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Identification and Characterization of Leishmania Major UDP-Sugar Pyrophosphorylase and Determination of Its Impact on UDP-Galactose Metabolism

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Identification and Characterization of Leishmania Major UDP-Sugar Pyrophosphorylase and Determination of Its Impact on UDP-Galactose Metabolism Identification and Characterization of Leishmania major UDP-sugar Pyrophosphorylase and Determination of its Impact on UDP-galactose Metabolism Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften Dr. rer. nat genehmigte Dissertation von Dipl.-Biochem. Sebastian Damerow geboren am 4. März 1978 in Braunschweig 2010 Referentin: Prof. Dr. Rita Gerardy-Schahn Korreferentin: Prof. Dr. Françoise Routier Tag der Promotion: 11. März 2010 meinen Großeltern Herbert & Lucie „It is not the strongest species that survives, nor the most intelligent, but the one most responsive to change.” Charles Darwin, 1809-1882 Table of Contents Table of Contents Zusammenfassung……... ………………………………………………………………………………..…………... 5 Summary………. …………………………………………………………………………………………….………… 7 CHAPTER 1 – General Introduction………. ................................................................................................... 8 1.1 Leishmania, Leishmaniasis and Leishmanicidals…………....…………………………………….… 8 1.2 Biology & Pathobiology………………………………………………………………………………….. 9 1.2.1 Hosts………………………………………………………………………………………….……...... 10 1.2.2 Life Cycle…………………………………………………………………………………...………... 10 1.2.3 Genome……………………………………………………………………………………………… 12 1.3 Leishmania cell surface and roles in pathogenicity……………………………………..….……... 13 1.3.1 GPI-anchored Proteins…………………………………………………………………………….. 14 1.3.2 LPG…………………………………………………………………………………………………….. 14 1.3.3 GIPLs…………………………………………………………………………………………………… 16 1.3.4 PPGs…………………………………………………………………………………………………… 17 1.4 From Monosaccharide to Glycan……………………………………………………………………... 17 1.4.1 Glycan assembly…………………………………………………………………………………… 18 1.4.2 UDP-galactose metabolism……………………………………………………………………… 19 1.4.3 UDP-glucose pyrophosphorylase………………………………………………………………... 19 1.4.4 UDP-galactose pyrophosphorylase…………………………………………………………….. 20 1.4.5 UDP-sugar pyrophosphorylase…………………………………………………………………… 21 1.4.6 Glycosomes………………………………………………………………………………………….. 22 1.5 Objectives………………………………………………………………………………………………….. 24 CHAPTER 2 – Deletion of UDP-glucose Pyrophosphorylase Reveals a UDP-glucose Independent UDP-galactose Salvage Pathway in Leishmania major …………………… 25 CHAPTER 3 – Leishmania major UDP-Sugar Pyrophosphorylase: The Missing Link in Galactose Salvage?................................................................................ 40 CHAPTER 4 – Evaluating the Role of UDP-sugar Pyrophosphorylase in the Protozoan Parasite Leishmania major………………………………………………………………………….59 CHAPTER 5 – General Discussion………………………………………………………………………………….. 73 5.1 UDP-glucose pyrophosphorylase deletion revealed a UDP-glucose independent galactose salvage pathway in L. major…………………………………………………………….. 74 5.2 In vitro characteristics of a plant-like UDP-sugar pyrophosphorylase from L. major………... 74 5.3 UDP-galactose metabolism in L. major……………………………………………………………… 77 5.4 Importance of galactosylation for growth, virulence and viability in L. major…………....... 78 References…………………………………………………………………………………………………………….. 82 Abbreviations………………………………………………………………………………………………….………. 91 Curriculum Vitae……………………………………………………………………………………………….……... 93 Danksagung…………………………………………………………………………………………………………… 94 Zusammenfassung Zusammenfassung Leishmanien sind obligat parasitäre Protozoen aus der Familie der Trypanosomatidae, die vor allem in tropischen und subtropischen Regionen, die weit verbreitete Krankheit Leishmaniose verursachen. Umhüllt und geschützt von einer dichten Glykokalyx, widerstehen sie extrem lebensfeindlichen Umwelteinflüssen. Die zelluläre Ummantelung des Parasiten besteht hauptsächlich aus den speziellen Lipophosphoglycanen (LPGs), den mucin-artigen Proteophosphoglycanen (PPGs) und den leichtkettigen Glycoinositolphospholipiden (GIPLs) und ist unentbehrlich für das Überleben im Darm seines Vektors, der Sandfliege, und für die Infektiosität im Säugetier, seines Reservoirs. Vor allem die Phosphoglycanstructuren von Leishmanien sind extrem galactosehaltig und setzen eine spezialisierte, enzymatische Maschinerie voraus, um die hohen Ansprüche der UDP-Galactose- Biosynthese zu decken. Die vor kurzem biochemisch charakterisierte L. major UDP-Glucose- Pyrophosphorylase (UGP), die äußerst spezifisch Glucose-1-Phosphat mit UTP umsetzt und UDP- Glucose und Pyrophosphat erzeugt, schien ein Schlüsselenzym der UDP-Galactose-Biosynthese zu sein, da UDP-Galactose (UDP-Gal) entweder über Epimerisierung von UDP-Glucose (UDP-Glc) oder durch Uridylyltransfer von UDP-Glc zu Galactose-1-Phosphat entstehen sollte (Leloir Stoffwechselweg). Eine gezielte Gendeletion der UGP (Δugp) zeigte allerdings nur geringfügigen Einfluss auf die Phosphoglykanbiosynthese, wobei galactosehaltige GIPLs überhaupt nicht betroffen waren. Dementsprechend war die Virulenz der Δugp Mutante nur moderat herabgesetzt. In Anbetracht dieser Datenlage war anzunehmen, dass Leishmanien einen zweiten, UDP-Glucose unabhängigen Stoffwechselweg nutzen können, was mit dem offensichtlichen Fehlen des Leloir-Stoffwechselwegs in Leishmanien in Einklang war, da bisher kein verantwortliches Gen (von GALT) im Leishmanien Genom annotiert werden konnte. Es konnte jedoch ein aus Pflanzen stammendes Homolog der UDP-Zucker Pyrophosphorylase (USP) in Leishmanien und einigen anderen Protisten identifiziert werden. Charakteristisch für diese neue Klasse von Enzymen (EC 2.7.7.64) war ihre Fähigkeit, eine Bandbreite an Monosaccharid-1- Phosphaten mit UTP zu aktivieren, wie Galactose-, Glucose-, Xylose-, L-Arabinose-, Galacturonsäure- und Glucuronsäure-1-Phosphat, um daraus das jeweilige UDP-Monosaccharid und Pyrophosphat zu formen. Es konnte gezeigt werden, dass dieses Enzym eine hohe Affinität zur Bindung von UTP zeigt und Galactose-1-Phosphat bevorzugt. Es zeigt einen gerichteten Bi-Bi Mechanismus, wobei zuerst UTP binden muss, gefolgt vom jeweiligen Monosaccharid-1-Phosphat, wodurch sehr wahrscheinlich die Erzeugung des UDP-Monosaccharids vorangetrieben wird. Somit stehen die in vitro gemessenen Eigenschaften der USP in Einklang mit der postulierten Funktion der Galactoseverwertung. Damit einhergehend ließ die erste Analyse einer L. major USP Gendeletionsmutante (Δugp) eine verminderte Galactosylierung der LPG Seitenketten vermuten. Darüber hinaus konnte gezeigt werden, dass bereits die heterozygote Deletion der USP in der Δugp Mutante ausreichend war, um die restliche LPG Expression dieses Stammes einzudämmen, und 5 Zusammenfassung demzufolge die Rolle der USP im UDP-Galactose Stoffwechsel verdeutlicht. Interessanterweise war es trotz mehrmaliger Versuche nicht möglich eine Doppelmutante (Δugp/Δusp) zu generieren, was darauf hindeutet, dass UDP-Gal und/oder UDP-Glc in L. major essentiell sind. Letztlich wurde die subzelluläre Lokalisation der USP und einiger anderer an der UDP-Glc/UDP-Gal Biosynthese beteiligter Enzyme aufgeklärt, wobei sich herausstellte, dass es bedeutende Unterschiede mit anderen Trypanosomatiden gibt, die, anders als L. major, ihre UDP-Zucker in spezialisierten Organellen, den Glycosomen, synthetisieren. Schlagwörter: Galactose, UDP-Zucker Pyrophosphorylase, LPG, Leishmanien 6 Summary Summary The protozoan parasites Leishmania spp., causing tropical and sub-tropical diseases called leishmaniases, are surrounded by a thick glycocalyx that protects them from the hostile environments in which they live. This cellular coat mainly consists of unique phosphoglycans, comprising the highly abundant lipophosphoglycan (LPG) and mucin-like proteophosphoglycans (PPGs), as well as low molecular weight glycoinositolphospholipids (GIPLs) and is indispensable for survival of the parasite in the insect vector and for establishment of infection in mammals. Leishmania phosphoglycans are extremely rich in galactose and require thus a specialized enzymatic machinery to cover the high demand on UDP-galactose (UDP-Gal) for biosynthesis. The recently biochemically characterized L. major UDP-glucose pyrophosphorylase (UGP), very specifically utilizing glucose-1-phosphate and UTP to form UDP-Glucose (UDP-Glc) and pyrophosphate, was supposed to be the key enzyme in UDP-Gal biosynthesis, either via subsequent epimerization of UDP- Glc or by uridylyl transfer from UDP-Glc to galactose-1-phosphate. Targeted gene deletion of UGP (Δugp), however, only partially affected the synthesis of the galactose rich phosphoglycans, while no alteration in the abundant galactose-containing GIPLs was found. Consistent with these findings, Δugp Leishmania virulence was only modestly affected. These data implied that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis and is consistent with the absence of GALT gene essential for the Leloir pathway in Leishmania genome. However, a homologue of the plant UDP-sugar pyrophosphorylase (USP) was found in Leishmania parasites and several other protists. Characteristic for this new class of enzyme (EC 2.7.7.64), L. major USP catalyzes the reaction of a broad pool of monosaccharide-1-phosphates, such as galactose-, glucose-, xylose-, L-arabinose-, galacturonic acid- or glucuronic acid-1-phosphate with UTP to form the respective UDP- monosaccharide and pyrophosphate. We have notably shown that this enzyme possesses a high affinity for UTP, favors Gal-1-P and proceeds via an ordered Bi-Bi substrate mechanism in which UTP binds first followed by the sugar monophosphate, and thus most likely promotes nucleotide sugar synthesis rather than their pyrophosphorolysis. The in vitro characteristics of USP are hence in perfect agreement
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