Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol

Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.     

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.     

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.     

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.     

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.     

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.     

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.     

Stimulation of antigen Thy-1 expression in mouse bone marrow cells by thymus extracts

The expression of antigen Thy-1 was studied in bone marrow cells of CBA line mice under the effect of thymus extracts. Extracts of the calf thymus--thymosine (fraction 5) and the preparation free of the Comsa factor were obtained by a combination of the Goldstein and Comsa extraction methods. The both extracts stimulate the expression of antigen Thy-1 in bone marrow cells. Incorporation of [14 C]sodium acetate into fragments containing antigen Thy-1 and absorbed by the column with anti-Thy-1-antibodies remains unchanged after stimulation. It is supposed that antigen Thy-1 ability to stimulate expression in bone marrow precursors of T-cells is not due to the synthesis of the antigen and is a property of one of the thymus factors with molecular mass of about 5000.     

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.     

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.     

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation

A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.     

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.     

Adaptive alteration in phospholipid composition of plasma membranes from a somatic cell mutant defective in the regulation of cholesterol biosynthesis

A somatic cell mutant (CR1) of a Chinese hamster ovary cell (CHO-K1) which has previously been shown to be defective in the regulation of cholesterol biosynthesis accumulates more cholesterol than the parental cell line in plasma membranes. Although such an increase in membrane cholesterol should lead to an increase in the order parameter of these membranes, as measured with an electron spin resonance spin probe, the order parameters of mutant and wild-type plasma membranes are identical- -apparently because of an adaptive alteration in membrane phospholipid composition. The phospholipid compositions of mutant and wild-type cell plasma membranes are compared and the mutant is shown to have a threefold higher level of oleic acid and a twofold lower level of phosphatidylethanolamine than the wild type. These results are consistent with model studies which show that these compositional changes lead to lower-order parameters for phospholipid dispersions.     

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.     

Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.     

Disappearance of a basic chromosomal protein from cells of a mouse temperature-sensitive mutant defective in histone phosphorylation

The amount of a basic nuclear protein which migrates a little slower than histone H1 in urea-acetic acid-polyacrylamide gel electrophoresis and a little faster than H1 in sodium dodecylsulfate-polyacrylamide gel electrophoresis, decreases when cells of a temperature-sensitive mutant, ts85, derived from a mouse carcinoma cell line, are incubated at the nonpermissive temperature (39°C). This protein appears again, when cells cultured at 39°C are shifted down to the permissive temperature, indifferent to the added cycloheximide. Neither in wild type nor in a revertant of ts85, the protein disappeared at 39°C. Since the ts85 cells were found to be defective in chromosome condensation and in the phosphorylation of histone H1 at 39°C (1,2), this basic protein may relate to the both events.