Understanding Human Drug Conjugating Enzymes; Regio- and Stereoselectivity in Sulfotransferase 1A3 and the UDP-Glucuronosyltransferases

Sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) are important detoxification enzymes and they contribute to bioavailability and elimination of many drugs. SULT1A3 is an extrahepatic enzyme responsible for the sulfonation of dopamine, which is often used as its probe substrate. A new method for analyzing dopamine-3-O-sulfate and dopamine-4-O-sulfate by high-performance liquid chromatography was developed and the enzyme kinetic parameters for their formation were determined using purified recombinant human SULT1A3. The results show that SULT1A3 strongly favors the 3-hydroxy group of dopamine, which indicates that it may be the major enzyme responsible for the difference between the circulating levels of dopamine sulfates in human blood. All 19 known human UGTs were expressed as recombinant enzymes in baculovirus infected insect cells and their activities toward dopamine and estradiol were studied. UGT1A10 was identified as the only UGT capable of dopamine glucuronidation at a substantial level. The results were supported by studies with human intestinal and liver microsomes. The affinity was low indicating that UGT1A10 is not an important enzyme in dopamine metabolism in vivo. Despite the low affinity, dopamine is a potential new probe substrate for UGT1A10 due to its selectivity. Dopamine was used to study the importance of phenylalanines 90 and 93 in UGT1A10. The results revealed distinct effects that are dependent on differences in the size of the side chain and on the differences in their position within the protein. Examination of twelve mutants revealed lower activity in all of them. However, the enzyme kinetic studies of four mutants showed that their affinities were similar to that of UGT1A10 suggesting that F90 and F93 are not directly involved in dopamine binding in the active site. The glucuronidation of β-estradiol and epiestradiol (α-estradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. The results show that there are clear differences in the regio- and stereoselectivities of UGTs. The most active isoforms were UGT1A10 and UGT2B7 demonstrating opposite regioselectivity. The stereoselectivities of UGT2Bs were more complex than those of UGT1As. The amino acid sequences of the human UGTs 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. Several mutants were constructed to identify the residues responsible for the activity differences. The results revealed that the residues between Leu86 and Tyr176 of UGT1A9 determine the differences between UGT1A9 and UGT1A10. Phe117 of UGT1A9 participated in 1-naphthol binding and the residues at positions 152 and 169 contributed to the higher glucuronidation rates of UGT1A10. In summary, the results emphasize that the substrate selectivities, including regio- and stereoselectivities, of UGTs are complex and they are controlled by many amino acids rather than one critical residue.

Zur lexikographischen Darstellung der Adjektivsyntax in neueren einsprachigen Wörtbüchern des Deutschen

The main objects of the investigation were the syntactic functions of adjectives. The reason for the interest in these functions are the different modes of use, in which an adjective can occur. All together an adjective can take three different modes of use: attributive (e. g. a fast car), predicative (e. g. the car is fast) and adverbial (e. g. the car drives fast). Since an adjective cannot always take every function, some dictionaries (esp. learner s dictionaries) deliver information within the lexical entry about any restrictions. The purpose of the research consisted of a comparison in relation to the lexical entries of adjectives, which were investigated within four selected monolingual German-speaking dictionaries. The comparison of the syntactical data of adjectives were done to work out the differences and the common characteristics of the lexical entries concerning the different modes of use and to analyse respective to assess them. In the foreground, however, were the differences of the syntactical information. Concerning those differences it had to be worked out, which entry is the grammatically right one respective if one entry is in fact wrong. To find that out an empirical analysis was needed, which based on the question in which way an adjective is used within a context as far as there are no conforming data within the dictionaries. The delivery of the correctness and the homogeneity of lexical entries of German-speaking dictionaries are very important to support people who are learning the German language and to ensure the user friendliness of dictionaries. Throughout the investigations it became clear that in almost half of the cases (over 40 %) syntactical information of adjectives differ from each other within the dictionaries. These differences make it for non-native speakers of course very difficult to understand the correct usage of an adjective. Thus the main aim of the doctoral thesis was it to deliver and to demonstrate the clear syntactical usage of a certain amount of adjectives.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.