Department of Immunoregulation

Three research fields in diseases, immune defense, and homeostasis are our main focus. The first examines how glycosylphosphatidylinositol-anchored proteins are synthesized, processed, transported and secreted, and how defects in these processes lead to the onset and pathology of diseases. The second examines how the pH of intracellular organelles is regulated and how these pH values regulate many biological functions. The third aims to identify the components and their functions in membrane contact sites between closely apposing organelles where lipids and ions are transported.

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. During the biosynthesis, an alkyl-lipid generated in the peroxisome is utilized. 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. Remodeling in the ER is necessary for efficient transport of GPI-APs to the Golgi by association with p24 family proteins. We found that these remodeling affects the sorting of GPI-APs because it alters the physical characteristics of the GPI-anchor. The genes in yellow boxes are currently known to be responsible for IGD and those in blue boxes are responsible for PNH.

1) Biogenesis, transport and remodeling of GPI-anchored proteins (GPI-APs).
Glycosylphosphatidylinositol (GPI) is a glycolipid that consists of phosphatidylinositol, glucosamine, mannoses and phosphoethanolamines. It 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 endoplasmic reticulum (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 by the GPI-anchor.

PIG genes are involved in the biosynthesis of the GPI-anchor in the ER. During this biosynthetic process, an alkyl-lipid generated in the peroxisome is utilized. Thereafter, GPI-APs are transported to the plasma membrane and are enriched in rafts. PGAP genes are involved in these latter 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. Remodeling in the ER is necessary for the efficient transport of GPI-APs to the Golgi via their association with p24 family proteins. We found that this remodeling process affects the sorting of GPI-APs because it alters the physical characteristics of the GPI-anchor. The genes in yellow boxes are currently known to be responsible for IGD, and those in blue boxes are responsible for PNH (see below).

Fig. 3 pH regulation of intracellular organelles The luminal pHs of organelles in secretory and endocytosis pathways are acidic as indicated in the boxes (the values are approximate).

2) Molecular genetics of GPI deficiencies.
In collaboration with our colleagues in England, we identified a disease called inherited GPI deficiency (IGD) that is caused by the PIGM mutation. PIGM is a mannosyltransferase-encoding gene that plays an essential role in GPI biosynthesis. The recent analysis of the exomes of patients with IGD with the next generation sequencer has already revealed twelve kinds of IGD to date. To further analyze IGDs, we are collaborating with many clinicians and scientists in Japan and abroad. As complete GPI deficiency is embryonically lethal, patients with IGD due to PIG gene mutations only have partial GPI deficiencies. The patients with IGD that is due to remodeling defects even have null mutations. The symptoms of IGD vary depending on the defective genes and the degree of defectiveness. The main symptoms are intellectual disability and seizures that are often accompanied by hyperphosphatasia, abnormal facial features, brachytelephalangy and sometimes other organ anomalies. IGD has been proven to be the main cause of Mabry syndrome/hyperphosphatasia mental retardation syndrome and CHIME syndrome. IGD has also been found in Early Onset Epileptic Encephalopathies such as Ohtahara syndrome and West syndrome. Good diagnostic markers for IGD are the presence of hyperphosphatasia and the decreased flow cytometric expression of granulocyte CD16, which is a GPI-AP . We will establish the diagnostic criteria by accumulating patient information and seek effective therapies.Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI deficiency that is caused by somatic mutation of the X-linked PIGA gene in hematopoietic stem cells. However, it was recently reported that PNH can also be caused by somatic mutation of one allele of the PIGT gene in combination with a germline mutation in the other allele. This showed that PIGA is not always the responsible gene in PNH, although this situation is rarely seen. PNH is a hematopoietic disease that is characterized by the expansion of clonal cells that are defective in GPI biosynthesis. As a result, abnormal erythrocytes lacking the GPI-APs CD59 and DAF/CD55 (which inhibit the activation of complement) predominate, leading to massive hemolysis due to excessive complement attack during infections and other events. We are presently analyzing the pathogenesis of PNH by seeking the mechanism behind the clonal expansion of GPI-deficient hematopoietic stem cells.

3) pH regulation in acidic organelles and its biological significance, and analysis of membrane contact sites between organelles.
Each intracellular organelle is compartmentalized by lipid bilayers and possesses a characteristic environment, proteins and lipids that allow it to fully exercise its functions. A major environment factor is pH. The lumens of organelles in the secretory and endocytosis pathways are known to have acidic environments. This lumen acidity is believed to play critical roles in many biological processes in cells, including the transport, processing, and glycosylation of proteins and lipids. It is also known to influence the morphology of organelles and to participate in the onset and pathology of diseases that are characterized by pH regulation defects. However, the mechanisms behind the latter abnormal phenotypes are largely not known. Recently, we reported for the first time the establishment of mutant cells in which the Golgi pH is dysregulated. Analysis of these cells led to the identification of the gene that bore the mutations and was responsible for the deranged functions of the mutant cells. This project aims to clarify the mechanisms and biological significance of acidic pH regulation by establishing and analyzing additional mutant cells that are defective in organelle pH regulation and to improve our understanding about how such mutations can lead to pathology, as this will facilitate the identification of effective drugs. As an offshoot from the above study, we are also analyzing the components and functions of the membrane contact sites between closely apposed organelle membranes where lipids and ions are transported.