Stimulation by dolichol phosphate-mannose of N-acetylglucosaminyl-lipid biosynthesis by membranes from class E Thy-1-negative mutant mouse lymphoma cells which are defective in dolichol phosphate-mannose biosynthesis

Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway.     

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.     

No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes.

Recent evidence shows that the mature Thy-1 surface glycoprotein lacks the C-terminal amino acids 113 to 143 predicted from the cDNA sequence and is anchored in the plasma membrane by a complex, phosphatidylinositol-containing glycolipid attached to the alpha-carboxyl group of amino acid 112. Here we studied the biosynthesis of Thy-1 in two previously described and two newly isolated Thy-1-deficient mutant cell lines. Somatic cell hybridization indicated that their mutations affected some processing step rather than the Thy-1 structural gene. The Thy-1 made by mutants of classes C, F, and H bound detergent but, in contrast to wild-type Thy-1, their detergent-binding moieties could not be removed by phospholipase C. In addition, tryptophan, which only occurs in position 124, was incorporated into Thy-1 of these mutants but not of wild-type cells. Last, the Thy-1 of wild-type but not mutant cells could be radiolabeled with [3H]palmitic acid. Together, these findings strongly suggest that mutants of classes C, F, and H accumulate a biosynthetic intermediate of Thy-1 which retains at least part of the hydrophobic C-terminal peptide. The Thy-1 of these mutants remained endoglycosidase H sensitive, suggesting that it accumulated in the rough endoplasmic reticulum or the Cis-Golgi. A different Thy-1 intermediate was found in a class B mutant cell line: the Thy-1 of this mutant was 2 kilodaltons smaller than the Thy-1 of other cell lines, did not bind detergent, and was rapidly secreted via a normal secretory pathway.     

Characterization of glycophospholipid intermediate in the biosynthesis of glycophosphatidylinositol anchors accumulating in the Thy-1-negative lymphoma line SIA-b.

Several mammalian mutant cell lines are deficient in the biosynthesis of glycophosphatidylinositol anchors for membrane proteins. When metabolically labeled with [3H]myo-inositol or [3H]mannose, two out of five mutant lines (SIA-b and EL4-f) accumulated abnormal lipids which remained undetectable in the corresponding parental cell lines. The most abundant glycolipid of SIA-b cells (named lipid X) was isolated and partially characterized using hydrofluoric acid, nitrous acid deamination, acetolysis, and exoglycosidase treatments alone or in combination. The partial structure for the carbohydrate moiety of lipid X is Man alpha-(X----)Man alpha-GlcN-inositol, X being a charged, HF-sensitive substituent (possibly phosphoethanolamine). Lipid X is largely resistant to phosphatidylinositol-specific phospholipase C treatment but can be rendered sensitive to the enzyme by treatment with methanolic NH3, which suggests the presence of an acyl chain on the inositol moiety. The lipid moieties of lipid X are heterogenous in that about 50% of headgroups remain bound to a lipid moiety after mild alkaline hydrolysis. Similarly, about 50% of the lipid moieties of Thy-1, a glycophosphatidylinositol-anchored surface glycoprotein, isolated from SIA, the parent of SIA-b cells or from EL4 lymphoma cells, are resistant to mild alkaline hydrolysis. Altogether the data suggest that the SIA-b mutant line lacks an enzyme acting late in the anchor glycolipid biosynthesis pathway.     

Thy-1-negative and Ly-1-negative variants of T cells produce interleukin 2 in response to mitogens

IL 2 production by T cell variants, which lack the Thy-1 or Ly-1 surface glycoproteins, was studied. Cross-linking of the Thy-1 molecule resulted in IL 2 production by the EL4 thymoma and by a T cell hybridoma, suggesting that Thy-1 may play a role in T lymphocyte triggering. To further study the functional role of this molecule, Thy-1-negative variants were selected and analyzed for IL 2 production in response to phorbol-12-myristate-13-acetate (PMA) or to Con A. It was demonstrated that in spite of their failure to express Thy-1, the Thy-1-negative clones were capable of IL 2 production. These results indicated that although Thy-1 cross-linking triggers cell activation, a signal provided by Thy-1 is not indispensable for cell activation by mitogens. The T cell tumor line LBRM331A5 responds synergistically to IL 1 and PHA by releasing IL 2. It was demonstrated that anti Ly-1 monoclonal antibodies and PHA co-stimulated LBRM331A5 cells, as did IL 1 plus PHA. Thus, anti Ly-1 antibodies mimic the effect of IL 1, suggesting a role for Ly-1 antigen in T cell activation, perhaps by serving as an IL 1 receptor or as an associated molecule. To further study the functional role of Ly-1 and its relation to IL 1 receptor, Ly-1-negative variants of the LBRM331A5 cell line were selected and analyzed for IL 2 production in response to PHA plus IL 1. It was demonstrated that the Ly-1-negative clones were capable of IL 2 production as efficiently as Ly-1-positive clones. These results indicate that the Ly-1 and IL 1 receptor are distinct molecules, which are involved in different activation pathways.     

Glutathione biosynthesis in murine L5178Y lymphoma cells

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg²⁺ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg²⁺ or Mn²⁺ but Ca²⁺ and K⁺ had no effect. Activation was inhibited by the phosphatase inhibitor, F^?. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg²⁺, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg²⁺ and was inhibited by F^?. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.     

Anchoring of membrane proteins via phosphatidylinositol is deficient in two classes of Thy-1 negative mutant lymphoma cells.

Recent evidence shows that mature Thy-1 glycoprotein lacks amino acids 113-143 predicted from the cDNA sequence and is anchored to the plasma membrane by a phosphatidylinositol-containing glycolipid attached to amino acid 112. Previously characterized Thy-1-deficient mutant lymphoma lines of complementation classes A and E were analysed. They make detergent binding Thy-1 precursors but, in contrast to wild-type, the detergent binding moiety cannot be removed by phospholipase C. Moreover, tryptophan which only occurs at position 124 is incorporated into mutant but not parental Thy-1. This suggests that the mutants make a Thy-1 precursor of 143 amino acids but fail to replace its C-terminal end by a glycolipid anchor.     

S49 mouse lymphoma cells are deficient in hypoxanthine transport

The rate of uptake of hypoxanthine in S49 cells was only about 2-5% of the rate of hypoxanthine transport observed in many other types of mammalian cells, and of the rate of uridine transport in this and other cell types. Part of the slow entry of hypoxanthine seems to be due to non-mediated permeation, but the remainder is saturable, strongly inhibited by uridine, nitrobenzylthioinosine and dipyridamole and not detectable in a nucleoside-transport-deficient mutant of S49 cells (AE1). The inhibition of hypoxanthine transport in S49 cells by nitrobenzylthioinosine resembles the inhibition of nucleoside transport in these and other mammalian cells, whereas it contrasts with the resistance of hypoxanthine transport to nitrobenzylthioinosine in all types of mammalian cells that have been investigated. We conclude that S49 cells lack the hypoxanthine transport system common to other types of cells and that hypoxanthine entry into these cells is mediated, although very inefficiently, by the nucleoside transporter. In contrast, adenine transport in S49 and AE1 cells was comparable to that in other types of cells.     

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.     

Abnormal lipid-linked oligosaccharides in class E Thy-1-negative mutant lymphomas.

The glycosylation defect of Thy-1-mutant lymphomas of the class E complementation group has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor of the asparagine-linked oligosaccharides of glycoproteins. Two major lipid-linked oligosaccharides were isolated from the mutant cells. Both oligosaccharides were smaller than the lipid-linkid oligosaccharides of wild-type lymphomas and, in contrast to the lipid-linked oligosaccharides isolated from wild-type cells, both were resistant to digestion with endoglycosidase H. The oligosaccharides of newly synthesized polypeptides in class E Thy-1-cells were also resistant to endoglycosidase H digestion, providing strong evidence that they are derived from the abnormal lipid-linked oligosaccharides.     

Peroxisomes and ether lipid biosynthesis in rat testis and epididymis

Plasmalogens are a main component of the spermatozoon membrane, playing a crucial role in their maturation. The initial steps in plasmalogen biosynthesis are catalyzed by two peroxisomal enzymes, dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase. The localization of both enzymes in the membrane of peroxisomes implies that plasmalogen-producing cells should contain this organelle. To unravel the putative source of spermatozoan plasmalogens we investigated which cell types in the testis and epididymis are endowed with peroxisomes. To this extent, testicular and epididymal tissue was analyzed at the protein and RNA levels by means of light and electron microscopical immunocytochemistry as well as by Western and Northern blotting. Proteins and mRNAs of peroxisomal enzymes, especially those of dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, were detected in the testis and epididymis. In the testis, peroxisomes were localized exclusively in Leydig cells and not in cells of the seminiferous tubules, implying that the latter do not contribute to the biosynthesis of plasmalogens of the sperm membrane. In contrast, peroxisomes could be clearly visualized in the epithelial cells of the epididymis. The results suggest that peroxisomes in epithelial cells of the rat epididymis play a pivotal role in the biosynthesis of plasmalogens destined for delivery to the sperm plasma membrane.     

Effects of physicochemical agents on murine epidermal Langerhans cells and Thy-1-positive dendritic epidermal cells

The possibility that Thy-1-positive dendritic epidermal cells (Thy-1+DEC) may contribute to the immunologic functions of murine epidermal cells (EC) prompted us to simultaneously assess the effects of certain immunomodulating physicochemical agents on both Thy-1+DEC and Ia-bearing Langerhans cells (LC). C3H/He mice received one of the following treatment modalities: UV-B irradiation (four consecutive days); psoralen plus UV-A (PUVA; three times a week for three consecutive weeks); topically and systemically applied glucocorticosteroids (GCS). Beginning 2 days after the last treatment, animals were sacrificed and the structure and surface marker expression of Ia+EC and Thy-1+DEC were assessed by immunohistologic means on epidermal sheet preparations from ear skin by using appropriate monoclonal antibodies. Whereas low-dose UV-B irradiation (4 X 100 or 200 J/m2) had little, if any, effect on either Ia+EC or Thy-1+DEC, high-dose UV-B (4 X 700 or 1000 J/m2) or PUVA treatment led to an almost complete disappearance of both surface characteristics. Immunoelectron microscopic studies revealed that in the case of LC, high-dose UV-B or PUVA treatment results in the disappearance of their anti-Ia reactivity but leaves their ultrastructural morphology intact. In sharp contrast, Thy-1+DEC escape ultrastructural detection after PUVA treatment and are greatly reduced in number after high-dose UV-B. Ia+EC continuously reappeared with both treatment modalities over a course of 4 to 6 wk, whereas even after 14 to 22 wk Thy-1+DEC were present only in negligible numbers. Similar to high-dose UV-B or PUVA therapy, administration of GCS resulted in the disappearance of both anti-Thy-1- and anti-Ia-reactive cells. Ultrastructural studies disclosed, however, that these steroid-induced alterations in the surface characteristics were accompanied by a dramatic reduction of the LC population but were not paralleled by morphologic changes of Thy-1+DEC. In the course of 7 wk after cessation of steroid treatment, the number of both Ia+EC and Thy-1+DEC had returned to normal values. The selective removal of either of these two dendritic epidermal cell populations by physicochemical agents may provide an excellent strategy to further clarify the functional properties of both LC and Thy-1+DEC.     

The synthesis and properties of T25 glycoprotein in thy-1-negative mutant lymphoma cells

The synthesis and properties of T25 glycoprotein which bears the serological specificity Thy-1 have been studied in mutants of cultured mouse lymphoma cells that do not express Thy-1 on their surface. Five complementation classes of mutant cells were previously characterized by somatic genetic analysis. Synthesis of abnormal T25 glycoproteins was detected in four classes of mutants. Each of these aberrant products was degraded more rapidly than T25 glycoprotein of wild-type cells. Defects in the oligosaccharide units of T25 glycoprotein were demonstrated in three classes of mutants. In one of these mutant classes, evidence for a general defect in glycosylation of cell surface glycoproteins was obtained. These data indicate that normal glycosylation of T25 glycoprotein is probably essential for the molecule to be incorporated into the plasma membrane and expressed on the cell surface.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.

Studies reported in the preceding paper (Trowbridge and Hyman, 1979) have demonstrated that Thy-1^? mutant lymphoma cells of the class E complementation group lack the normal high molecular weight lipid-linked oligosaccharide, but instead accumulate two smaller species termed I and II. This paper reports studies which elucidate the structures of lipid-linked oligosaccharides I and II. By subjecting oligosaccharides radiolabeled with ³H-mannose, ³H-glucose or ³H-glucosamine to methylation, acetolysis, periodate oxidation and exoglycosidase digestion, the structures were shown to be: where R = GlcNac B1,4(3) GlcNAc. A comparison of I and II with lipid-linked oligosaccharides from normal Chinese hamster ovary cells indicates that both I and II are normal biosynthetic intermediates. On the basis of these data we suggest that the defect in the class E mutant cells is the lack of an α1,3 mannosyltransferase involved in the conversion of the Man₅GlcNAc₂ lipid-linked oligosaccharide to the Man₆GlcNAc₂ intermediate. It is also impossible that the same enzyme is involved in conversion of the Glc₃Man₅GlcNAc₂ lipid-linked oligosaccharide to Glc₃Man₆GlcNAc₂. The latter reaction, however, has not yet been demonstrated in normal cells.     

Detection of a Thy-1 precursor in human T lymphoma cell lines

In an attempt to gain insight into the biosynthesis and processing of human Thy-1, rabbit antisera (R alpha TP) were raised against a synthetic peptide (TP) of 13 amino acid residues of identical amino acid sequence to that of residues 110-122 of the putative Thy-1 precursor deduced from the cDNA sequencing. Immunoblotting assay showed that the IgG fraction of R alpha TP (R alpha TP-IgG) recognized the synthetic peptide without demonstrable cross-reactivity with isolated human brain Thy-1 and detergent-solubilized membrane proteins of human brain cells. Immunohistochemical studies showed that both R alpha TP-IgG and HB-2S-1 (a monoclonal antibody which reacts with both membrane-bound Thy-1 on viable cells and detergent-solubilized Thy-1) stained cells of two human T lymphoma cell lines (HUT-78 and HUT-102) but they did not stain cells of a human B lymphoma cell line (Raji). In contrast, in cell surface indirect immunofluorescence assay, HB-2S-1 reacted with HUT-78 and HUT-102 cells while R alpha TP-IgG did not. Taken together, these data indicate the existence of a precursor form of human Thy-1 which is processed prior to anchoring to the cell surface.