RESUMO
Precision cell-selective surface glycan remodeling is of vital importance for modulation of cell surface dynamics, tissue-specific imaging, and immunotherapy, but remains an unsolved challenge. Herein, we report a switchable enzymatic accessibility (SEA) strategy for highly specific editing of carbohydrate moieties of interest on the target cell surface. We demonstrate the blocking of enzyme in the inaccessible state with a metal-organic framework (MOF) cage and instantaneous switching to the accessible state through disassembly of MOF. We further show that this level of SEA regulation enables initial guided enzyme delivery to the target cell surface for subsequent cell-specific glycan remodeling, thus providing a temporally and spatially controlled tool for tuning the glycosylation architectures. Terminal galactose/N-acetylgalactosamine (Gal/GalNAc) remodeling and terminal sialic acid (Sia) desialylation have been precisely achieved on target cells even with other cell lines in close spatial proximity. The SEA protocol features a modular and generically adaptable design, a very short protocol duration (ca. 30â min or shorter), and a very high spatial resolving power (ability to differentiate immediately neighboring cell lines).
Assuntos
Membrana Celular/enzimologia , Polissacarídeos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Aptâmeros de Nucleotídeos/química , Biocatálise , Membrana Celular/química , Ativação Enzimática , Galactose/química , Galactose/metabolismo , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Estruturas Metalorgânicas/química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Imagem Óptica/métodos , Polissacarídeos/química , Propriedades de SuperfícieRESUMO
BACKGROUND: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology - particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. OBJECTIVES: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. METHODS: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. RESULTS: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. CONCLUSION: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label-free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.
Assuntos
Inibidores Enzimáticos/química , Galactose Oxidase/química , Galactose/química , Ensaios de Triagem em Larga Escala , Adsorção , Biocatálise , Galactose Oxidase/antagonistas & inibidores , Ouro/química , Halogenação , Peróxido de Hidrogênio/química , Monossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Extra-cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 degrees C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four-day-old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one-step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS-PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl-galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH(4)(+), Na(+), Mg(2+), K(+), and glycerol were not. The Michaelis-Menten constant (K(m)) for galactose was estimated to be 16.2 mM, while maximal velocity (V(max)) was 0.27 micromol of H(2)O(2) . ml(-1) . min(-1).
Assuntos
Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Galactose Oxidase/isolamento & purificação , Galactose Oxidase/metabolismo , Metabolismo dos Carboidratos , Cobre , Meios de Cultura , Fermentação , Fusarium/crescimento & desenvolvimento , Galactose Oxidase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Magnésio , Manganês , Dodecilsulfato de Sódio/farmacologia , Especificidade por Substrato , Tensoativos/farmacologia , TemperaturaRESUMO
The development of an indirect anaerobic electrochemical regeneration of galactose oxidase (GOase) allows the prevention of the undesired production of the enzyme inhibitor hydrogen peroxide, which is generated under aerobic regeneration conditions during synthetic applications of GOase. The pH optimum for the electrochemical regeneration of GOase with polyethyleneglycol-modified ferrocene mediators in carbonate buffer is 10.8. Total turnover numbers achieved by either electrochemical or aerobic regeneration of GOase are almost the same. The electrochemical regeneration is half as fast as the aerobic regeneration. It is not necessary to work under anaerobic conditions, because at pH 10.8 the aerobic regeneration of GOase is prevented. The enzyme can be stabilized most effectively by immobilization on an aminopropylated polysiloxane (DELOXAN) via the glutaric dialdehyde procedure with good activity yields up to 37%. Buffers containing amino groups proved to be fatal for long-term GOase stability.
Assuntos
Eletroquímica/métodos , Galactose Oxidase/química , Galactose Oxidase/metabolismo , Soluções Tampão , Eletroquímica/instrumentação , Galactose Oxidase/antagonistas & inibidores , Peróxido de Hidrogênio/química , Membranas Artificiais , Oxigênio/química , Oxigênio/metabolismo , Xilitol/químicaRESUMO
Processing of trans-2-phenylcyclopropylmethanols 5 and 6 by the monocopper/tyrosine radical enzyme galactose oxidase led to mechanism-based inactivation with a partition ratio, (k(cat) + k(inact))/k(inact), of approximately 1 and a primary deuterium isotope effect, k(inact(H))/k(inact(D)), of 3.2. The data are consistent with a radical mechanism for galactose oxidase with a short lived ketyl radical anion intermediate.
Assuntos
Galactose Oxidase/metabolismo , Sondas Moleculares , Catálise , Galactose Oxidase/antagonistas & inibidores , CinéticaRESUMO
Galactose oxidase, a mononuclear copper enzyme, oxidizes primary alcohols to aldehydes using molecular oxygen. A unique type of cross-link between tyrosine 272, an active-site copper ligand, and cysteine 228 provides a modified tyrosine radical site believed to act as a one-electron redox center. Substrate analogs incorporating a primary thiol group in place of the primary alcohol group in normal substrates (RCH2OH) have been studied as active-site mechanistic probes. Thiol sulfur coordinates to the active-site copper, leading to enzyme inactivation in a time- and concentration-dependent manner. The mechanism of inactivation involves redox chemistry related to the active-site redox centers, though inactivation does not proceed through the rate-determining hydrogen atom abstraction step that occurs in alcohol oxidation. Thiols are therefore classified as active-site-directed redox inactivators. The thiol analog of galactose, 6-Thio-Me-Gal, is also turned over by the enzyme, albeit at a much reduced rate, indicating that the energetics of turnover is changed significantly. Thiols constitute a particularly good model of the ground state enzyme-substrate complex. The Michaelis complex for thiol substrate analogs is stabilized at least 200-fold compared to the analogous alcohol substrates, whereas the transition state of H atom abstraction is destabilized, presumably due to a slight increase in distances of reacting atoms and weakening of hydrogen-bonding interactions due to the larger atomic radius of sulfur compared to that of oxygen.
Assuntos
Galactose Oxidase/química , Compostos de Sulfidrila/química , Sítios de Ligação , Catálise , Cobre/química , Inibidores Enzimáticos/química , Radicais Livres , Fusarium/enzimologia , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Sulfeto de Hidrogênio/química , Oxirredução , Especificidade por SubstratoRESUMO
The intracellular galactose oxidase from Dactylium dendroides was purified to homogeneity with a 64% yield. The enzyme is a glycoprotein (7.7% neutral sugars, 1.7% aminosugars) with 72,000 Da of molecular mass. The enzyme showed nonlinear double reciprocal plots with O2 and D-galactose, suggesting cooperative binding for both substrates. The intracellular galactose oxidase catalyzes the oxidation of galactose derivatives and dihydroxyacetone but not of glycerol, glycolaldehyde, beta-hydroxipyruvate, and allyl alcohol which are substrates for the extracellular enzyme. Compared with the extracellular galactose oxidase, the intracellular enzyme showed higher carbohydrate content and sensitivity to diethyldithiocarbamate.
Assuntos
Fungos/enzimologia , Galactose Oxidase/isolamento & purificação , Carboidratos/análise , Quelantes/farmacologia , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Cinética , Peso Molecular , Oxigênio/metabolismo , Especificidade por SubstratoRESUMO
Patterns of oxidation of dilute solutions of desialylated fetuin and submaxillary mucin by galactose oxidase have been examined. A significant portion (20-40%) of the terminal galactosyls exposed on the glycoproteins, which theoretically were expected to be accessible to the enzyme, was not oxidized. In comparison, galactosyls in oligosaccharides released from completely desialylated glycoproteins were oxidized more effectively with an apparently lower degree of crypticity to the enzyme. Partial desialylation usually resulted in a reduction of both the rate and the final level of substrate oxidation. A second cycle of oxidation of a desialylated substrate earlier oxidized by galactose oxidase and then reduced by NaB3H4 revealed a selectivity in the pattern of galactosyl oxidation. The same galactosyl residues oxidized in the first cycle were again the most susceptible to oxidation in the second cycle, leaving unmodified the same fraction of galactosyls throughout both cycles. The relevance of these results to the application of the galactose oxidase-NaBH4 procedure for detecting and measuring desialylated glycoconjugates in solution and in biological membranes is discussed.
Assuntos
Assialoglicoproteínas , Galactose Oxidase/metabolismo , Glicoproteínas/metabolismo , Animais , Bovinos , Fetuínas , Galactose Oxidase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Mucinas/metabolismo , Pronase/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.
Assuntos
Galactose Oxidase/metabolismo , Metilgalactosídeos/metabolismo , Metilglicosídeos/metabolismo , Fungos Mitospóricos/enzimologia , Sítios de Ligação , Ligação Competitiva , Cobre , Cianetos/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Fluoretos/farmacologia , Galactose Oxidase/antagonistas & inibidores , Cinética , Metaloproteínas/metabolismoRESUMO
The chemical constituents of coal have not been fully characterized in relation to the incidence of coal workers' pneumoconiosis (CWP). In this study two soft coals obtained from mines in which workers had high and low incidences of CWP were leached with aqueous base and acid to remove their acidic and basic components. The results suggest that humic substances similar to those found in soil are present in the coal samples. Further, differences in the quantity of material removed ant its metal-binding and enzyme inhibitory activity are related to disease incidence.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Carvão Mineral/análise , Galactose Oxidase/antagonistas & inibidores , Substâncias Húmicas/análise , Metais , Benzopiranos/análise , Cromatografia por Troca Iônica , Minas de Carvão , Depressão Química , Dianisidina/antagonistas & inibidores , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Metais/metabolismo , Pennsylvania , Peroxidases/antagonistas & inibidores , Pneumoconiose/etiologia , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , UtahRESUMO
Galactose oxidase is a metalloenzyme containing a single copper atom per molecule. The mechanism of action of galactose oxidase is studied in this paper by investigating substrate specificity and activation by peroxidase, and probing the copper site by electron spin resonance (ESR) spectroscopy. Line-shape simulation of ESR spectra are also reported and a comparison is made between observed and simulated spectra for galactose oxidase. A comparison is also reported for the enzyme from various commercial sources and enzyme isolated from a fungus in this laboratory. The results of this investigation suggest that the copper is in an environment of four in-plane nitrogens with axial symmetry.