Dissertations / Theses on the topic 'Β-Glucuronidases'

Abstract: Conjugated carbohydrates, such as proteoglycans, play pivotal roles in a number of diverse biological processes. Their structural complexity encodes information that regulates intercellular communication, recognition events and cellular activity. As such, the biosynthesis and catabolism of these complex molecules requires the tight regulation of a large number of enzymes. Two enzymes that are of particular interest in the metabolism of glycosaminoglycans are exo-β-glucuronidase and the endo-β-glucuronidase, heparanase. These two glycosidases play an important role in the catabolism of glycosaminoglycan chains from extracellular matrix and basement membrane proteoglycans. Over-expression of these enzymes has been reported in a number of tumours, and they have been implicated in tumour metastasis and angiogenesis. Thus, there is considerable interest in the characterisation of these enzymes for the development of novel enzyme inhibitors. The work described in this thesis is divided into two parts. The first part describes the cloning, expression and characterisation of a novel heparanase from the pathogenic bacterium Burkholderia pseudomallei. In addition to the enzyme’s hydrolase activity against heparan sulfate, the B. pseudomallei heparanase was also found to have significant exo-β-glucuronidase activity. This is the first report of a bacterial enzyme that is capable of cleaving heparan sulfate via a hydrolase mechanism. In the second part of the work, the X-ray crystal structure of human β-glucuronidase was used to design novel enzyme inhibitors. A number of computational tools such as GRID, molecular docking and de novo ligand design were employed to generate a library of substituents that could be introduced at the C-1, C-2 and/or C-3 positions of specific carbohydrate templates that would potentially improve ligand binding to the enzyme. A number of 1-thioglucuronides, with substituted benzylic or aliphatic aglycon moieties, identified by the structure-based ligand design process, were chosen for synthesis. The central glycosidation step used selective diethylamine-mediated de-S-acetylation of methyl 2,3,4,5-tetra-O-acetyl-1-S-acetyl-1-thio-β-D-glucuronate, and subsequent reaction of the generated thiolate with an activated acceptor molecule. In the case of the substituted benzyl halide acceptors, glycosidations gave solely the β-glycosides, whereas glycosidation with substituted propyl halide acceptors was found to produce α/β-anomeric mixtures. The inhibitory activity of the prepared thioglucuronides was evaluated in vitro using a fluorogenic enzyme assay, with the most potent inhibitor showing activity down to 10-5 M against the test enzyme, bovine β-glucuronidase.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith University. Institute for Glycomics.
Griffith Health
Full Text

Glycosaminoglycans (GAGs) are the most prevalent hetero-polysaccharides in the body. They are usually attached to a protein core, forming proteoglycans (PGs). PGs play crucial roles in a wide range of biological processes including cell growth, migration and differentiation. Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and the extracellular matrix (ECM), which surrounds cells and is formed by a network of fibrous proteins, glycoproteins and proteoglycans. In mammalian cells heparan sulfate (HS) is cleaved by a single β-glucuronidase: heparanase. Cleavage of HS-chains changes the integrity of the ECM and releases a variety of bioactive molecules such as growth factors, cytokines and enzymes. Heparanase is implicated in cancer and inflammation and inhibition of heparanase has been shown to reduce tumour metastasis and angiogenesis. Heparanase is therefore regarded as a promising target for anti-cancer and anti-inflammatory drug design. As the three dimensional structure of human heparanase is still not available, the progress in specific inhibitor development has been impeded.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
Full Text

Biopharmaceutical manufacturing is a rigorous and expensive process. Due to the medicinal nature of the product, a high purity level is required and several expensive purification steps must be utilized. Cost-effective production and purification is essential for any biopharmaceutical product to be successful and development of the fastest, most economical, and highest-yielding purification scheme is a constant engineering challenge. Commercial-scale purification schemes currently revolve around the use of multiple chromatography steps for the purification of biopharmaceutical products. Chromatography has many shortcomings including high cost, limited throughput, and complex scale up. The goal of this research was to develop an alternative, non-chromatography purification step for the separation of an acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco with high yield and purity. Aqueous two-phase extraction (ATPE) is a powerful technique for separation and purification of proteins, and has the potential to replace an expensive chromatography step for the initial purification of recombinant proteins. ATPE enables high levels of target protein recovery and concentration while removing large amounts of impurities from the initial extract. Fractional factorial designs and response surface methodology were used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4 % (w/w) PEG/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process.
Master of Science

Résumé : La prééclampsie (PE) est un désordre de la grossesse caractérisée par une dysfonction endothéliale faisant en sorte que l’endothélium devient moins sensible aux signaux de vasodilatation. La réponse provoquée par la liaison de la sérotonine au sous-type de récepteur S[indice inférieur 2] entraîne la libération de molécules aux propriétés vasoconstrictrices, qui, par une boucle de rétroaction positive, entraîne la libération de davantage de sérotonine par les plaquettes. Cette boucle amplifie la réponse et contribue ainsi à l’hypertension présente chez les femmes ayant une PE. Précédemment, il a été démontré par notre laboratoire que le Bisphénol A (BPA) s’accumulait davantage dans le placenta des femmes avec PE en comparaison aux femmes normotensives. Cette accumulation pourrait découler d’une perturbation de sa métabolisation qui impliquerait notamment la β-glucuronidase (GUSB). Des études chez les animaux ont quant à elles démontré que le BPA pouvait inhiber l’activité de la monoamine oxydase (MAO) à forte dose. Nous avons étudié l’effet du BPA à faible concentration (10 ng/ml) sur la MAO-A des cellules placentaires et démontré que le BPA inhibait la MAO-A de façon significative sans affecter son expression protéique. Afin d’expliquer l’accumulation particulière du BPA chez les femmes PE, nous avons comparé l’activité spécifique et l’expression protéique de la β-glucuronidase (GUSB) placentaire en utilisant un devis cas-témoins. Une tendance non significative suggère que la GUSB pourrait partiellement contribuer à l’accumulation du BPA chez les femmes PE. Nous avons étudié la relation entre la concentration sérique maternelle de BPA et la concentration à laquelle le fœtus est exposé par régression linéaire et corrélation de Spearman. Un tel modèle ne pourrait être utilisé pour déterminer de façon quantitative l’exposition fœtale. En revanche, en vue de la forte corrélation entre ces deux variables, une haute concentration sérique maternelle de BPA devrait se refléter par une haute exposition fœtale. Cette corrélation implique aussi que le métabolisme placentaire ne joue pas un rôle significatif dans la protection du fœtus. Le BPA pourrait ainsi contribuer à l’hypertension chez les femmes PE présentant une dysfonction endothéliale en inhibant la MAO-A et ainsi, favorisant la hausse de sérotonine circulante. Cette étude suggère les bases d’un mécanisme par lequel le BPA s’accumulerait davantage chez les femmes PE et affecterait ainsi la MAO-A placentaire et potentiellement, la MAO-A fœtale vu ses propriétés physico-chimiques.
Abstract : Preeclampsia (PE) is an hypertensive disorder of pregnancy characterized by a generalized endothelial dysfunction where the response to vasodilatation signals is compromised. The binding of serotonin to its S[subscript 2] receptor subtype 2 releases vasoconstrictor molecules which, by a positive retroaction loop, stimulates the release of more serotonin from platelets. This positive retroaction loop stimulates the vasoconstriction of blood vessels and contributes to the hypertension in women with PE. Previously, we showed that Bisphenol A (BPA) accumulates more in the placenta of women with PE than in normotensive women. This accumulation may be the result of an impaired metabolization due to the action of the β-Glucuronidase (GUSB). Animal studies showed that BPA at high dose could lower the activity of the monoamine oxidase A (MAO-A), an enzyme implicated in the metabolism of serotonin. We studied the impact of BPA at low dose (10 ng/ml) in trophoblastic primary cells and showed that even at low dose, BPA can lower its activity without affecting the protein expression. To determine if GUSB could be the cause of the BPA accumulation in women with PE, we studied its activity and protein expression in placental biopsies from women with and without PE. A nonsignificant tendency showed that the GUSB activity and protein expression were higher in women with PE. To study the impact of placental metabolism in the fetal exposure, we studied the relation between maternal and fetal concentrations of BPA with linear regression analysis and Spearman’s correlation. We showed that maternal BPA could not precisely predict the fetal exposure and that the placental metabolism is probably limited in light of the strong correlation between both variables. This strong correlation also implied that high maternal exposure would result in high fetal exposure. This study shows that the accumulation of BPA in preeclamptic women could contribute to maternal hypertension by interacting with serotonin levels. This accumulation could partially be attributed to a higher GUSB, but other factors are probably implicated. The strong correlation between maternal and fetal exposure implies that the placental metabolism of BPA is limited and does not protect the fetus significantly. This study suggests the basis of a mechanism explaining the abnormal accumulation of BPA in the placenta in women with PE and its impact on the placental MAO-A and potentially, the foetal MAO-A because of its physico-chemical properties.

La mucopolisacaridosi tipus VII (MPS VII) és una malaltia monogènica molt rara inclosa en el grup de malalties lisosòmiques. Està causada per la manca d’activitat β-­‐ glucuronidasa, un enzim lisosòmic involucrat en la via de degradació de glicosaminoglicans. Aquesta alteració congènita causa una disfunció del sistema lisosòmic que provoca una acumulació anormal als lisosomes i la disrupció de l’homeòstasi de la cèl·lula. La malaltia presenta diferents graus de severitat clínica que van des de la mort prenatal fins a pacients amb formes lleus i una esperança de vida de 20 o 30 anys. Entre els casos que presenten un inici postnatal de la malaltia, la forma més severa de MPS VII es caracteritza per hepatomegàlia, esplenomegàlia, anomalies esquelètiques, desenvolupament tardà i retard mental, entre altres símptomes. Actualment els tractaments per als pacients de MPS VII són intervencions per pal·liar els símptomes de la malaltia, però no disposen de cap tractament curatiu. La teràpia gènica és una estratègia prometedora de cara a trobar una cura per a malalties monogèniques. Entre els vectors de teràpia gènica disponibles, els virus adeno-­‐associats (AAV) presenten certes característiques que els fan molt atractius per a la teràpia gènica: permeten la transducció tant de cèl·lules en divisió com quiescents, proporcionen una expressió del transgèn a llarg termini, no poden replicar-­‐se de manera autònoma sense un virus accessori i, a més, les infeccions naturals dels AAV no són patogèniques. Diferents serotips d’AAV s’han utilitzat com a vectors de teràpia gènica en estudis preclínics. Entre ells, els serotips AAV9 i AAVrh10 són els que presenten una capacitat de transducció més gran, així com una gamma més àmplia de tipus cel·lulars que poden transduir, especialment al sistema nerviós central. El serotip AAVrh10 no és d’origen humà. Per això, el fet d’utilitzar-­‐lo com a vector de teràpia gènica podria evitar la neutralització per part dels factors immunològics específics contra AAV, factors que són presents en el sèrum humà després d’infeccions d’AAV naturals. Tanmateix, el reconeixement creuat per part d’anticossos generats contra AAV2, el serotip humà més comú, podrien interferir en el resultat de la teràpia. En aquest treball es proposa una estratègia de teràpia gènica per a MPS VII basada en una única injecció intratecal d’un vector AAVrh10 que conté el gen de la β-­‐ glucuronidasa, testada en ratolins MPS VII adults joves. Es mostra que l’administració del vector al líquid cefaloraquidi (LCR) per punció lumbar, una tècnica molt poc invasiva, permet la transducció d’estructures del sistema nerviós central (SNC) utilitzant una dosi de vector més baixa que mitjançant injecció intravenosa. A més, el drenatge del vector del LCR cap al torrent sanguini comporta la transducció d’òrgans somàtics com el fetge. Això genera una font de β-­‐glucuronidasa que aconsegueix una activitat enzimàtica en sèrum comparable a la de ratolins sans. L’expressió sostinguda de l’enzim recombinant per part de les cèl·lules transduïdes, així com la correcció creuada deguda a la secreció de l’enzim, aconsegueixen la correcció de les característiques bioquímiques i histopatològiques de la malaltia, tant al SNC com als òrgans somàtics. Aquesta correcció a nivell cel·lular condueix a una millora significativa de les capacitats físiques, cognitives i emocionals dels ratolins MPS VII i aconsegueix doblar la seva esperança de vida.
Mucopolysaccharidosis type VII (MPS VII) is an ultrarare monogenic lysosomal storage disease. It is caused by the lack of β-­‐glucuronidase activity, a lysosomal enzyme involved in the degradation pathway of glycosaminoglycans. This inborn genetic alteration causes a dysfunction of the lysosomal system that entails abnormal lysosomal storage and disruption of cell homeostasis. The disease presents a range of clinical severity among patients, from death in utero to a life expectancy of up to 20 or 30 years for the milder forms. The severe form of MPS VII among cases with postnatal disease onset is characterized by hepatosplenomegaly, skeletal abnormalities, developmental delay and mental retardation, among other symptoms. Currently, the only treatments for MPS VII patients are interventions to alleviate the symptoms, but no curative treatment is available. Gene therapy is a promising therapeutic approach to find a cure for monogenic diseases. Among the available gene delivery vectors, adeno-­‐associated viruses (AAVs) present several features that make them attractive for gene therapy strategies: they are able to transduce dividing and quiescent cells, they provide long term expression of the transgene, they are not able to autonomously replicate without a helper virus, and wild type AAV infections are not pathogenic. Different AAV serotypes have been used as gene therapy vectors in preclinical studies. Among them, AAV9 and AAVrh10 are the serotypes which show greater transduction capacity and a broader range of cell-­‐type specific tropism, particularly in the central nervous system. The use of AAVrh10, a non-­‐human serotype, may avoid the neutralization by anti-­‐AAV immune factors present in human sera after natural AAV infections. However, cross-­‐reactivity with antibodies raised against AAV2, the most common human AAV serotype, may still interfere in the therapeutic outcome. In this work, we propose a gene therapy strategy for MPS VII based on a single intrathecal injection of an AAVrh10 coding for the β-­‐glucuronidase gene, tested in young adult MPS VII mice. We show that vector delivery to the CSF by lumbar puncture, a poorly invasive technique, allows the transduction of CNS structures using a lower vector dose than by intravenous delivery. In addition, the drainage of the vector from the CSF to the bloodstream results in transduction of somatic organs such as liver, thus providing a systemic β-­‐glucuronidase source that achieves serum enzymatic activity comparable to wild type mice. The sustained recombinant enzyme expression by AAV-­‐transduced cells, and the cross-­‐correction provided by enzyme secretion, attains the correction of biochemical and histopathological hallmarks of the disease in CNS and somatic organs. This correction at the cellular level leads to a significant improvement of physical, cognitive and emotional characteristics of MPS VII mice and a doubling of the MPS VII mouse life span.

Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016
The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment
The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)

Cette étude avait pour but la mise au point d'un protocole de transformation génétique de l'épinard (Spinacia oleracea L. ) et son application à l'introduction du gène de la nitrate réductase de tabac. De tels résultats doivent permettre l'étude de l'impact de ce gène sur le métabolisme de l'azote de l'épinard ainsi que l'obtention de plantes à teneur réduite en nitrate. Au cours de l'expérimentation, le transfert direct utilisant le canon à particules et le transfert indirect via les agrobactéries ont été développés. Transfert direct: l'application de cette technique à différents explants a permis l'obtention de massifs cellulaires et de bourgeons. Ces structures présentent l'activité béta-glucuronidase. Cependant, aucune plante transgénique n'a été actuellement obtenue. Transfert via agrobacterium: la sensibilité de l'épinard aux souches sauvages d'Agrobacterium tumefaciens a été démontrée par l'obtention de tumeurs de type crown gall et de tumeurs avec pointes racinaires. Par contre, l'utilisation de souches désarmées a été sans succès, probablement en raison de l'incapacité de la maîtrise des conditions de régénération à partir des cellules transformées. L’infection de jeunes plantes décapitées avec la souche A4 GUS d'A. Rhizogènes a induit la formation de racines de type hairy root. A partir de ces racines, des plantes ont été néoformées par les voies organogène et embryogène. La mise en évidence d'opines et les analyses d'activité beta-glucuronidase, d'amplification d'une séquence du gène GUS par PCR et d'hybridation de type southern blot ont démontré la nature transgénique des plantes obtenues. Ces plantes sont fertiles et donnent une descendance R1 GUS+ L’application de cette méthode a l'introduction du gène nitrate réductase a conduit a l'obtention de pousses feuillées présentant le caractère NR+ après analyse PCR.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática.
Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

博士
國立中興大學
生物科技學研究所
107
Gut bacterial β-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced β-D-glucuronides. These reactions can have deleterious consequences when they reverse xenobiotic metabolism. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the substrate preference(s) of individual enzymes must be known. Herein, we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. In an attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in TIM barrel loop 3 (or the 2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow both the prediction of the functional roles of GUSs and the design of selective inhibitors. Furthermore, we demonstrated how charge, conformation, and substituents of several uronic iminocyclitols and analogues contribute to the inhibitory potency and selectivity for gut bacterial GUSs by using crystallographic and biochemical methods. Uronic isofagomine was a potent inhibitor (Ki up to 4 nM) unlike uronic deoxynojirimycin that showed a less potent inhibition (Ki up to 930 nM), indicating that the positive charge at the anomeric position, but not endocyclic oxygen position, was favorable to form electrostatic interactions with the two catalytic glutamates of GUSs. Moreover, D-glucaro-δ-lactam displayed good inhibitions (up to 280 nM) for bacterial GUSs owing to the half-chair conformation that favored several polar contacts and a charge-dipole interaction. Additionally, C6-alkylated uronic isofagomine displayed selective inhibition for opportunistic bacterial GUSs, which was attributed to the hydrophobic interactions between the propyl group and the loop 5 residues of the GUSs.In contrast, N1-alkylated analogue was a non-selective and moderate inhibitor for GUSs, which was explained by a steric hindrance between the alkyl group of the inhibitor and the catalytic acid/base glutamate of GUSs. In summary, we identified the responsible group of GUSs for the xenobiotic-induced toxicity, and developed a selective inhibitor targeting these GUSs.

This thesis describes engineering studies with the Escherichia coli β-glucuronidase enzyme (E. coli β-GUS) that catalyzes the hydrolysis of D-glucuronic acids (glycone) that are conjugated through a β-O-glycosidic linkage to an aglycone. The enzyme is specific for the glucuronic acid component and will tolerate a variety of aglycones. A point mutation was known to convert β-GUS into a glucuronylsynthase, that is an enzyme capable of the synthesis of glucuronide conjugates. The long-term aim of the present research was to change the donor sugar specificity of the glucuronylsynthase from a glucuronyl donor to a glucosyl donor allowing the synthesis of glucosides. The approach taken to achieve the long-term aim was to first alter the specificity of β-GUS and to then convert the variants to a synthase. There were two approaches taken to altering the substrate specificity of β-GUS. The first approached was to use site saturation mutagenesis to alter key residues that are located at the active site. A more effective approach involved using directed evolution to generate variants with altered specificity. A selection of these variants were converted to (putative) synthetic enzymes and tested for activity. The structure-guided site saturation mutagenesis of 9 sites was carried out in an attempt to alter the substrate specificity of β-GUS. The choice of the residues to be altered was made with the aid of the structure of β-GUS with a bound substrate analogue. Nine codons in the β-GUS gene were randomised to create libraries that contained all possible amino acid of residues in and / or near the active site. Mutants were assayed using substrates presenting 5 different glycones. Of the positions randomised, most glycosyl binding residues were found not to tolerate amino acid substitutions, suggesting they are essential for β-GUS function while majority of non-glycosyl binding residues were found to tolerate amino acid substitutions – but not with good activity One of the common dogmas of directed evolution is the idea that evolvability is related to stability. We set out to test this idea while evolving substrate specificity. Other workers had generated a more thermostable variant of β-GUS. In parallel, we evolved 1) the native enzyme (GUS-WT) and 2) the thermostable (GUS-TR3337) variant of β-GUS. Mutant libraries of both GUS-WT and GUS-TR3337 were created under identical conditions and had the same distributions of mutations. After five rounds of evolution, the catalytic efficiency (kcat/Km) of the best mutant of the wild type parent for pNP-glucoside was increased ~307-fold while the best mutant of the thermophilic parent demonstrates a ~4-fold increased kcat/Km over the best mutant of wild type parent. Selected mutants from both libraries were characterised with regard to conformational properties and stability and these investigations, combined with kinetic data, provided valuable information about how thermostability promotes the ease of protein evolvability. The initial characterisation of β-GUS variants was done with crude lysates. It was observed that the GUS-WT and GUS-TR3337 variants lost their newly evolved activity after purification. It was eventually determined that the BugBusterTM reagent used to lyse cells prior to screening, had affected the directed evolution campaign. The n-octyl β-D-thioglucopyranoside (OTG) presents in the BugBusterTM reagent, was very similar in structure to pNP-glucoside and was identified as a competitive inhibitor that suppresses glucosidase activity of wild-type β-GUS, indicating that it can be bound in the active site. In response to the addition of OTG in the screening assay, OTG enhanced the glucosidase activity of selected mutants. This observation highlights the potential pitfall in the use of commercial reagents to lyse cells for enzymes with glycosidase activities. However, the evolution in the presence of OTG gives some insight into how an enzyme might evolve to be regulated by an effector molecule – OTG, in this case. Finally, improved cell lysis variants were converted to glucuronylsynthase variants by introducing the site specific mutation. Their glycosynthase ability was tested using a similar protocol developed by McLeod Group for assaying glucuronylsynthase activity. Unfortunately, glycosynthase activity was not observed with α-D-glucuronyl fluoride donor and steroid accepters. Time constraints did not allow other substrate to be tested.

碩士
國立臺灣大學
化學研究所
107
Gastrointestinal microbiota plays an important role in shaping many metabolic pathways. One example is glucuronidation of xenobiotics, which occurs in the liver to conjugate hydrophobic compounds with glucuronic acid (a monosaccharide) to increase their solubility and then facilitate rapid excretion. Microbiome-encoded β-glucuronidases (GUSs) that catalyze the hydrolytic removal of glucuronic acid are known to be critical to regenerate bioactive compounds and recycle endogenous molecules. However, these enzymes also represent the main cause of xenobiotics-induced toxicity. To develop potent and selective inhibitors for gut bacterial GUSs, herein we report the chemical synthesis of several iminosugars that often display satisfying potency. Among them, uronic-type isofagomine (UIFG) can be protonated at physiological pH to be a good mimicry of the transition state in β-glucuronidase-catalyzed reactions. We designed and synthesized several UIFG derivatives according to previous structure-activity relationship studies of GUS inhibitors, including N- and C6-substituted UIFGs. The preliminary results indicated that the substitution at C6 increased the affinity and selectivity at the same time. Also, we developed an efficient synthesis of C6-aminomethyl UIFG which can be further diversified by combinatorial chemistry to search for more potent and selective inhibitors.

碩士
嘉南藥理科技大學
藥物科技研究所
98
B-glucuronidase (BG) is both an attractive reporter gene and a promising prodrug-converting enzyme for cancer therapy.。Imaging of BG activity in living animals would be a convenient method to monitor reporter gene expression and BG prodrug treatments. In this report, we developed a 124I tyramine-difluoromethylphenol glucuronide probe (124I-TrapG) that could be converted by BG to release a trapping 124I-difluoromethylphenol moiety to crosslink any nucleophile moiety on cells. Enhanced signals were shown in CT26/mBG-eB7 tumors in our micro-PET imaging studies, however CT26 tumors are not, indicate 124I-Trap could present the distribution and expression of BG in vitro and in vivo. According to the result, we could use molecular imaging probes and micro-PET to detect the expression of BG in tumor cells. Also application in clinic, we could trace the tumor progression, or estimate the result of anticancer therapy in future.

博士
國立臺灣大學
化學研究所
104
In this thesis, two validated drug targets are focused for structural studies, i.e., human β-galactoside-binding lectins (galectins) and bacterial β-glucuronidases (GUSs), which have been known as key players in tumor progression and drug metabolism, respectively. Because the primary sequence and tertiary structure were remarkably conserved among the galectin and GUS families, developing potent and selective inhibitors for specific members has become a longstanding challenging. We particularly applied multi-disciplinary approaches for structure-based drug development, such as synthetic chemistry, X-ray crystallography, isothermal titration calorimetry, and NMR spectroscopy. To date, TD139 (3,3ʹ-deoxy-3,3ʹ-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol- 1-yl)-thio-digalactoside) representing the most potent inhibitor for galectin-3, one of the most prominent galectin family members involved in several pathological processes, has been approved for the clinical trial of idiopathic pulmonary fibrosis. However, the full structural information concerning subsites A–E of galectin and the interactions with TD139 are not currently available. In the first part of this thesis, we studied the binding contributions of these subsites in galectins-1, -3, and -7 with several sialylated/sulfated disaccharides and three thio-digalactoside (TDG) derivatives, including TDG, TD139, and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio- digalactoside). Surprisingly, we found that the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterized dual binding modes demonstrate how binding potency, reflected in decreased Kd values of the TDG-derived inhibitors from μM to nM levels, is improved. The resulting information offers insights into the development of selective inhibitors for individual galectins. In the second part, we focused on microbial GUSs, which interfere with xenobiotic detoxification and thus impact human health. Currently several selective inhibitors (such as ASN 03273363) for bacterial GUSs are known to rely on the unique loop that is located near the enzyme active site and is found only in microbial enzymes. However, there are two crucial problems associated with ASN 03273363. One is that the bacterial loop exhibits significant sequence variation, whilst the other is the presence of more than half of bacterial GUSs lacking this loop (NL-GUSs). To solve these problems, we developed a series of uronic isofagomine (IFG) derivatives as transition-state analogues, which directly interact with the conserved catalytic residues of GUSs. The results indicated that substituents introduced at the C1-position play an important role in determining the selectivity between bacterial and mammalian GUSs. Moreover, a combinatorial method was applied to rapidly generate and screen a variety of uronic IFG derivatives. Among them, an irreversible inhibitor was identified to target the non-catalytic cysteine that is conserved in several NL-GUSs, but is absent in both the human and loop-containing bacterial GUS. The selectivity is up to 2–3 orders of magnitude greater for bacterial NL-GUSs (IC50 in the range of nM) than for HsGUS (IC50 of μM).

碩士
高雄醫學大學
香粧品學系碩士班
104
Excessive reactive oxygen species (ROS) and free radicals impact on human health and furthermore cause aging, diseases or cancer. Patients with cancer chemotherapy not only increase in free radical generation and lipid peroxidation but also suffer from many serious side-effects. Chemotherapy-induced diarrhea (CID) is a common side-effect with patients, caused by the cytotoxic effects of cancer chemotherapy, e.g., irinotecan (CPT-11). It affects the quality of patients’ life and makes patients delay or stop cancer chemotherapy. The main factor of CID is Escherichia coli β-glucuronidase (eβG) cause epithelial cells damaged in intestinal. The aim of this study is to develop some lead compounds from natural products which could be ingredients for specific inhibition of eβG to release CID. In the preliminary screen data, the methanolic extract of the root of Neolitsea acuminatissima (NAR) showed anti-eβG effect and without affecting human β-glucuronidase (hβG) activity. The phytochemistry, the anti-eβG effect, and the mechanism involved in the improvement of molecular biology of NAR has been studied rarely. Therefore, NAR was selected as the candidate. In the results, three new compounds, demethoxydaibucarboline A (1), methoxyneolitacumone A (2), and neolitacumome E (14), along with 12 known compounds were isolated from NAR by silica gel column chromatography and their structures were spectroscopically determined. Among them, compounds 1 and quercetin (7) showed anti-eβG activity with inhibition ratio of 70-80%, meanwhile, they also showed DPPH- and ABTS- radicals scavenging activities. Compound 7 showed significant DPPH- and ABTS- radicals scavenging activities in a concentration-dependent manner with SC50 values of 10.2 ± 1.4 and 7.8 ± 2.1 μM, respectively. The anti-eβG and antioxidant activities of compound 7 which is related with the structure skeleton of flavonoid. In summary, compounds 1 and 7 are not only eβG inhibitors but also antioxidant to be ingredients in cosmetics, dietary supplement, or medicine.

The demand for O-glucuronides as potential therapeutic products and biomarkers continue to increase. However, large-scale synthesis of O glucuronides remains a challenge for the industry, and has prompted the development of an alternative synthetic routes. This dissertation extends from a previous enzyme engineering work that had introduced a site-specific mutation E504G in β glucuronidase (β-GUS), resulting in a functional glucuronylsynthase (Syn). However, the synthetic activity of Syn is low and leaves ample scope for improvement. The work described in this thesis aims to produce a more efficient glucuronylsynthase using different enzyme engineering approaches. Two separate strategies were employed to achieve our objective. The first strategy engages a two-step process where the β-GUS is first engineered to have higher activity in the presence of excess substrate; 10–20 times its Km. This is followed by site-specific mutation E504G to convert the β-GUS variant into a Syn. The second strategy engineers the glucuronylsynthase directly. Chapter 3 describes the attempt to improve the activity of the native enzyme in the presence of t-BuOH, a solvent that was found to improve the chemistry of the glucuronylsynthase chemoenzymatic reaction. The engineering attempt produced a potential variant with a mutation at its C-terminal region, L561S, that is more active in the presence of the solvent. This mutation appears to be a determinant mutation. Biophysical characterization of the enzyme revealed that this improvement is not due to increased stability in t-BuOH, while our analysis of the crystal structure suggests that the mutation improved the activity by increasing loop flexibility at the C-terminal region. Subsequently, I incorporated E504G into the β-GUS variant, but this did not translate into a better glucuronylsynthase variant. Chapter 4 describes the second strategy. Two mutations, H162Q and Y160G, at the N-terminal region were found to boost the synthetic activity but this was not accompanied by improvement in their thermostability nor solvent stability. However, combining the results from the biophysical characterization experiments and observations from the structural examination on 3K4D, it can be inferred that the mutations promote glucuronylsynthase activity by modulating the active site of the Syn so that it would favour the glucuronyl donor substrate. Therefore, these mutations would serve as concrete starting points for further evolution program of the Syn. Chapter 5 explores the potential reason that could account for the lack of success in transposing the potency of L561S to the glucuronylsynthase system. The work here is driven by the hypothesis that translational misincorporation introduced contaminating wild-type during enzyme expression. Essentially, this chapter highlights the potential pitfall of the glucuronylsynthase system and describes potential strategies to avoid this pitfall. Finally, Chapter 6 builds upon the results from β GUS engineering and explores the mutational tolerance of β GUS. Its mutational tolerance is compared with another enzyme that is structurally less complex (β-lactamase, TEM-1). In addition, its mutational tolerance with different substrate is also compared. This exercise attempts to provide insights into the elements that would drive the adaption process of the β-GUS. Consequently, we expect this study to facilitate future directed evolution studies of the β-GUS and the glucuronylsynthase.

博士
高雄醫學大學
醫學研究所
101
β-glucuronidase (βG) has been used as tumor marker and prodrug-converting enzyme for selective chemotherapy. The ability to monitor βG activity in patients would be ideal to access the efficacy of βG-mediated drug release and generate personalized prodrug therapy. However, whole-body imaging of βG activity for medical usage is not yet available. In order to overcome this problem, we conjugated fluorecein (FITC), near infrared dye (IR-820) and radioactive isotope (124I-tyramine) to a difluoromethylphenol-glucuronide moeity (TrapG) to form FITC-TrapG, NIR-TrapG and 124I-TrapG, respectively, for noninvasively monitoring βG activity by optical and positron emission tomography (PET) imaging. Both of the FITC-TrapG, NIR-TrapG and 124I-TrapG specifically trapped on βG-expressing cell/tumor in vitro and in vivo. βG-activated TrapG showed bystander labeling activity, cytotoxicity and did not interfere with βG activity. To exam whether βG expression is proportional to the efficacy of βG prodrug therapy, we use NIR-TrapG to image the βG activity in mice bearing variant human tumors and the βG expression in Colon205 tumor is higher than SW620 tumor. After the βG prodrug (9ACG) treating, the mean size of Colon205 tumor is smaller than SW620 tumor. Thus the βG activity-based trapping probe may provide an available tool for the optimization of prodrug targeted therapy and personalized therapy.

博士
國立陽明大學
微生物及免疫學研究所
99
Traditional chemotherapy represents an important treatment option for many types of cancer, however, low specificity and severe side effects limited the therapeutic index. Selective activation of anti-neoplastic prodrugs by antibody-directed enzyme prodrug therapy (ADEPT) or gene-directed enzyme prodrug therapy (GDEPT) can reduce systemic drug exposure and increase cancer treatment specificity. In this study, we examined different strategies to improve the therapeutic index of cancer therapy. In the first part, we tethered human, murine or E. coli β-glucuronidase (hβG, mβG and eβG, respectively) on the surface of tumor cells and examined their in vitro and in vivo efficacy for the activation of glucuronide prodrugs (9ACG and HAMG) of 9-aminocamptothecin and p-hydroxy aniline mustard. Measurement of in vitro cytotoxicity demonstrated that surface mβG possessed the best catalytic activity for hydrolysis of 9ACG and HAMG. The tumoral growth of EJ cells with membrane displayed mβG was successfully delayed or suppressed by 9ACG or HAMG treatment, respectively. Although, mβG displayed good anti-tumor activity in the GDEPT model, hβG is still an attractive enzyme for selective prodrug activation. It is sequestered in lysosomes from circulating glucuronides and should display low immunogenicity in patients to allow multiple rounds of treatment. But its catalytic activity is relatively low as compared to mβG or eβG at the pH values (6.8~7.2) found in the interstitial tumor space. Therefore, in the second part, we sought to enhance the utility of hβG for cancer treatment by increasing hβG activity with directed evolution. hβG was tethered on the plasma membrane of fibroblasts to allow high-throughput screening of enzymatic activity under defined environmental conditions. Combining directed evolution with flow cytometric screening allowed the isolation of an hβG variant (S2) exhibiting 60-fold higher activity (kcat/Km) than wild-type hβG at pH 7.0. The S2 variant also generated cytotoxic drugs from two structurally disparate glucuronide prodrugs an order of magnitude more effectively than wild-type hβG. In the third part, we constructed humanized immunoenzymes by fusing a humanized single-chain antibody (scFv) of mAb CC49 to wild-type hβG or S2 (hcc49-hβG or hcc49-S2, respectively) that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG and more effectively killed TAG-72 positive cancer cells exposed to the anticancer glucuronide prodrug HAMG. Treatment of tumor-bearing mice with hcc49-S2 followed by HAMG treatment significantly delayed tumor growth as compared to hcc49-hβG. Taking together, our studies demonstrated that increased conversion of glucuronide prodrugs in the tumor microenvironment by murine or enhanced hβG represent promising approaches to improve the therapeutic impact of GDEPT and ADEPT.

碩士
國立陽明大學
放射醫學科學研究所
95
Part One: Objectives: β-glucuronidase has been widely used as a prodrug-activating enzyme for cancer therapy. The ability to monitor β-glucuronidase activity in living animals would further allowed both imaging and therapy of cancer. This study demonstrated that radioiodinated phenolphthalein glucuronide (*I-PhG) was a promising probe to monitor the membrane-anchored β-glucuronidase (mβG) gene expression in vitro and in vivo. Methods: The radioiodinated glucuronide conjugate 124/131I-PhG was prepared with high radiochemical purity (>95%). The accumulation and metabolism of 131I-PhG in mβG gene expressing CT26/mβG colon carcinoma cells and the wild-type CT26 cells were studied. About 5x106 CT26/mβG and CT26 cells were inoculated in the right and left flanks of BLAB/c mice. The biodistribution study and scintigraphic imaging of 124/131I-PhG were performed with tumors-bearing mice at 7-10 days post tumor implantation. Result: The hydrophilic 131I-PhG was metabolized by the β-glucuronidase to produce mainly 131I-Ph and a trace amount of 131I-iodide in the CT26/mβG cell culture medium. The radioactivity retained in CT26/mbG cells owing to the retention of lipophilic metabolite, 131I-Ph, in the cell membrane increased rapidly with time and reached plateau (46.2%) after 240 min incubation. The 131I-PhG was stable in the CT-26 cell culture, no 131I-Ph was found and only 2.0% radioactivity was accumulated in the cells after 240 min incubation. The biodistribution studies showed higher radioactivity accumulations in CT26/mβG tumors than those in CT-26 tumors in the whole experimental periods. The results of microPET imaging were consistent with those of biodistribution. The production of lipophilic metabolite *I-Ph from deglucuronidation of hydrophilic 131I-PhG might account for the high retention of radioactivity in abdomen region. Conclusions: These results demonstrate that radioiodinated PhG can systemically monitor the membrane-anchored β-glucuronidase in vitro and in vivo. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic. Part Two: Objectives: IUdR is long regarded as a highly potential tumor therapeutic drug and radiosensitizer. But IUdR is not very stable in vivo and high tumor uptake cannot be achieved easily. We prepare more lipophilic [*I]IUdR-3',5'-diacetyl ester ([*I]IUdR-(OAc)2) and [*I]IUdR -3',5'-dibenzoyl ester ([*I]IUdR-(OBz)2) as prodrugs of [*I]IUdR and evaluate their potency in hepatoma radiotherapy. Methods: Starting from IUdR and acetic anhydride or benzoic anhydride, IUdR-(OAc)2 (or IUdR-(OBz)2) was obtained. After reacting with hexabutylditin followed by radioiodination, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 can be prepared. [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 mixed with lipiodol (act as vehicle) followed by sterile filtration with 0.22 micrometer membrane filter to afford [131I]IUdR/lipiodol, [131I]IUdR-(OAc)2/lipiodol and [131I]IUdR-(OBz)2/lipiodol injections. The release of IUdR or prodrugs from lipiodol was examined. The cellular uptake and DNA incorporation ratio of [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 in N1S1 hepatoma cells has been determined after incubation for different time periods. Results: The overall chemical yields in preparing Bu3SnUdR-(OAc)2 and Bu3SnUdR-(OBz)2 were 40 and 30%, individually. The radiochemical yield and radiochemical purity of [131I]IUdR-(OAc)2 and [131I]IUdR -(OBz)2 were both higher than 95%. The release rates of the two prodrugs from lipiodol were slower than that of IUdR, indicating the higher lipophilicity of the prodrugs. When incubated with [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2, the cellular uptakes and the incorporation of radioactivity into DNA all increased steadily within 24 h, but the DNA incorporation ratio of [131I]IUdR was higher than the two lipophilic prodrugs. Conclusions: Due to the higher lipophilicity of the two prodrugs, their release from lipiodol was slower than that of IUdR. Since lipiodol as carrier will stay longer in hepatocyte carcinoma than in normal liver, both [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 will retain longer in hepatoma, higher tumor uptake, higher DNA incorporation ratio of [131I]IUdR and higher radiation dosimetry to hepatoma cells can thus be achieved.