1) Biogenesis, transport and remodeling of GPI-anchored proteins (GPI-APs).
Glycosylphosphatidylinositol (GPI) is a glycolipid that consists of phosphatidylinositol, glucosamine, mannoses and phosphoethanolamines, and acts as a lipid anchor for various plasma-membrane proteins. GPI-APs play important roles in host self-defense, intercellular signal transduction, and other important processes. In addition, some GPI-APs function as receptors for certain viruses and toxins. The GPI-anchor is widely distributed and conserved in various eukaryotes and is essential for the development of higher animals, as well as for the growth of yeasts and protozoan parasites. The modification of proteins due to the attachment of the GPI-anchor functions as a protein localization and sorting signal. Our current project is to identify and clarify the functions of all the genes involved in the biosynthesis of the GPI-anchor in the ER (PIG genes; PhosphatidylInositol glycan) and in the sorting and localization of GPI-APs after their anchorage with GPI (PGAP genes; Post GPI-Attachment to Proteins). We expect that these studies will reveal why many proteins are modified with the GPI-anchor.
Fig. 1 GPI-anchor biosynthesis and the transport/remodeling of GPI-APs.
PIG genes are involved in the biosynthesis of the GPI-anchor in the ER. Thereafter, GPI-APs are transported to the plasma membrane and enriched in rafts. PGAP genes are involved in these later processes. PGAP1 and PGAP5, which localize in the ER, and PGAP2 and PGAP3, which localize in the Golgi, are involved in the lipid or glycan remodeling of the GPI-anchor. We found that the remodeling affects the sorting of GPI-APs because it alters the physical characteristics of the GPI-anchor.
2) Molecular genetics of acquired (paroxysmal nocturnal hemoglobinuria, PNH) and inherited GPI deficiencies.
PNH is an acquired hematopoietic stem cell disorder in which clonal cells that are defective in GPI biosynthesis are expanded. As a result, abnormal erythrocytes that lack CD59 and DAF/CD55 predominate. CD59 and DAF/CD55 are widely distributed GPI-anchored proteins that inhibit the activation of complement on the host cell surface, and their absence on erythrocytes makes these cells very sensitive to complement and lysis during infections and other events. We are proposing a three-step model of PNH pathogenesis. Step 1 involves the generation of GPI-deficient hematopoietic stem cells due to the somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. In this step, GPI-deficient cells not only survive, but they also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase the chance that additional genetic mutations are acquired, which leads to Step 3, where a subclone bearing the growth phenotype is generated (Fig. 2). We identified HMGA2 as the candidate gene for Step 3.
Along with our colleagues in England, we have also identified a novel disease that is characterized by venous thrombosis and seizures, and is caused by a GPI deficiency that has been inherited in an autosomal recessive manner. The patients have a point mutation in the promoter of PIG-M, a mannosyltransferase-encoding gene that plays an essential role in GPI biosynthesis. The point mutation severely reduces PIG-M expression and leads to partial GPI deficiency. While complete GPI deficiency is lethal, partial GPI deficiency could be caused by a partial defect in one of the GPI biosynthesis genes, and the symptoms may vary depending on the defect.
Fig. 2 Pathogenesis of PNH
Step 1 involves the generation of GPI-deficient hematopoietic stem cells due to the somatic mutation of the PIG-A gene.
Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. In this step, GPI-deficient cells survive and proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase the chance that additional genetic mutations occur.
Step 3 involves the generation of a subclone bearing the growth phenotype.
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Fig. 3 Structure of the GPIs of Pathogens |
3) Glycolipid biosynthesis in pathogens and its use in drug development.
Our research focuses on elucidating the biosynthesis of GPIs in mycobacteria and Trypanosoma brucei. T. brucei is the causative agent of African sleeping sickness while mycobacteria cause a number of diseases, including tuberculosis. GPIs are located on the cell surface of these pathogens and appear to play key roles in their evasion of host immune attack. In particular, GPI-like molecules found in mycobacteria have anti-inflammatory activities and are thought to be important for the establishment of the infection. We aim first to identify the genes that are involved in these GPI biosynthetic pathways, after which we can create and characterize gene deletion/overexpression mutants. This research will help us to understand the roles these GPI molecules play at the molecular level in cell surface structure maintenance and host immune response modulation. We are also seeking to determine the key enzymes of the biosynthetic pathways and develop a high-throughput screening system that will help us to identify lead compounds in drug libraries.