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.     

Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.     

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.     

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.     

A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) -deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS.

The development of a system for the detection of somatic cell mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8) deficiency in L5178Y mouse lymphoma cells is described. The selection of mutant cells was not influenced by the concentration of the selective agent 6-thioguanine (6-TG). In addition, all the mutants selected, spontaneous as well as induced ones, showed a complete loss of HGPRT activity. In reconstruction experiments, in which mutant cells were mixed with wild-type cells, the recovery of mutant cells was only markedly influenced when wild-type cells were seeded in a cell density ten times higher than the one, 5-10(4) cells/ml, used in subsequent induction experiments. X-irradiation and treatment with ethyl methanesulfonate (EMS) increased in the mutation rate above the spontaneous background. A clear-cut dose-dependent mutagenic effect after exposure to X-rays was measured. The rate of induced mutations at the HGPRT locus in lymphoma cells was 1-3-10(-7) per R, as determined after exposures of 200, 300, 400, 500 and 600 R. The time the cells needed to express their mutations was much longer than 48 h. Further study of this phenomenon showed that the optimal expression time for TGr-resistant mutants in L5178Y cells was 6 to 7 days. No indication for a dose-dependent effect on the optimal expression of the mutants was found.     

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.     

Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.     

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.     

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.     

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.     

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 leader polypeptide of Theiler's murine encephalomyelitis virus is required for the assembly of virions in mouse L cells

Deletion of the entire leader polypeptide of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) results in the production of an attenuated virus that grows in baby hamster kidney (BHK) cells but cannot grow at all in mouse L-929 cells. This study examined the reasons for the failure of dl-L, the GDVII variant that lacks the leader polypeptide, to grow in mouse cells. At low multiplicities of infection, it was difficult to detect any viral proteins in mouse cells. However, levels of positive- and negative-strand RNA molecules were only moderately reduced in these infections. Viral RNA showed no major defect in translatability, as the mutant viral RNA was nearly as efficient as that of the wild-type (WT) virus in directing protein synthesis in vitro in assays using extracts prepared from mouse L cells. Viral protein synthesis was detected in dl-L-infected mouse cells as multiplicities of infection were increased and approached the levels observed in WT infections. Despite this, there was a total lack of virus production in high-multiplicity infections, and this was found to correlate with the failure of viral proteins and early virion precursors to assemble into virions in mouse cells. Thus, the inability of dl-L to grow in mouse cells reflects complex effects on various stages of the virus infection but is primarily a defect in virus assembly.     

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

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.     

Dynamics and longevity of the glycolipid-anchored membrane protein, Thy- 1

Thy-1 and a number of other proteins are anchored to the outer hemi-leaflet of membranes by a glycolipid moiety containing ethanolamine phosphate, mannose, glucosamine, and phosphatidylinositol. They nevertheless have the striking property of being able to transduce signals across the plasma membrane. We here demonstrate, for the BW5147 murine T lymphoma, that (a) greater than 90% of Thy-1 is at the cell surface, (b) Thy-1 is about one order of magnitude less concentrated in coated pits than the transferrin receptor or H-2 antigens, (c) Thy-1 undergoes at most very limited endocytosis or diacytosis, and (d) Thy-1 has an unusually slow turnover rate. Several similar observations have also been made for a second glycolipid-anchored protein, the T cell activating protein. Thus, the absence of cytoplasmic and trans-membrane domains may result in lipid-anchored proteins being confined to the cell surface and being free from constraints which affect the turnover of transmembrane proteins.     

Procyclin null mutants of Trypanosoma brucei express free glycosylphosphatidylinositols on their surface

Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.     

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.     

The choice of cell fate in the epidermis of Drosophila

In Drosophila, neural precursors are formed in a spaced pattern separated by intervening epidermal cells. Segregation of neural and epidermal lineages relies on cellular interactions. Failure of this cell communication, as in the mutants Notch (N), Delta, and shaggy, results in most or all of the cells becoming neural. Cells mutant for N and shaggy, but not Delta, autonomously adopt the neural fate when adjacent to wild-type cells in mosaics. Furthermore, wild-type cells adopt the epidermal fate if adjacent cells express a lower level of N activity than themselves, but produce neural precursors if adjacent cells express a higher level of N activity. This shows that there is competition between the cells and that the N protein is required for the mechanism whereby the cells choose between alternative fates. It also suggests that N acts as a receptor for an inhibitory signal emanating from the neural precursors.