Closing the crucial genetic gap in plasmalogen biosynthesis

Our body synthesizes a surprisingly rich variety of fat compounds called lipids. We need these compounds not only to store energy, but also to build the walls around our cells and walls inside cells and in our brain, so-called biological membranes. Metabolites formed from these lipids by special enzymes also play an important role in our body to communicate messages with biochemical signals. A disturbance of this complex mixture results in diseases which sometimes may be very severe. Although plasmalogens are among the most abundant phospholipid classes in our body its biosynthesis is still not fully understood at the genetic level. In particular, we still do not know which gene is responsible for introducing a special feature to these lipids, the vinyl ether double bond. Therefore our knowledge on the physiological significance of this bond is limited even though it gives plasmalogens the chemical characteristics the name is derived from, the liberation of an aldehyde in the plasma of cells. It is the goal of the present project to close this gap in our knowledge. We want to assign a gene to the enzyme which introduces the double bond into these lipids. We will use an unconventional approach which in our preceding project was successful to assign a sequence to another enzyme in ether lipid metabolism, alkylglycerol monooxygenase. We will select candidate genes by bioinformatic methods from the databases covering all proteins of mammalian organisms, and test them with a special cell culture system and the use of synthetic lipids carrying a fluorescence label. As a result of the project we expect the characterization of the gene responsible for the introduction of the double bond into plasmalogens. With this information a whole new set of research questions can be addressed. For the first time we will be able look into databases to look for hints of associations of this gene with diseases. We can get information on the enzyme carrying out the reaction, we can try to produce and study it to design drugs altering its activity. We can change its activity in model organisms and study the outcome to get information on the physiology and pathophysiology of this enzyme.

In this project we assigned a gene to an important biosynthetic reaction in mammalian lipid metabolism. This reaction is required to form one of the most abundant lipid classes in our brain and in immune cells, the so-called plasmalogens. Although the human genome project has been completed several years ago, we still do not know the function of approximately three thousand of our approximately twenty thousand protein-coding genes. This lack of knowledge hampers our efforts to draw conclusions from studies of genetic association of diseases, or to look for mechanisms and treatment of rare inherited diseases in newborns. The results of this work reduce the number of genes with unknown function by one, describing the role of a gene previously called transmembrane protein of unknown function number 189 to a step in plasmalogen formation. In the disease dementia of the Alzheimer type, plasmalogens are lower than in healthy people, and this might contribute to the disease. Smoking depletes plasmalogens from the lungs which are required as surfactant, a kind of lubricant. This depletion is thought to contribute to smoking-related lung disease. The identification of the this gene for the synthesis of plasmalogens is an important prerequisite for future studies on the way to develop drugs to manipulate this pathway. This knowledge enables us to predict a three-dimensional structure of the enzyme which then allows to selected by algorithms chemicals that might fit into this structure in a way potentially inhibiting its enzymatic reaction. The identification of this gene is also a prerequisite for the study of the importance of this reaction for the metabolism of cells and organs. It allows to attenuate or augment the amount of enzyme in a cell or organism and to look for the consequences of these alterations. As a first step on the way to identifying the gene, we developed methods to better measure the enzymatic reaction it performs. For this we used special fat molecules which have properties similar to natural fats, but can easily be detected since they have a strong fluorescence. We produced the special fat molecules we required in cultured cells with defined deficits in fat metabolism and purified them from these cultures. Another important feature required for our success was the public databases which store an enormous amount of data concerning the amount of messenger RNA for all known genes made by cell lines and by mouse tissues. A straight forward correlation analysis of the enzymatic activity we had measured in cultured cells and in mouse tissues with the data from public databases about the amount of messenger RNA in all these cells and tissues readily led us to the identification of the gene.