Synthesis of unsaturated aminopyranosides as possible transition state mimics for glycosidases

Four unsaturated aminopyranosides have been prepared as possible transition-state mimics targeted towards carbohydrate processing enzymes. The conformations of the protonated aminosugars have been investigated by molecular modelling and their ability to inhibit alpha- and beta-glucosidases and an a-mannosidase have been probed. Two targets proved moderate inhibitors of alpha-glucosidases from Brewer's yeast and Bacillus stearothennophilus.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Trypanosoma cruzi recognition by macrophages and muscle cells: perspectives after a 15-year study

Macrophages and muscle cells are the main targets for invasion of Trypanosoma cruzi. Ultrastructural studies of this phenomenon in vitro showed that invasion occurs by endocytosis, with attachment and internalization being mediated by different components capable of recognizing epi-or trypomastigotes (TRY). A parasitophorus vacuole was formed in both cell types, thereafter fusing with lysosomes. Then, the mechanism of T. cruzi invasion of host cells (HC) is essentially similar (during a primary infection in the abscence of a specific immune response), regardless of wether the target cell is a professional or a non-professional phagocytic cell. Using sugars, lectins, glycosidases, proteinases and proteinase inhibitors, we observed that the relative balance between exposed sialic acid and galactose/N-acetyl galactosamine (GAL) residues on the TRY surface, determines the parasite's capacity to invade HC, and that lectin-mediated phagocytosis with GAL specificity is important for internalization of T. cruzi into macrophages. On the other hand, GAL on the surface to heart muscle cells participate on TRY adhesion. TRY need to process proteolytically both the HC and their own surface, to expose the necessary ligands and receptors that allow binding to, and internalization in the host cell. The diverse range of molecular mechanisms which the parasite could use to invade the host cell may correspond to differences in the available "receptors"on the surface of each specific cell type. Acute phase components, with lectin or proteinase inhibitory activities (a-macroglobulins), may also be involved in T. cruzi-host cell interaction.

Studies toward new anti-cancer strategies based on alpha-mannosidase inhibition.

Changes in the glycosylation pattern of cellular glycoproteins constitute a hallmark in human cancer and influence tumor progression, suggesting that inhibitors of selected glycosidases may control cancer progression. Following the studies on swainsonine, a natural inhibitor of Golgi alpha-mannosidase II, which highlighted the inhibition of cellular mannosidases as a potential innovative approach for the treatment of cancer, several dihydroxylated pyrrolidines and analogues were developed as new potent inhibitors of alpha-mannosidases II able to induce antiproliferative effects in human cancer cells.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

Synthesis and glycosidase inhibitory profiles of functionalised morpholines and oxazepanes

In this work libraries of morpholines and oxazepanes have been prepared via the reductive amination reaction between dialdehydes, derived from carbohydrates, and a range of amines. In this way, functionalised morpholines and oxazepanes have been prepared that include N-alkylated derivatives, disaccharide analogues, and ester containing derivatives. The abilities of these functionalised morpholines and oxazepanes to inhibit a broad panel of glycosidase enzymes, that are associated with a range of diseases, have been probed and in this way new inhibitors of a range of glycosidases, but particularly β-d-galactosidase derived from Bovine kidney, have been discovered. N-Alkyl morpholines demonstrated the best inhibition profiles for this enzyme and derivatives (15a)–(15d) acted as non-competitive inhibitors with IC50 values of 55.1–88.6 μM. Within this study, some preliminary structure–activity relationships are proposed, and it is demonstrated that N-substituted morpholines display better inhibitory profiles for the enzymes analysed than any of the N-substituted oxazepanes.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.

Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed. (C) 2009 Elsevier Inc. All rights reserved.

Purificação e caracterização parcial de uma B-D glicosidade de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Effect of long-term treatment with insulin and/or acarbose on glomerular basement membrane thickening in alloxan-diabetic rats

Acarbose is a competitive inhibitor of the intestinal alpha-glycosidases, that can delay absorption of intestinal carbohydrates causing their malabsorption. In the present paper we studied the effects of insulin, acarbose and their association on glomerular basement membrane thickening in alloxan-diabetic rats. Twenty-five male and female Wistar rats, approximately 3 months old at the beginning of the experiment, were assigned randomly to each of five experimental groups: normal control rats, alloxan-diabetic control rats, alloxan-diabetic rats treated with acarbose, alloxan-diabetic rats treated with insulin, and alloxan-diabetic rats treated with insulin plus acarbose. Alloxan was administered in a single iv dose of 42 mg/kg body weight. Insulin was given subcutaneously at doses of 18 to 30 IU/kg corrected daily on the basis of glycosuria and ketonuria. Acarbose was given mixed with rat chow in a dose of 50 mg/100 g chow. Body weight, water and food intake and diuresis, as well as blood and urine glucose were determined after 1, 3, 6, 9, and 12 months of treatment. Glomerular basement membrane (GBM) thickening was determined by electron microscopy at the same times. Clear clinical and laboratory signs of severe diabetes, with blood glucose levels above 200 mg/dl and urine glucose above 3000 mg/dl, were observed in all alloxan-diabetic control rats, in all periods of follow-up, whereas administration of insulin or acarbose reduced the blood glucose levels of treated groups. The most satisfactory control of blood and urine glucose was observed in animals treated with both insulin and acarbose. However, diarrhea was observed in diabetic rats treated with acarbose associated or not with insulin. GBM thickening was correlated with age in all groups. Beginning at six months after diabetes induction, the GBM of untreated diabetic rats was significantly thicker (mean +/- SEM, 4.446 +/- 0.45 mm) than that of normal rats (2.977 +/- 0.63 mm). Both insulin and acarbose prevented GBM thickening and their combination induced thickening similar to the age-dependent thickening observed for normal rats of the same age. We conclude that acarbose when combined with insulin may be a good option in the control of diabetes and its renal complications.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

Imobilização multipontual covalente de xilanases: seleção de derivados ativos e estabilizados

Pós-graduação em Biotecnologia - IQ

Kristallisation der Arbutin-Synthase und der Strictosidin Glukosidase - zwei Enzyme aus dem sekundären Glykosidstoffwechsel von Rauvolfia serpentina

Kristallisation der Arbutin-Synthase und der Strictosidin Glukosidase - zwei Enzyme aus dem sekundären Glykosidstoffwechsel von Rauvolfia serpentina Die vorliegende Arbeit befasst sich mit der Kristallisation und der strukturellen Auswertung der Arbutin-Synthase (AS) und der Strictosidin Glukosidase (SG). Beide Enzyme stammen aus der Medizinalpflanze Rauvolfia serpentina. Für die Kristallisation der Arbutin-Synthase wurden ca. 2500 verschiedene Beding-ungen experimentell untersucht. Für einige dieser Experimente wurde das Enzym molekularbiologisch und chemisch verändert. Trotzdem konnten keine Kristalle erhalten werden. Die bei diesen Veränderungen erhaltenen Ergebnisse wurden anhand von Vergleichen mit Strukturen anderer Glykosyltransferasen der gleichen Familie analysiert. Bei der Reinigung der AS konnte mit verschiedenen Trennsystemen nie eine homogene Lösung produziert werden. Der wahrscheinliche Grund für diese schlechte Isolierbarkeit, und damit der wahrscheinliche Grund für die schwierige Kris-tallisation, liegt in der überdurchschnittlich hohen Anzahl an Cysteinen in der Proteinsequenz. Mit den Aminosäuren Cys171, Cys253 und Cys461 wurden drei Cysteine gefunden, die einem Strukturvergleich nach an der Proteinoberfläche liegen und möglicherweise durch Quervernetzungen mit anderen Proteinmolekülen ein heterogenes Gemisch bilden, das nicht geordnet kristallisieren kann. Durch gezielte Mutationen dieser drei Aminosäuren könnte die Kristallisation zukünftig ermöglicht werden. Für die SG waren bereits Bedingungen bekannt bei denen nicht vermessbare Enzymkristalle (Nadeln) wuchsen. In weit gefächerten Versuchen konnten diese Kristalle jedoch nicht zu 3D-Wachstum angeregt werden. Es wurden mit einem HTS-Screening neue Bedingungen zur Kristallisation gefunden. Anschließend konnten die native Struktur und der Strictosidin/Enzym-Komplex vermessen und aufgeklärt werden. Die SG gehört zur Familie 1 der Glukosidasen (GH-1) und besitzt die in dieser Familie konservierte (beta/alpha)8-Barrel-Faltung. Im Vergleich mit 16 bekannten Glykosidasen der Familie GH-1 wurde die Substratbindung untersucht. Dabei wurde die in der Familie konservierte Zuckerbindung vorgefunden, jedoch große Unterschiede in der Aglykonbindung entdeckt. Es wurden Bedingungen für die Konformationsänderung des Trp388 erkannt. Diese Konformationsänderung dirigiert den Aglykonteil des Substrates auf verschiedene Seiten der Substratbindungstasche und teilt so die Familie GH-1 in zwei Gruppen.

Synthese von MUC1-Glycopeptid-Konjugaten mit fluorierten Analoga des Thomsen-Friedenreich-Antigens

Krebserkrankungen gehen oft mit der Überexpression von mucinartigen Glycoproteinen auf der Zelloberfläche einher. In vielen Krebserkrankungen wird aufgrund der fehlerhaften Expression verschiedener Glycosyltransferasen das transmembranständige Glycoprotein MUC1, mit verkürzten Glycanstrukturen, überexprimiert. Das Auftreten der verschiedenen tumor-assoziierten Antigene (TACA) korreliert meist mit dem Fortschreiten des Krebs und der Metastasierung. Daher stellen TACAs interessante Zielmoleküle für die Entwicklung einer aktiven Tumorimmuntherapie zur spezifischen Behandlung von Adenokarzinomen dar. In dieser Arbeit galt das Interesse dem epithelialen Mucin MUC1, auf Basis dessen ein synthetischer Zugang zu einheitlichen Antitumorvakzinen, welche aus mucinanalogen Glyco-peptid¬konjugaten des MUC1 und Carrierproteinen bestehen, hergestellt werden sollten.rnUm eine tumorspezifische Immunantwort zu erhalten, müssen die selbst schwach immunogenen MUC1-Antigene über einen nicht-immunogenen Spacer mit einem geeigneten Trägerprotein, wie Tetanus Toxoid oder Rinderserumalbumin (BSA), verbunden werden. rnDa ein Einsatz von Glycokonjugaten in Impfstoffen durch die metabolische Labilität der O-glycosidischen Bindungen eingeschränkt ist, wurden hierzu erstmals fluorierte Vetreter von MUC1-analogen Glycopeptiden verwendet, in denen das Kohlenhydrat-Epitop durch den strategischen Einbau von Fluor¬atomen gegenüber einem raschen Abbau durch Glycosidasen geschützt werden soll. Dazu wurden auf Basis des literaturbekannten Thomsen-Friedenreich-Antigens Synthesestrategien zur Herstellung eines 2’F- und eines 2’,6’-bisfluorierten-Analogons erarbeitet. rnSchlüsselschritte in der Synthese stellten neben der elektrophilen Fluorierung eines Galactalvorläufers auch die -selektive 3-Galactosylierung des TN-Antigen-Bausteins zum 2’F- und 2’,6’-bisfluorierten-Analogons des TF-Disaccharids dar. Durch entsprechende Schutzgruppentransformationen wurden die beiden Derivate in entsprechende Glycosyl¬amino-säure-Bausteine für die Festphasensynthese überführt.rnNeben den beiden Analoga des TF-Antigens wurde auch erstmals ein 2F-Analogon des 2,6-Sialyl-T-Antigens hergestellt. Dazu wurde der entsprechende 2’F-TF-Baustein mit Sialinsäure-xanthogenat nach bereits bekannten Syntheseprotokollen umgesetzt. Aufgrund von Substanzmangel konnte die Verbindung nicht zur Synthese eines MUC1-Glycopeptid-Analogons herangezogen werden.rnDer Einbau der hergestellten Glycosylaminosäure-Bausteine erfolgte in die aus 20 Amino-säuren bestehende vollständige Wiederholungseinheit aus der tandem repeat-Sequenz des MUC1, wobei die entsprechenden Glycanseitenketten stets in Position 6 eingeführt wurden. Um die erhaltenen Glycopeptide für immunologische Studien an Carrier-Proteine anbinden zu können und so ggf. zu funktionsfähigen Impfstoff-Konjugaten zu gelangen, wurden diese stets N-terminal mit einem nicht-immunogenen Triethylenglycol-Spacer verknüpft. Die anschließende Funktionalisierung mit Quadratsäurediethylester erlaubte die spätere chemoselektive Konjugation an Trägerproteine, wie Tetanus Toxoid oder BSA.rnIn ersten immunologischen Bindungsstudien wurden die synthetisierten BSA-Glycopeptid-Konjugate mit Serum-Antikörpern aus Vakzinierungsstudien von MUC1-Tetanus Toxoid-Konjugaten, die (i) eine natürliche TF-Antigenstruktur und (ii) ein entsprechendes TF-Antigenderivat mit Fluorsubstituenten an C-6 des Galactosamin-Bausteins und C-6’ des Galactoserests tragen, untersucht.rn