Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones.

To aid in studies of human glycoinositol-phospholipid (GPI) anchor pathway biochemistry in normal and affected paroxysmal nocturnal hemoglobinuria cells, GPI anchor-defective human K562 cell lines were derived by negative fluorescent sorting of anti-decay-accelerating factor (DAF) monoclonal antibody-stained cells either following or in the absence of ethylmethylsulfonate pretreatment. The resulting cloned cells showed deficiencies of both DAF and GPI-anchored CD59, some (designated group A) exhibiting total absence and some (designated group B) exhibiting approximately 10% levels of surface expression of the two proteins. In heterologous cell fusions, group A clones complemented defective Thy-1 expression by class A, B, C, E, and I Thy-1-negative lymphoma lines, but not H or D lines, the latter of which is defective in the Thy-1 structural gene. In contrast, group B clones complemented all previously described GPI anchor pathway-defective lymphoma classes. Immunoradiomatic assays of cells and supernatants and 35S biosynthetic labeling showed that group A cells degraded DAF protein while group B cells secreted it but failed to attach a GPI anchor structure. [3H]Man labeling of intact cells and UDP-[3H]GlcNAc and GDP-[3H]Man labeling of broken cell preparations demonstrated that group A cells failed to synthesize GlcNAc- and GlcN-PI (GPI-A and -B) as well as more polar mannolipids, whereas group B cells showed accumulation of GlcNAc-PI with approximately 10-fold diminished levels of GlcN-PI and more polar mannolipids. The failed assembly of GlcNAc-PI in group A cells and the reduced conversion of this intermediate to GlcN-PI in group B cells indicates that the former harbors a defect in UDP-GlcNAc transferase or in assembly of its PI acceptor, while the latter harbors a defect in GlcN-PI deacetylase activity.     

Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine.

A number of mammalian cell surface proteins are anchored by glycoinositol phospholipid (GPI) structures that are preassembled and transferred to them in the endoplasmic reticulum. The GPIs in these proteins contain linear ethanolamine (EthN)-phosphate (P)-6ManManManGlcN core glycan sequences bearing an additional EthN-P attached to the Man residue (Man 1) proximal to GlcN. The biochemical precursors of mammalian GPI anchor structures are incompletely characterized. In this study, putative [3H]Man-labeled GPI precursors were obtained by in vitro GDP-[3H] Man labeling of HeLa cell microsomes and by in vivo [3H]Man labeling of class B and F Thy-1 negative murine lymphoma mutants known to accumulate incomplete GPIs. The high performance liquid chromatography-purified in vitro and accumulated in vivo GPI products were structurally analyzed by nitrous acid deamination, hydrofluoric acid, trifluoroacetic acid hydrolysis, biosynthetic labeling, and exoglycosidase treatment. The data were consistent with a biosynthetic scheme in which Man and EthN-P are added stepwise to the developing glycan. Several additional points were demonstrated: 1) putative mammalian GPI precursors contain incomplete core glycans corresponding to those in previously characterized trypanosome GPI precursors. 2) The proximal EthN-P found in mature mammalian GPI anchor structures is added to Man 1 prior to incorporation of Man 2 and Man 3. 3) Glycans in the incomplete GPIs that accumulate in classes B and F lymphoma mutants consist of Man2- and Man3GlcN in which EthN-P is linked to Man 1. 4) Distal EthN-P linked to the 6-position of Man, characteristic of the complete GPI core, is found both in a subsequent GPI species with the glycan sequence EthN-P-6ManMan(EthN-P----)ManGlcN and in a more polar GPI product.     

Hydrophilic anchor-deficient Thy-1 is secreted by a class E mutant T lymphoma

To investigate the mechanism of glycophospholipid anchoring of the surface antigen Thy-1, we have undertaken a comparative biosynthetic study using a wild-type Thy-1+ murine T lymphoma (BW5147) and a mutant T lymphoma (class E) that synthesizes Thy-1 but fails to express it on the plasma membrane. Labelling experiments with D-[2-3H]mannose demonstrate that, unlike the wild type, the mutant cells are secreting large amounts of Thy-1 and that the secreted molecules are hydrophilic. Moreover, unlike the wild type, they fail to incorporate [3H]palmitic acid into Thy-1. Both wild-type and mutant cells do incorporate labeled galactose and fucose into Thy-1. We conclude that the lack of surface expression of Thy-1 by this mutant results from the failure to add anchor components to Thy-1.     

The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein

Thy-1 glycoprotein is a member of a class of proteins which are anchored to the plasma membrane via a covalently bound glycophospholipid. The biosynthesis and anchoring of Thy-1 were investigated in a family of wild-type and mutant (complementation groups A, B, C, E, and F) T lymphomas. The mutants all synthesize Thy-1 but fail to express it on the cell surface. Analysis of the size of D-[2-3H]mannose-labeled dolichol-linked oligosaccharides showed that the class E mutant is the only cell line which does not synthesize dolichol-P-P-Glc3Man9GlcNAc2. Turnover and possible secretion of Thy-1 by mutant T lymphoma cells were documented in D-[2-3H]mannose pulse-chase experiments. The turnover of [3H]Thy-1 for all wild-type cells is considerably slower than for the mutant cells. Class B and E cells release appreciably more [3H]Thy-1 than wild-type cells. Additional experiments were performed to determine the electrophoretic mobility and hydrophobicity of cell-associated and released forms of Thy-1 labeled overnight with [3H]mannose. All wild-type and class A, C, E, and F mutant cells contain a major Triton X-114 binding species of cell-associated [3H]Thy-1. All extracellular [3H]Thy-1 was almost exclusively hydrophilic. The presence of two Thy-1 anchor components, ethanolamine and palmitate, was investigated. Biosynthetic labeling with [3H]palmitic acid showed that all of the wild-type cells but none of the mutants incorporated this anchor precursor into Thy-1. In [3H]ethanolamine-labeling experiments, incorporation was detected in the Thy-1 of all wild-type cells and in two mutants, S1A-b and T1M1-c. Based on the above studies, the phenotype of Thy-1 negative T lymphoma mutants can be re-evaluated. In classes A and F, dolichol-linked oligosaccharides appear normal and no anchor is detected. In class B, dolichol-linked oligosaccharides appear normal, a partial anchor may be present, and a substantial amount of Thy-1 is released. In class C, dolichol-linked oligosaccharides appear normal and a partial anchor may be present. In class E, truncated dolichol-linked oligosaccharides are formed, no anchor is detected, but a substantial amount of newly synthesized Thy-1 is released. These observations are discussed with reference to the possibility that the lesions which characterize the mutants pertain to the biosynthesis of the glycophospholipid moiety of Thy-1.     

Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants

The glycosyl phosphatidylinositol (GPI) anchors that attach certain proteins to membranes are preassembled by sequential addition of glycan components to phosphatidylinositol (PI) before being transferred to nascent polypeptide. A cell-free system consisting of trypanosome membranes has been reported to catalyze GPI biosynthesis (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800; Menon, A. K., Schwarz, R. T., Mayor, S., and Cross, G. A. M. (1990) J. Biol. Chem. 265, 9033-9042). We now describe conditions for studying the initial steps of GPI biosynthesis in extracts of murine lymphoma cells. Two chloroform-soluble products, tentatively identified as [6-3H]GlcNAc-PI and [6-3H]GlcN-PI were generated during incubations of EL4 cell lysates with UDP-[6-3H]GlcNAc. The involvement of PI in the reaction was established by the sensitivity of the products to hydrolysis by PI-specific phospholipase C and the finding that the addition of exogenous PI to the incubation stimulated the reaction. The minor, more polar product was sensitive to nitrous acid cleavage and was converted to the major product, as judged by TLC, after treatment with acetic anhydride. The glycolipids generated in lymphoma extracts appeared to be the same as the products produced in parallel incubations with trypanosome membranes. Analysis of available lymphoma mutants deficient in Thy-1 surface expression revealed that extracts of the class A, C, and H mutants are completely defective in synthesizing GlcNAc-PI and GlcN-PI.     

The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells.

Stimulation of murine T cells by engagement of the multi-component T cell antigen receptor or by cross-linking the Thy-1 molecule leads to a similar response characterized by lymphocyte activation and lymphokine production. The early biochemical events induced by engaging these molecules also are similar and begin with activation of a tyrosine kinase pathway and tyrosine phosphorylation of a comparable set of substrates. Previous work demonstrates that the protein tyrosine kinase p60fyn is associated with the antigen receptor and therefore it may participate in the tyrosine phosphorylations that are observed with antigen receptor signaling. In this study we demonstrate that the Thy-1 molecule is also associated with p60fyn in a murine T cell hybridoma and in murine thymocytes. The interaction is independent of antigen receptor expression. Thy-1 is a member of the class of molecules anchored to the plasma membrane by a glycophosphatidylinositol (GPI) group. The association of Thy-1 with p60fyn is dependent on the GPI linkage, since cleavage of the GPI anchor disrupts the interaction. The association of Thy-1 and p60fyn suggests a means by which Thy-1 cross-linking leads to tyrosine phosphorylation and T cell activation.     

Atypical mannolipids characterize Thy-1-negative lymphoma mutants.

Essentially all eukaryotic cells, including murine lymphomas, express surface proteins, such as Thy-1, which are anchored by a phosphoinositol mannolipid. Putative mannolipid anchor precursors can be detected in these cells. Six distinct Thy-1-negative lymphoma mutants lack complete mannolipids, and three mutants synthesize atypical mannolipids. The absence of complete mannolipids can account for the lack of expression of multiple mannolipid-anchored proteins and may also account for the lack of lipid anchoring in the human disease paroxysmal nocturnal hemoglobinuria. Structural information on the mannolipids of wild-type and mutant cells indicates that anchor biosynthesis in these cells may involve both transmembrane flip-flop of intermediates and a deacylation step.     

Class F Thy-1-negative murine lymphoma cells are deficient in ether lipid biosynthesis

The glycosyl phosphatidylinositol (PI) membrane anchors of several proteins contain 1-alkyl-2-acyl-glycerophosphoinositol. Although this PI analog has never been found free in cells, the presence of "alkyl-PI" as a component of some membrane anchors suggests its existence. The resistance of ether linkages to cleavage by mild alkali treatment was used to detect possible alkyl chains in the [3H]inositol-labeled phospholipids of several murine lymphoma cell lines which normally express the glycosyl PI-anchored protein Thy-1. One lipid, which arose from alkaline hydrolysis of PI and had mobility on thin layer chromatography similar to lyso-PI, was detected in all wild-type cell lines. Analysis of the base-stable inositol lipids of several lymphoma lines that are deficient in Thy-1 surface expression because of defective biosynthesis of the glycosyl PI membrane anchor revealed that the putative alkyl-PI was missing in the class F mutant. The levels of both the ethanolamine- and choline-containing plasmalogens were also decreased 10-fold in these cells, suggesting a general defect in the production of ether lipids. The activity of the peroxisomal form of dihydroxyacetonephosphate acyltransferase, which catalyzes the first step of ether lipid biosynthesis, was found to be 10-fold decreased relative to the wild-type level. Unlike previously described Chinese hamster ovary cell mutants deficient in ether lipids (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5170-5174), the class F Thy-1- cells contain intact functional peroxisomes. Attempts to restore the putative alkyl-PI to the class F mutants by alkylglycerol supplementation were unsuccessful, despite concomitant restoration of the much larger plasmenylethanolamine pool, suggesting that there are some differences in the biosynthesis of this PI analog and plasmalogens that are presently not understood. Although the deficiencies in ether lipids and surface expression of Thy-1 in the class F mutants could also be due to separate mutations, our findings raise the possibility that alkyl-PI exists in animal cells and may be an obligate precursor for the biosynthesis of the glycosyl-PI membrane anchor of Thy-1.     

The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.     

The glycophospholipid anchor of Thy-1. Biosynthetic labeling experiments with wild-type and class E Thy-1 negative lymphomas

The Thy-1 antigen of the surface of lymphocytes and neurons is anchored to the plasma membrane via a glycophospholipid moiety. In contrast, the Thy-1 synthesized by the class E Thy-1 negative mutant lymphoma is secreted as a hydrophilic species. The present investigation uses the approach of biosynthetic labeling to investigate further the structure of the intracellular Thy-1 of wild-type cells and the secreted Thy-1 of these mutant cells. In the wild-type cells, Thy-1 can be labeled with [3H] mannose, [3H]galactose, [3H]fucose, [3H]ethanolamine, and [3H]palmitic acid. In the latter two cases the label is recovered almost exclusively in a detergent-binding Pronase fragment of the protein. The incorporated label is in the form of [3H]ethanolamine, or [3H]palmitate and stearate, respectively. Reductive methylation of biosynthetically labeled Thy-1 and a nonradioactive sample of Thy-1 shows that [3H]ethanolamine is incorporated equally into two residues of ethanolamine, only one of which has a free amino group. A single residue of glucosamine with a free amino group is also detected. Each of the sugar precursors is incorporated with extensive conservation of chemical identity. In the class E cells, each of the labeled sugars but neither [3H]ethanolamine nor [3H]palmitate is incorporated into Thy-1. The anchor moiety therefore appears to be entirely missing, although N-linked oligosaccharide processing is essentially normal. We postulate that the anchor deficiency in the mutant cells results from a biosynthetic lesion.     

Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria

Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.     

Thy-1, a versatile modulator of signaling affecting cellular adhesion, proliferation, survival, and cytokine/growth factor responses

Thy-1 is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored protein involved in T cell activation, neurite outgrowth, apoptosis, tumor suppression, wound healing, and fibrosis. To mediate these diverse effects, Thy-1 participates in multiple signaling cascades. In this review, we discuss Thy-1 signaling primarily in non-immunologic cell types, including neurons, mesangial cells, ovarian cancer cells, nasopharyngeal carcinoma cells, endothelial cells, and fibroblasts. We review the current literature regarding Thy-1 signaling via integrins, protein tyrosine kinases, and cytokines and growth factors; and the roles of these signaling pathways in cellular adhesion, apoptosis, cell proliferation, and cell adhesion and migration. We also discuss the role of Thy-1 localization to lipid rafts, and of the GPI anchor in Thy-1 signaling. Ongoing research on the mechanisms of Thy-1 signaling will add to our understanding of the diverse physiologic and pathologic processes in which Thy-1 plays a role.     

Identification of defects in glycosylphosphatidylinositol anchor biosynthesis in the Thy-1 expression mutants.

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.     

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae

Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copper-resistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copper-resistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21delta and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21delta hog1delta strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21delta cells and in ssk1delta las21delta cells but not in sho1delta las21delta cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21delta strain. The constitutive activation was canceled by introducing the sskl mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Slnl branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.     

Biochemical background of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by paroxysms of intravascular hemolysis. A considerable part of erythrocytes in patient blood is susceptible to autologous complement activation because of the deficiency of CD59, which is a glycosylphosphatidylinositol (GPI)-anchored protein and inhibits the formation of the membrane attack complex (MAC) of complement. The deficiency of CD59 is derived from the inability of GPI-anchor synthesis. Although more than 10 proteins are involved in the GPI-anchor synthesis, the mutation of only one protein, PIG-A, causes the defect in about 200 patients with PNH who have been analyzed. The reason why only PIG-A causes the deficiency of GPI anchor is due to the location of its gene on X chromosome. The clonal stem cell mutated with PIG-A gene in the bone marrow loses the capability of the synthesis of GPI-anchor. The mutation of PIG-A gene alone, however, seems to be insufficient to account for the survival of the PIG-A-deficient cells in the bone marrow. Thus, a fraction of the mutant stem cells probably gain a survival advantage by some additional changes, either additional mutations or changes in immunological circumstances. The release of the surviving cells into blood stream results in a clinical syndrome with PNH.     

Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells.

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.     

Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells

Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.     

Possible pathogenic role of the transmembrane isoform of CD160 NK lymphocyte receptor in paroxysmal nocturnal hemoglobinuria

PIGA mutations in paroxysmal nocturnal hemoglobinuria (PNH) patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency. Herein, we report the constitutive expression of the transmembrane CD160 (CD160-TM) activating receptor on non PIGA-mutated PNH patients circulating NK cells. In healthy individuals, only the GPI-anchored isoform of CD160 receptors is expressed on the circulating NK lymphocytes, while the transmembrane isoform appears after ex vivo activation. Similarly to CD160-GPI, we identified CD160-TM as a receptor for the MHC class I molecules. We demonstrate that PNH patients NK lymphocytes spontaneously produce significant amounts of IFN-γ that is inhibited by anti-CD160-TM or anti-MHC class I mAbs. These results indicate that circulating NK cells from PNH patients exhibit a self-MHC class I molecule reactive effector function, which could be mediated through the recruitment of CD160-TM receptor. Our data provide new insights regarding the possible role of CD160-TM on PNH patients NK lymphocytes and in the pathogenesis of the disease.     

DNA methylation alters chromatin structure and regulates Thy-1 expression in EL-4 T cells

Thy-1 exhibits marked differences in expression in various tissues in many species; therefore, it is of interest to define possible mechanisms that may regulate Thy-1 expression. We produced Thy-1 negative variants of the murine T cell lymphoma EL-4 by mutagenesis with ethylmethanesulfonate (EMS), negative selection with anti-Thy-1 monoclonal antibodies (mAb) plus complement, and fluorescence-activated cell sorting (FACS). Thy-1 surface negative (Thy-1-) mutants produced in this manner were shown to produce no detectable Thy-1 mRNA, but contained an intact Thy-1 gene as determined by Southern blotting. 5' CG sequences, which had been demethylated in the parent EL-4 clone, were completely methylated in the EMS-induced Thy-1-variant. In addition, a DNase I hypersensitive site that mapped to the 5' end of the Thy-1 gene in EL-4 was absent in the Thy-1- variant. Treatment of this Thy-1- clone with 5-azadeoxycytidine (5-dAZA) resulted in re-expression of surface Thy-1, demethylation of the 5' CG sequences, and regeneration of the DNase I hypersensitive site. These studies indicate that methylation of certain critical DNA sequences in the 5' region of the Thy-1 gene can alter local chromatin structure and regulate expression of this gene.     

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.