Alkylglycerol Monooxygenase in Dictyostelium discoideum

Our body synthesizes a fascinating complexity of fat species (lipids) which we require to build membranes in our body, to store energy, and to synthesize signalling molecules which regulate the response of our body to a big variety of physiological needs. One class of these lipids are ether-linked glycero-phospholipids. These compounds occur throughout our body in many different forms but their roles and the enzymes required for their metabolism are poorly understood. It is known that ether- linked glycero-phospholipids are essential for brain- and semen development and that they protect the eye from cataract. Alkylglycerol monooxygenase is the only enzyme capable of cleaving the ether bond in these ether- linked glycero-phospholipids, leading to their irreversible degradation. The reaction catalyzed by this enzyme had been first described in 1964, but it took until 2010 when we were able to assign a sequence to this labile membrane protein within the preceding project. This discovery enabled the use of the tools of modern molecular biology to detect and to manipulate the expression of the gene encoding this enzyme and to interpret data of associations of this gene with diseases in humans. To our surprise, gene association studies in humans and in the frog Xenopus laevis point to a role of alkylglycerol monooxygenase in cellular differentiation during embryonic development. From preparatory experiments and from these gene association studies we have set up a hypothesis how alteration of alkylglycerol monooxygenase might exert these effects. This includes the variation of the concentration of intracellular, ether-linked phosphatidylinositols followed by a modulation of phosphorylation signalling cascades affecting the wnt signalling pathway. In the present project we want to test this hypothesis in the social amoeba Dictyostelium discoideum. This eukaryotic model organism contains homologues of alkylglycerol monooxygenase and of the proteins constituting the signalling cascades included in our hypothesis. This organism can be easily manipulated genetically since it contains only one set of chromosomes and has a comparatively simple genome. Enough material for biochemical studies can be easily prepared, and well established methods to precisely quantify the cellular differentiation are at hand. To test our hypothesis, we will generate strains of Dictyostelium discoideum lacking or overexpressing alkylglycerol monooxygenase, and will characterize their behaviour along the differentiation to spores as well as the impact of these alterations on as many of the fat species as we can characterize, i.e. their lipidome. We will then mutate the suspected signalling proteins in these strains and look whether they interact with alkylglycerol monooxygenase to alter differentiation. With these experiments we anticipate to characterize, for the first time, how alterations of alkylglycerol monooxygenase affect a biological system and explain in a mechanistic way the associations of alkylglycerol monooxygenase with cellular differentiation.

Alkylglycerol monooxygenase is an enzyme involved in the metabolism of a special class of fat constituting substances, the ether lipids. Although abundant throughout our body, ether lipids have not been investigated to an extent comparable to other lipids. Platelet activating factor, a potent inflammatory mediator, belongs to the class of ether lipids. Alkylglycerol monooxygenase is the only enzyme known to be capable of cleaving the ether bond in alkylglycerol lipids. This enzyme had been described already in 1964 and it had been found to require tetrahydrobiopterin, a compound synthesized in our body with structural similarity to the vitamin folic acid. In preceding work funded by the fwf we were able to characterize the gene encoding this enzyme, which enabled us to manipulate this activity in model organisms to investigate its potential physiological role. Dictyostelium discoideum is a slime mold living in soils, which has been extensively used as a model organism for biochemical research. Several basic biochemical mechanisms had been first detected in this mold which later turned out to be important for human cells too. In the present project we wanted to get an insight into the impact of altering alkylglycerol monooxygenase on the behaviour of the slime mold Dictyostelium discoideum. We generated strains of this organism with almost zero alkylglycerol monooxygenase activity as well as one with strongly increased alkylglycerol monooxygenase activity and observed the impact of these manipulations on the behaviour of the mold in different settings in the laboratory. These include the ability of the single cell forms of the organism to cooperate to form a fruiting body, the ability to migrate towards food or light, as well as the ability to feed on bacteria offered as a bacterial lawn. Surprisingly, for most of the tests we made with the organism we observed no influence of the activity of alkylglycerol monooxygenase on the behaviour. Only the ability to feed on bacteria was impaired by the lack of the alkylglycerol monooxygenase to cleave the ether bond of alkylglycerols. When we fed the organism with a food broth instead of bacteria, no difference in the growth rate was observed. We characterized also the biochemical behaviour of alkylglycerol monooxygenase from Dictyostelium discoideum. This organism synthesizes an isomer of the above mentioned tetrahydrobiopterin, a folate like substance required for the enzymatic activity. The isomer occurring in Dictyostelium discoideum is called tetrahydrodictyopterin. We found that tetrahydrodictyopterin is more active as cofactor of the Dictyostelium enzyme as compared to tetrahydrobiopterin, which is formed in animals including humans. These data suggest that tetrahydrodictyopterin is formed by Dictyostelium discoideum as a hydroxylation cofactor, rather than only as antioxidative substance as previously suggested.