephtalate) (PBAT). The inhibitory activity was tested against Aspergillus niger , Penicillium sp., and Sclerotinia sclerotiorum , microorganisms that frequently degrade food packaging. The use of chitin as carbon source in liquid medium induced T. asperellun to produce N-acetylglucosaminidase, ß-1,3-glucanase, chitinase, and protease. The presence of T. asperellun cell wall degradating enzymes (T-CWD) immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. The enzymatic activity of T-CWD was stronger on S. sclerotiorum than on the Aspergillus or Penicillum isolates tested. These results suggest that T-CWD can be used in a free or immobilized form to suppress fungi that degrade food packaging.
Assuntos
Acetilglucosaminidase/farmacologia , Antifúngicos/farmacologia , Quitinases/farmacologia , Enzimas Imobilizadas/farmacologia , Proteínas Fúngicas/farmacologia , Fungos/efeitos dos fármacos , Glucana 1,3-beta-Glucosidase/farmacologia , Trichoderma/enzimologia , Acetilglucosaminidase/química , Antifúngicos/química , Parede Celular/efeitos dos fármacos , Quitinases/química , Enzimas Imobilizadas/química , Embalagem de Alimentos , Conservação de Alimentos , Proteínas Fúngicas/química , Fungos/crescimento & desenvolvimento , Glucana 1,3-beta-Glucosidase/química , Hidrólise , Trichoderma/químicaRESUMO
The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFalpha and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70,000 and pI approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization.
Assuntos
Acetilglucosaminidase/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Lectinas/isolamento & purificação , Lectinas/metabolismo , Paracoccidioides , Acetilglucosamina/metabolismo , Acetilglucosaminidase/química , Acetilglucosaminidase/isolamento & purificação , Animais , Anticorpos Antifúngicos/imunologia , Western Blotting , Parede Celular/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Lectinas/química , Lectinas/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Paracoccidioides/enzimologia , Paracoccidioides/metabolismo , Mapeamento de Peptídeos , Ligação Proteica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-beta-N-acetylglucosaminide as substrate were of 0.13 mM: and 1.95 U mg(-1) protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 degrees C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-beta-N-acetylglucosaminide and p-nitrophenyl-beta-D-N,N'-diacetylchitobiose.
Assuntos
Acetilglucosaminidase/química , Acetilglucosaminidase/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Streptomyces/enzimologia , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Especificidade por SubstratoRESUMO
OBJECTIVE: Using the murine sponge model, we investigated the temporal relationship between angiogenesis, leukocyte accumulation and endogenous generation of the pro-inflammatory chemokines CXCL1-3/KC and CCL2/JE. Furthermore, the effects of exogenous administration of these chemokines were studied. METHODS: Angiogenesis in the implants was assessed by measuring the hemoglobin content (vascular index) and leukocyte accumulation quantified by evaluating MPO and NAG enzyme activities. RESULTS: A progressive increase in hemoglobin content and in enzymatic activities was observed during the whole period. The levels of CXCL1-3/KC and CCL2/JE in the implants peaked at days 7 and 1, respectively. Exogenous administration of CXCL1-3/KC (100 ng/day intra-implant) applied at days 1-3 resulted in increased neovascularization and macrophage accumulation. Intra-implant injections of CCL2/JE (100 ng/day) also resulted in increased angiogenesis and macrophage accumulation. CONCLUSIONS: These results demonstrated that the chemokines, CXCL1-3/KC and CCL2/JE, are generated within the sponge compartment and that neovascularization and inflammatory cells influx can be modulated by exogenous administration of the chemokines.
Assuntos
Quimiocina CCL2/biossíntese , Quimiocinas CXC/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Neovascularização Patológica , Acetilglucosaminidase/química , Animais , Quimiocina CXCL1 , Quimiocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hemoglobinas/metabolismo , Imuno-Histoquímica , Inflamação , Cinética , Leucócitos/citologia , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Peroxidase/metabolismo , Proteínas Recombinantes/química , Fatores de TempoRESUMO
The effect of tunicamycin, an inhibitor of protein N-glycosylation, was studied in non-growing mycelium of Trichoderma harzianum induced to secrete N-acetyl-beta-D-glucosaminidase by the addition of N-acetylglucosamine. Tunicamycin (30 microg ml(-1)) had no significant effect on growth of the fungus, or on the total protein secreted or specific activity of N-acetyl-beta-D-glucosaminidase. However, in the presence of the inhibitor an underglycosylated form of the enzyme was produced. The apparent molecular masses for this and the native enzyme were 110 and 124 kDa, respectively. Both forms of the enzyme showed the same optimum pH and temperature, but the underglycosylated form was more sensitive to inactivation by both high temperature (60 degrees C) and the proteolytic enzyme trypsin.
Assuntos
Acetilglucosaminidase/metabolismo , Antibacterianos/farmacologia , Trichoderma/efeitos dos fármacos , Tunicamicina/farmacologia , Acetilglucosamina/farmacologia , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/química , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Temperatura , Trichoderma/enzimologiaRESUMO
After a 10.5-fold purification, beta-N-acetyl-D-glucosaminidase (EC 3.2.1.52) produced by Aspergillus niger 419, showed the following main characteristics: maximum activity at 65 degrees C, pH 4.5; K(m) and kcat using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate, 0.2 mM and 0.93 x 10(4) min-1, respectively; Ea, 30.5 kJ/mol; molecular mass, 131,000 Da; pI 4.4. The activity after heating for 15 min at 70, 75 and 80 degrees C was 70, 28 and 13% of that found at 65 degrees C, respectively. The enzyme was active in reaction mixtures containing glycerol, ethanol, methanol, propan-2-ol, acetone or dioxan. The presence of Sr2+ or Ca2+ enhanced the activity, while it was inhibited by Cu2+ and Fe3+. The enzyme was highly specific for p-nitrophenyl N-acetyl-beta-D-glucosaminide and no activity was found when p-nitrophenyl derivatives of N-acetyl-beta-D-galactosaminide, beta-D-galactopyranoside and beta-D-N,N'-diacetylchitobiose were tested as substrates. Due to its thermostability, specificity and resistance to different organic solvents, the enzyme might be a potentially useful tool for the analysis and production of oligosaccharides.