Kazuhito Ohishi

iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS.

TACE Trafficking The cytokine tumor necrosis factor (TNF) is a major driver of inflammation and contributes to the immune pathology seen in a variety of diseases, including inflammatory bowel disease, rheumatoid arthritis, and sepsis. Soluble TNF is produced by cleavage of its ectodomain by the ADAM family metalloprotease, TNFα-converting enzyme (TACE). However, the molecular regulation of TACE is not understood (see the Perspective by Lichtenthaler). Adrain et al. (p. 225) and McIlwain et al. (p. 229) now show that the rhomboid family member iRhom2 interacts with TACE in macrophages and is required for its proper intracellular trafficking and activation. In the absence of iRhom2, TACE was not released from the endoplasmic reticulum, and active protease did not reach the cell surface. Because of an inability to produce TNF, iRhom2-deficient mice were more resistant to lipopolysaccharide-induced septic shock but could not adequately control a Listeria monocytogenes infection. A pseudoprotease is required for the proteolytic cleavage of the proinflammatory cytokine tumor necrosis factor. Innate immune responses are vital for pathogen defense but can result in septic shock when excessive. A key mediator of septic shock is tumor necrosis factor–α (TNFα), which is shed from the plasma membrane after cleavage by the TNFα convertase (TACE). We report that the rhomboid family member iRhom2 interacted with TACE and regulated TNFα shedding. iRhom2 was critical for TACE maturation and trafficking to the cell surface in hematopoietic cells. Gene-targeted iRhom2-deficient mice showed reduced serum TNFα in response to lipopolysaccharide (LPS) and could survive a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to control the replication of Listeria monocytogenes. Our study has identified iRhom2 as a regulator of innate immunity that may be an important target for modulating sepsis and pathogen defense.

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.

Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI‐MT‐I) have different substrate specificities and are targets of species‐ specific inhibitors of GPI biosynthesis. GPI‐MT‐I, however, has not been molecularly characterized. Characterization of GPI‐MT‐I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI‐MT‐I and the gene responsible, PIG‐M. PIG‐M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG‐M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

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.

PIG-B, a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor.

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy‐terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post‐translationally linked to protein. We cloned a human gene termed PIG‐B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG‐B encodes a 554 amino acid, ER transmembrane protein with an amino‐terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy‐terminal portion of 470 amino acids within the ER lumen. A mutant PIG‐B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG‐B resides on the lumenal side of the ER membrane. The PIG‐B gene was localized to chromosome 15 at q21‐q22. This autosomal location would explain why PIG‐B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X‐linked PIG‐A gene.

DPM2 regulates biosynthesis of dolichol phosphate‐mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate

Biosynthesis of glycosylphosphatidylinositol and N‐glycan precursor is dependent upon a mannosyl donor, dolichol phosphate‐mannose (DPM). The Thy‐1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis. The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis. Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells. DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1. Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase. Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2. Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2.

Functional competence of T cells in the absence of glycosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption of the Pig-a gene

T lymphocytes express various glycosylphosphatidylinositol (GPI)‐anchored surface proteins, such as Thy‐1 and Ly‐6A. However, functional contribution of GPI‐anchored proteins in T cell activation is as yet poorly understood. Here we report the generation of mutant mice deficient in the expression of GPI‐anchored molecules exclusively in their T cells. We established mice carrying three identically oriented lox‐P sites within the Pig‐a gene, which encodes a component essential for the initial step of GPI anchor biosynthesis. These mice were crossed with mice carrying the Cre recombinase gene driven by the T cell‐specific p56lck proximal promoter. Offspring carrying both the lox‐P ‐containing Pig‐a gene and the Cre transgene exhibited almost complete loss of the surface expression of GPI‐anchored molecules on peripheral T cells. Interestingly, those T cells deficient in GPI‐anchored mole cules were capable of responding to T cell receptor stimulation in vitro and in vivo. These results indicate that T cells lacking the expression of GPI‐anchored molecules are functionally competent in exerting TCR‐mediated immune responses.

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin

Aerolysin of the Gram‐negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The α‐toxin of Gram‐positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)‐anchored proteins and generate pores in the cells plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI‐anchored proteins, but nevertheless bound aerolysin poorly and was 10‐fold less sensitive than wild‐type cells. A cDNA of N‐acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N‐glycan is involved in the binding. Removal of mannoses by α‐mannosidase II was important for the binding of aerolysin. In contrast, the α‐toxin killed GnTI‐deficient and wild‐type CHO cells equally, indicating that its binding to GPI‐anchored proteins is independent of N‐glycan. Because SL bound to wild‐type but not to GnTI‐deficient cells, and because a hybrid toxin consisting of SL and the α‐toxin killed wild‐type cells 10‐fold more efficiently than GnTI‐ deficient cells, SL with its binding site for N‐glycan contributes to the high binding affinity of aerolysin.

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.

GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits

Glycosylphosphatidylinositol (GPI) anchor is a membrane attachment mechanism for cell surface proteins widely used in eukaryotes. GPIs are added to proteins posttranslationally by a complex enzyme, GPI transamidase. Previous studies have shown that human and Saccharomyces cerevisiae GPI transamidases are similar and consist of five homologous components: GAA1, GPI8, PIG-S, PIG-T, and PIG-U in humans and Gaa1p, Gpi8p, Gpi17p, Gpi16p, and Cdc91p in S. cerevisiae. We report that GPI transamidase of Trypanosoma brucei (Tb), a causative agent of African sleeping sickness, shares only three components (TbGAA1, TbGPI8, and TbGPI16) with humans and S. cerevisiae but has two other specific components, trypanosomatid transamidase 1 (TTA1) and TTA2. GPI transamidases of both bloodstream form (growing in mammalian blood) and procyclic form (growing in tsetse fly vector) of the parasite have the same five components. Homologues of TTA1 and TTA2 are present in Leishmania and Trypanosoma cruzi but not in mammals, yeasts, flies, nematodes, plants, or malaria parasites, suggesting that these components may play unique roles in attachment of GPI anchors in trypanosomatid parasites and provide good targets for antitrypanosome drugs.