Σε αυτή τη μελέτη εξατάσαμε κατά πόσον οι φλεγμονώδεις κυτοκίνες ρυθμίζουν την έκφραση της δισακχαριδάσης της ψηκτροειδούς παρυφής των εντεροκυττάρων, σουκράσης-ισομαλτάσης(ΣΙ), στην κυτταρική σειρά Caco2. Επίσης εξετάσαμε την πιθανότητα οι κυτοκίνες να ρυθμίζουν την έκφραση και άλλων ειδικών πρωτεϊνών των εντεροκυττάρων, χρησιμοποιώντας την λακτάση σαν πρότυπο. Τέλος εξετάσαμε την πιθανότητα καταστολής του γονιδίου της ΣΙ στα εντεροκύτταρα των λαχνών in vivo, κατά την τοπική φλεγμονώδη αντίδραση της νόσου του Crohn. Για τις μελέτες μας χρησιμοποιήσαμε τεχνικές κυτταροκαλλέργειας, βιοσυνθετικής σήμανσης-ανοσοκαθίζησης-ηλεκτροφόρησης, ανάλυση προστασίας ριβονουκλεάσης, ανοσοϊστοχημεία και ανάλυση υβριδισμού in situ. Τα αποτελέσματα δείχνουν ότι η IL-6 και η IFN-γ προκαλούν ελάττωση, και ο TNFα αύξηση της ΣΙ στα Caco2 κύτταρα. Η σύνθεση τηε λακτάσης δεν επηρεάζεται. Υπάρχει μια εκσεσημασμένη και ειδική ελάττωση της γονιδιακής έκφρασης της ΣΙ στα εντεροκύτταρα των λαχνών ειλεού νόσου του Crohn με οξεία φλεγμονή, σε σύγκριση με παρακείμενο μη φλεγμαίνοντα ειλεό και με φυσιολογικό ειλεό. Τα αποτελέσματα παρέχουν απόδειξη για ένα μέχρι τώρα άγνωστο μηχανισμό ανεπάρκειας δισακχαριδάσης στην εντερική φλεγμονή. / In the present study we examined whether inflammatory cytokines regulate the expression of the enterocyte brush border disaccharidase sucrase-isomaltase(SI), in the Caco2 cell line. We also examined the possibility that inflammatory cytokines regulate the expression of other enterocyte-specific proteins, using lactase as a prototype. Last we examined the possibility that SI gene expression is down-regulated in villous enterocytes in vivo during the local inflammatory response of Crohn´s disease. For our studies we used techniques including cell culture, biosynthetic labeling-immunoprecipitation-electrophoresis, ribonuclease protection assay, immunocytochemistry and in situ hybridization anlysis. The results show that IL-6 and IFN-γ mediate a decrease, whereas TNFα mediates an incrase in SI synthesis in Caco2 cells. Synthesis of lactase is not affected. There is a marked and specific decrease in SI gene expression in villous enterocytes in acutely inflammed Crohn´s ileum, as compared to adjacent uninflammed ileum and normal ileum. The results provide evidence for a previously unrecognized mechanism for disaccharidase deficiency in intestinal inflammation.

Although carbohydrates are a principal component of the human diet, the mechanism of the final stages of starch digestion is not fully understood. One approach to treating metabolic diseases such as type II diabetes, obesity, and congenital sucrase isomaltase deficiency is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals: the release of free glucose. MGAM and SI consist of two catalytic subunits: N-terminal and C-terminal, with overlapping, but variant substrate specificities. The objective of this thesis is to increase the understanding of the differential substrate specificity seen in the catalytic subunits of SI and MGAM. Through inhibitor studies, the structural and biochemical differences between the enzymatic subunits are explored, illustrating that each individual catalytic subunit can be selectively inhibited. In Chapter 3, homology models of ctSI and ctMGAM-N20 are presented, giving insight into the residues hypothesized to impact substrate specificity, enhancing our understanding of the functionality of these enzymatic subunits and overlapping substrate specificity. The structural implications of mutations seen in ntSI in CSID patients and the potential functional and structural implications are discussed in Chapter 4 in addition to the prevalence of SNPs in the SI gene in different populations. The mammalian α-glucosidases are compared to the 3 Å structure of CfXyl31, a Family 31 glycoside hydrolase from Cellulomonas fimi. Comparison to Family 31 glycoside hydrolases of known structure gives rise to possible mutations proposed to mimic ntMGAM α-glucosidase activity. Through inhibitor studies, homology models, examining mutations found in disease states such as congenital sucrase isomaltase deficiency, and investigating a bacterial family 31 glycoside hydrolase from Cellulomonas fimi, the active site characteristics and substrate specificities of SI and MGAM are better understood.

Amadori-Produkte werden spontan während der ersten Phase der Maillard-Reaktion aus reduzierenden Zuckern und Aminen wie Lysin gebildet. Sie entstehen während der Erhitzung von Lebensmitteln und in vivo. Der enzymatische Abbau solcher spontan gebildeten Produkte ist Thema dieser Arbeit. Ein Teil untersuchte die Rolle von Oligosaccharid-Amadori-Produkten während der Verdauung von Kohlenhydraten im Dünndarm. Aufgrund ihrer strukturellen Ähnlichkeit mit bekannten Glycosidase-Inhibitoren wurde eine hemmende Wirkung der Amadori-Produkte auf die Kohlenhydratverdauung vermutet. Der andere Teil beschäftigte sich mit Fructosamin-3-kinase (FN3K) und dessen verwandtem Enzym Fructosamin-3-kinase-related Protein (FN3K-RP) aus humanen Erythrocyten. Diese Ketosaminkinasen werden als Proteinreparaturenzyme betrachtet, sogar als enzymatische Verteidigung gegen Glykierung in vivo diskutiert. Durch ihre Reaktion entstehen jedoch auch hoch-reaktive 1,2-Dicarbonylverbindungen, die weitere Proteinschäden bewirken können. Noch ist nicht klar, ob die Ketosaminkinasen die pathophysiologischen Folgen der Glykierung verhindern oder fördern. In dieser Arbeit wurde die Substratspezifität von Ketosaminkinasen mit einer Reihe von Amadori-Produkten untersucht. Damit könnten Inhibitoren zur weiteren Enzymcharakterisierung oder sogar für pharmazeutische Anwendungen identifiziert werden. Außerdem wurde die Variabilität der Enzymaktivitäten von Mensch zu Mensch in einer Kohorte von 100 Probanden untersucht. Als Modell für die menschliche Kohlenhydratverdauung im Dünndarm wurden Caco-2-Zellen als Monolayer etabliert. Deren Sucrase-Isomaltase kann die alpha-glycosidische Bindung in Amadori-Produkten von Maltose und Maltotriose mit Lysin und auch in Maltulose hydrolysieren. Trotz der Aminogruppe hemmen diese Amadori-Produkte die Maltosehydrolyse nur schwach als konkurrierende Substrate. Lactulosyllysin konnte nicht durch die Lactase der Caco-2-Zellen hydrolysiert werden. Tagatosyllysin und die Heyns-Produkte Glucosyllysin und Mannosyllysin hemmten die Lactosehydrolyse schwach. Alle beobachteten Hemmeffekte sind wahrscheinlich zu schwach, um während der Verdauung in vivo bedeutsam zu sein. Für FN3K konnte Desoxypiperidinofructose als kompetitiver Inhibitor identifiziert werden (Kic 0,006 mM). FN3K zeigte nur geringe Selektivität gegenüber Amadori-Produkten verschiedener Amine, ausgenommen aromatischer Amine. FN3K-RP war in Erythrocyten wesentlich aktiver als FN3K, auch wenn die Aktivität nicht selektiv inhibiert werden konnte. Beide Enzymaktivitäten unterscheiden sich unter den 100 Probanden, mit einer Spannweite von 3 bis 12 mU/g Hämoglobin für FN3K und 60 bis 135 mU/g Hb für FN3K und FN3K-RP zusammen. Es scheint eine Verbindung zwischen der Ketosaminkinase-Aktivität in Erythrocyten und Nierenerkrankungen, familiär auftretendem Diabetes mellitus, sowie familiär aufgetretenen Herzinfarkten oder Schlaganfällen zu bestehen, wie orientierende Auswertungen zeigten. Deshalb ist eine genauere Untersuchung der physiologischen Bedeutung der Ketosaminkinasen nötig. / Amadori products are formed spontaneously from reducing sugars and amines, e.g. lysine, during the first phase of the Maillard reaction. They occur in heated food and in vivo. The thesis focuses on the enzymatic degradation of such spontaneously formed compounds. One part of this work investigated the faith and impact of oligosaccharide derived Amadori products during small intestinal carbohydrate digestion. Due to their structural similarity with known glycosidase inhibitors, an inhibitory action of Amadori products towards carbohydrate digestions was assumed. The other part dealt with fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) from human erythrocytes. Such ketosamine kinases are regarded as protein repair enzymes, maybe even an enzymatic defence against glycation in vivo. While deglycating protein bound Amadori products, however, they produce highly reactive 1,2-dicarbonyl compounds, which can lead to further protein damage. It is unclear, whether the ketosamine kinase action prevents or supports the pathophysiological effects of glycation. This work studied the substrate specifity of ketosamine kinases with a variety of Amadori products, which could result in inhibitors for further enzyme characterisation or even pharmaceutical uses. Further, the variability of both enzyme activities in a cohort of 100 subjects was examined. As a model for human small intestinal carbohydrate digestion, a Caco-2 cell monolayer was employed. Their sucrase-isomaltase is able to hydrolyse the alpha-glucosidic linkage in Amadori products of maltose and maltotriose with lysine, as well as in maltulose. Despite their amino group, those amadori products inhibited maltose hydrolysis merely weakly as competing substrates. Lactulosyl lysine on the other hand could not be hydrolysed by Caco-2 lactase. Tagatosyl lysine and the Heyns products glucosyl lysine and mannosyl lysine showed weak inhibition of lactose hydrolysis. All observed inhibitory effects are probably too weak to be of importance during carbohydrate digestion in vivo. Deoxypiperidinofructose was identified as a competitive inhibitor of FN3K (Kic 0,006 mM). FN3K acted rather non-specific towards Amadori products of different amines, except aromatic amines. FN3K-RP showed much higher activity in erythrocytes than FN3K, although its activity could not be inhibited selectively. Both enzyme activities vary among 100 subjects, with a range of 3 to 12 mU/g hemoglobin for FN3K and 60 to 135 mU/g hb for FN3K and FN3K-RP together. Relations of ketosamine kinase activity in erythrocytes with renal diseases, familial diabetes mellitus and familial cardiovascular events seem to exist. Thus, investigating the physiological impact of ketosamine kinases is necessary.

Reproduction de : Thèse d'exercice : Médecine : Nancy 1 : 2006. / Titre provenant de l'écran-titre.

Type 2 diabetes is a major health concern worldwide. One of its complications is postprandial hyperglycemia, i.e., high blood glucose concentrations, caused by glucose fast release from dietary polysaccharides into the bloodstream after meals. α-Glucosidase inhibitor drugs reduce postprandial hyperglycemia by inhibiting maltase/glucoamylase (MGAM) and sucrase/isomaltase (SI). MGAM and SI transform polysaccharides into absorbable monosaccharides, and inhibiting them delays monosaccharide release into the blood. The three commercially available α-glucosidase inhibitors are limited by their absorption abilities, inhibition efficacies, and side effects, which highlights the need for more specific α-glucosidase inhibitors. Because enzymes catalyze their reactions by tightly binding to their cognate transition states (TS), TS analogs can be powerful inhibitors and potential drugs. The measurement and interpretation of kinetic isotope effects (KIEs) is the only method that can directly determine TS structures on large molecules. In this work, methods to prepare radioisotopically labelled maltose were developed, as well as methods to measure KIEs on acid- and enzyme-catalyzed maltose hydrolysis. However, the methods developed did not achieve the required precision for TS analysis. Also, KIEs were calculated computationally for a model reaction of maltose hydrolysis. / Thesis / Master of Science (MSc)

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Amadori-Produkte werden spontan während der ersten Phase der Maillard-Reaktion aus reduzierenden Zuckern und Aminen wie Lysin gebildet. Sie entstehen während der Erhitzung von Lebensmitteln und in vivo. Der enzymatische Abbau solcher spontan gebildeten Produkte ist Thema dieser Arbeit. Ein Teil untersuchte die Rolle von Oligosaccharid-Amadori-Produkten während der Verdauung von Kohlenhydraten im Dünndarm. Aufgrund ihrer strukturellen Ähnlichkeit mit bekannten Glycosidase-Inhibitoren wurde eine hemmende Wirkung der Amadori-Produkte auf die Kohlenhydratverdauung vermutet. Der andere Teil beschäftigte sich mit Fructosamin-3-kinase (FN3K) und dessen verwandtem Enzym Fructosamin-3-kinase-related Protein (FN3K-RP) aus humanen Erythrocyten. Diese Ketosaminkinasen werden als Proteinreparaturenzyme betrachtet, sogar als enzymatische Verteidigung gegen Glykierung in vivo diskutiert. Durch ihre Reaktion entstehen jedoch auch hoch-reaktive 1,2-Dicarbonylverbindungen, die weitere Proteinschäden bewirken können. Noch ist nicht klar, ob die Ketosaminkinasen die pathophysiologischen Folgen der Glykierung verhindern oder fördern. In dieser Arbeit wurde die Substratspezifität von Ketosaminkinasen mit einer Reihe von Amadori-Produkten untersucht. Damit könnten Inhibitoren zur weiteren Enzymcharakterisierung oder sogar für pharmazeutische Anwendungen identifiziert werden. Außerdem wurde die Variabilität der Enzymaktivitäten von Mensch zu Mensch in einer Kohorte von 100 Probanden untersucht. Als Modell für die menschliche Kohlenhydratverdauung im Dünndarm wurden Caco-2-Zellen als Monolayer etabliert. Deren Sucrase-Isomaltase kann die alpha-glycosidische Bindung in Amadori-Produkten von Maltose und Maltotriose mit Lysin und auch in Maltulose hydrolysieren. Trotz der Aminogruppe hemmen diese Amadori-Produkte die Maltosehydrolyse nur schwach als konkurrierende Substrate. Lactulosyllysin konnte nicht durch die Lactase der Caco-2-Zellen hydrolysiert werden. Tagatosyllysin und die Heyns-Produkte Glucosyllysin und Mannosyllysin hemmten die Lactosehydrolyse schwach. Alle beobachteten Hemmeffekte sind wahrscheinlich zu schwach, um während der Verdauung in vivo bedeutsam zu sein. Für FN3K konnte Desoxypiperidinofructose als kompetitiver Inhibitor identifiziert werden (Kic 0,006 mM). FN3K zeigte nur geringe Selektivität gegenüber Amadori-Produkten verschiedener Amine, ausgenommen aromatischer Amine. FN3K-RP war in Erythrocyten wesentlich aktiver als FN3K, auch wenn die Aktivität nicht selektiv inhibiert werden konnte. Beide Enzymaktivitäten unterscheiden sich unter den 100 Probanden, mit einer Spannweite von 3 bis 12 mU/g Hämoglobin für FN3K und 60 bis 135 mU/g Hb für FN3K und FN3K-RP zusammen. Es scheint eine Verbindung zwischen der Ketosaminkinase-Aktivität in Erythrocyten und Nierenerkrankungen, familiär auftretendem Diabetes mellitus, sowie familiär aufgetretenen Herzinfarkten oder Schlaganfällen zu bestehen, wie orientierende Auswertungen zeigten. Deshalb ist eine genauere Untersuchung der physiologischen Bedeutung der Ketosaminkinasen nötig. / Amadori products are formed spontaneously from reducing sugars and amines, e.g. lysine, during the first phase of the Maillard reaction. They occur in heated food and in vivo. The thesis focuses on the enzymatic degradation of such spontaneously formed compounds. One part of this work investigated the faith and impact of oligosaccharide derived Amadori products during small intestinal carbohydrate digestion. Due to their structural similarity with known glycosidase inhibitors, an inhibitory action of Amadori products towards carbohydrate digestions was assumed. The other part dealt with fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) from human erythrocytes. Such ketosamine kinases are regarded as protein repair enzymes, maybe even an enzymatic defence against glycation in vivo. While deglycating protein bound Amadori products, however, they produce highly reactive 1,2-dicarbonyl compounds, which can lead to further protein damage. It is unclear, whether the ketosamine kinase action prevents or supports the pathophysiological effects of glycation. This work studied the substrate specifity of ketosamine kinases with a variety of Amadori products, which could result in inhibitors for further enzyme characterisation or even pharmaceutical uses. Further, the variability of both enzyme activities in a cohort of 100 subjects was examined. As a model for human small intestinal carbohydrate digestion, a Caco-2 cell monolayer was employed. Their sucrase-isomaltase is able to hydrolyse the alpha-glucosidic linkage in Amadori products of maltose and maltotriose with lysine, as well as in maltulose. Despite their amino group, those amadori products inhibited maltose hydrolysis merely weakly as competing substrates. Lactulosyl lysine on the other hand could not be hydrolysed by Caco-2 lactase. Tagatosyl lysine and the Heyns products glucosyl lysine and mannosyl lysine showed weak inhibition of lactose hydrolysis. All observed inhibitory effects are probably too weak to be of importance during carbohydrate digestion in vivo. Deoxypiperidinofructose was identified as a competitive inhibitor of FN3K (Kic 0,006 mM). FN3K acted rather non-specific towards Amadori products of different amines, except aromatic amines. FN3K-RP showed much higher activity in erythrocytes than FN3K, although its activity could not be inhibited selectively. Both enzyme activities vary among 100 subjects, with a range of 3 to 12 mU/g hemoglobin for FN3K and 60 to 135 mU/g hb for FN3K and FN3K-RP together. Relations of ketosamine kinase activity in erythrocytes with renal diseases, familial diabetes mellitus and familial cardiovascular events seem to exist. Thus, investigating the physiological impact of ketosamine kinases is necessary.