Norimitsu Inoue

Clostridium perfringens Enterotoxin Utilizes Two Structurally Related Membrane Proteins as Functional Receptors in Vivo

Human and mouse cDNAs showing homology to theClostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTMaccession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239–1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression ofCPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/orRVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE.

Dissecting and manipulating the pathway for glycosylphos-phatidylinositol-anchor biosynthesis.

The pathway for glycosylphosphatidylinositol-anchor biosynthesis consists of at least 10 reaction steps. Many of the genes encoding the enzymes and regulators involved in this pathway have been recently cloned and their products characterised. These studies have revealed the common and also different characteristics of glycosylphosphatidyl-inositol biosynthesis enzymes in different organisms, leading to the development of species-specific inhibitors of the pathway.

The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1.

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N‐acetylglucosamine (GlcNAc) from UDP‐GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG‐A and PIG‐C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG‐H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG‐L, which is involved in the second step in GPI synthesis, GlcNAc‐PI de‐N‐acetylation, did not associate with the isolated complex. The protein complex had GPI–GlcNAc transferase (GPI–GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized ∼100‐fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI–GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI–GnT may be relevant to selective usage of PI and/or regulation.

Defective Glycosyl Phosphatidylinositol Anchor Synthesis and Paroxysmal Nocturnal Hemoglobinuria

Publisher Summary The glycosyl phosphatidylinositol (GPI) anchor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to the nascent peptides in the ER. If the peptides are not GPI anchored, they are not expressed on the cell surface due to intracellular retention, degradation, and secretion. The human disease, paroxysmal nocturnal hemoglobinuria (PNH), is caused by this mechanism. PNH is a hematopoietic stem cell disorder characterized by the presence of abnormal cells of various hematopoietic cell lineages that are deficient in the surface expression of GPI-anchored proteins. The abnormal red blood cells of patients with PNH are sensitive to the hemolytic action of complement due to the deficient surface expression of decay accelerating factor and CD59, which are GPI-anchored complement inhibitors. The first step of GPI anchor biosynthesis is deficient in abnormal blood cells from patients with PNH. The X-linked gene PIG-A is cloned and somatically mutated in abnormal cells from patients with PNH. Due to its X-chromosomal location, a single inactivating mutation results in a loss of GPI anchor synthesis, even in a female hematopoietic stem cell, if it occurs in the active allele of the PIG-A gene. More than 60 patients with PNH from various countries were analyzed and the PIG-A gene was responsible for PNH in all of them. The X-chromosomal location of the PIG-A gene would also account for this uniformity of the responsible gene among the 10 or so genes involved in GPI anchor synthesis. Patients with PNH have one or more mutant hematopoietic stem cell clones that are deficient in GPI anchor synthesis due to somatic mutations in the X-linked gene, PIG-A. The somatic mutations of PIGA are widely distributed in the coding regions and splice sites, indicating that they occur at random sites.

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2

Glycosylphosphatidylinositols (GPIs) are attached to the C‐termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI‐N‐acetylglucosaminyltransferase (GPI‐GnT), which transfers N‐acetylglucosamine from UDP‐ N‐acetylglucosamine to phosphatidylinositol. GPI‐GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG‐A, PIG‐H, PIG‐C and GPI1. Here, we report that GPI‐GnT requires another component, termed PIG‐P, and that DPM2, which regulates dolichol‐phosphate‐mannose synthase, also regulates GPI‐GnT. PIG‐P, a 134‐amino acid protein having two hydrophobic domains, associates with PIG‐A and GPI1. PIG‐P is essential for GPI‐GnT since a cell lacking PIG‐P is GPI‐anchor negative. DPM2, but not two other components of dolichol‐phosphate‐mannose synthase, associates with GPI‐GnT through interactions with PIG‐A, PIG‐C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI‐GnT; however, the enzyme activity is enhanced 3‐fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI‐GnT and imply co‐regulation of GPI‐GnT and the dolichol‐phosphate‐mannose synthase that generates a mannosyl donor for GPI.

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8.

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a proteins C‐terminal GPI attachment signal peptide with a pre‐assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG‐S and PIG‐T. To determine roles for PIG‐S and PIG‐T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG‐S and PIG‐T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG‐S and PIG‐T form a protein complex with GAA1 and GPI8, and that PIG‐T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG‐T and PIG‐S, respectively.

Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol

Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases.Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutantPig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen inPig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F.

A HOMOLOGUE OF SACCHAROMYCES CEREVISIAE DPM1P IS NOT SUFFICIENT FOR SYNTHESIS OF DOLICHOL-PHOSPHATE-MANNOSE IN MAMMALIAN CELLS

Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates. It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor. In yeasts it also donates one mannose to the O-linked oligosaccharide. The yeastDPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum. We cloned human and mouse homologues of DPM1, termedhDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase. Human and mouse DPM1cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells. Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant. In contrast, hDPM1 andmDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1restored both mutants. Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.

Analysis of PIG‐A gene in a patient who developed reciprocal translocation of chromosome 12 and paroxysmal nocturnal hemoglobinuria during follow‐up of aplastic anemia

The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow‐up of AA. All metaphases in CD59‐deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59‐sufficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single‐base deletion mutation in the PIG‐A gene of this patients peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation.

MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic Male Germ Cells

During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.