1) GPI-anchor biosynthesis and transport/remodeling of GPI-anchored proteins (GPI-APs)
The GPI (glycosylphosphatidylinositol)-anchor is a glycolipid consisting of phosphatidylinositol, glucosamine, mannose and phosphoethanolamine that acts as a lipid anchor for various plasma-membrane proteins. GPI-APs play critical roles in a variety of important processes, including host self-defense and intercellular signal transduction. Some GPI-APs also function as receptors for certain viruses and toxins. The GPI-anchor is widely distributed and conserved in eukaryotes and is essential for the proper development of higher animals and growth of yeasts and protozoan parasites. The modification of proteins due to the attachment of the GPI-anchor serves as a protein localization and sorting signal. We are currently seeking to identify and clarify the functions of all genes involved in the biosynthesis of the GPI-anchor in the ER (known as PIG genes after phosphatidylinositol glycan) and the sorting and localization of GPI-APs after their anchorage with GPI in the ER (known as PGAP genes after 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 transport/remodeling of GPI-APs
PIG genes are involved in the biosynthesis of the GPI-anchor and its binding to proteins in the ER. Thereafter, GPI-APs are transported to the plasma membrane, where they accumulate in rafts. PGAP genes are involved in the latter processes. PGAP1 and PGAP2/3, which are localized in the ER and Golgi, respectively, are involved in the lipid remodeling of the GPI-anchor. We found that this remodeling affects the sorting of GPI-APs because it alters the physical characteristics of the GPI-anchor.
2) Molecular genetics of acquired and inherited GPI deficiencies
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder in which clonal cells 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 make these cells very sensitive to complement-mediated lysis during infections and other events. We have reported that the gene responsible for PNH is PIG-A, which is the catalytic subunit of the first enzyme in GPI biosynthesis. We are proposing a three-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by 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, they also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate increases 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 are seeking to identify the cytotoxic cells and their targets, which plays a role in immunological selection. We are also seeking to identify which additional genetic mutation leads to the subclone with the growth phenotype in Step 3.
Our colleagues in England and we have also identified a novel disease that is characterized by venous thrombosis and seizures and is caused by 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. By making a model mouse, we are currently investigating the pathogenesis of this inherited GPI deficiency.
Fig. 2 Pathogenesis of PNH
Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by 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. The elevated proliferation rate increases the chance of additional genetic mutations being acquired.
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) GPI biosynthesis in pathogens and the identification of GPI-biosynthesis-inhibiting drugs
Our research here aims to elucidate 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 located on the cell surface of these pathogens appear to play key roles in their evasion of the host's immune defenses. In particular, GPIs found in mycobacteria have anti-inflammatory activities and are thought to be important for the establishment of the infection. We are seeking to identify genes involved in these GPI-biosynthetic pathways, after which we will create and characterize gene deletion mutants. These experiments will reveal the importance of these GPI molecules in the host immune response at the molecular level. We are also seeking to identify the key enzymes of the GPI biosynthetic pathways, after which we wish to develop a high-throughput screening system with which we can identify lead compounds from drug libraries.