Fas/APO-1 and TNF receptor 1 share a common signaling motif in their cytoplasmic tail called the ''death domain.'' Using the death domain as bait in the yeast two-hybrid system, several death domain-containing proteins that participate in cell death signaling have been identified, Here we report the isolation of a novel protein, sentrin, which interacts with Fas/APO-1 and TNF receptor 1 but not with FADD/MORT1 or CD40, Two-hybrid interaction assays reveal that sentrin associates only with the signal-competent forms of Fas/APO-1 or TNF receptor 1 death domains, Sentrin is a novel protein of 101 amino acids with homology to ubiquitin, Nedd8, and a Saccharomyces cerevisiae protein, Smt3, When overexpressed, sentrin provides protection against both anti-Fas/APO-1 and TNF-induced cell death.

The defect in the biosynthesis of the glycosyl-phosphatidyl inositol (GPI) anchor in paroxysmal nocturnal hemoglobinuria (PNH) appears to be in the initial steps. In biosynthetic studies using [3H]mannose, abnormal granulocytes of eight patients, and B lymphocytes transformed by Epstein-Barr virus of six different patients synthesized dolichyl phosphoryl mannose, but little or no later mannosylated intermediates. When fused with murine cell lines known to be deficient at different biosynthetic steps of the GPI anchor, the GPI-anchor-deficient granulocytes of 21/21 patients and lymphocytes from 6/6 patients complemented all murine cell lines except those of class A; cells of this class are not able to add N-acetylglucosamine to phosphatidylinositol. These studies indicate that the defect in GPI- anchor synthesis in PNH is early in the pathway, and is the same as that of class A mutants, but may be partial in some patients, resulting in the production of small amounts of mannosylated intermediates.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.

To investigate the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in the granulocytes of paroxysmal nocturnal hemoglobinuria (PNH), the glycolipids of granulocytes from PNH patients and normal volunteers were biosynthetically labeled with [3H]mannose in the presence of tunicamycin. Extracted glycolipids were examined by thin- layer chromatography and compared with known biosynthetic intermediates. Normal granulocytes consistently showed [3H]mannose incorporation into the complete GPI core, several GPI biosynthetic intermediates, and dolichol phosphate mannose (DPM). The granulocytes of 10 patients with PNH that had no expression of CD55 and CD59 on greater than 95% of the cells were able to incorporate [3H]mannose into DPM, but were not able to incorporate detectable amounts into the complete GPI core. THus, PNH granulocytes do not synthesize detectable amounts of the complete GPI core and this defect likely accounts for the absence of GPI-linked membrane proteins on hematopoietic cells in this syndrome.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.

A number of eukaryotic proteins are anchored to the membrane by glycosylphosphatidylinositol (GPI), of which the core structure is conserved from protozoan to mammalian cells. Here, we used a panel of thymoma mutants, which synthesize Thy-1 but cannot express it on the cell surface, to study the GPI biosynthetic pathway in mammalian cells. These mutants have been assigned into six complementation classes (A, B, C, E, F, H) by the technique of somatic cell hybridization. Using a combination of metabolic labeling and chemical/enzymatic tests, the biosynthetic defects were mapped to four different steps. Class A, C, and H mutants cannot transfer N-acetylglucosamine (GlcNAc) to a phosphatidylinositol acceptor, suggesting that the first step of GPI synthesis is regulated by at least three genes. The Class E mutant does not synthesize dolichol-phosphate-mannose, the donor for the first mannose residue transferred to the GPI core, and thus cannot form any mannose-containing GPI precursors. Class B and F mutants are defective in the addition of the third mannose residue or ethanolamine phosphate, respectively, to the elongating GPI core. Our findings have implications for the biosynthesis and attachment of the mammalian GPI anchor.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.