A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.

In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 °C and 32 °C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 °C and 32 °C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.

The major membrane proteins of Paramecium are anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI). The expression of these GPI-proteins, the surface antigen (SAg) and the surface GPI-proteins (SGPs), is temperature-dependent, different sets are expressed at 23°C and at 32 °C. To characterize the GPI-anchor lipid moieties of these proteins, a new strategy of biosynthetic radiolabeling was developed. Cells of Paramecium primaurelia, grown at 23°C or at 32 °C, were fed with [(14)C]-labeled cyanobacteria. The paramecia metabolized the cyanobacteria lipids and synthesized fatty acids with longer and more unsaturated chains. The SAg and SGPs from [(14)C]-labeled paramecia, were purified and the lipid moieties of their GPI-anchors were cleaved by a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and identified as ceramides. The GPI-anchor ceramides, from the SAg and SGPs expressed at both temperatures, contained long-chain bases which did not display variations detectable upon thin layer chromatography analysis. In contrast, the amide-linked fatty acid component varied: palmitic acid was identified as the major amidelinked fatty acid in the GPI-protein anchors from paramecia grown at 23°C, while at 32°C a C(14) fatty acid could be the prominent fatty acid. This modulation in the fatty acid composition could playa role in the antigenic variation process.

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

Using a strategy based upon specific features of membrane proteins linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, we have studied in the strain 513 of Paramecium primaurelia the glycosylphosphatidylinositol (GPI) proteins both in their membrane-bound and soluble forms. 35S-Labeling associated with bacterial phosphatidylinositol-specific phospholipase C treatment of purified cilia allowed the identification of soluble GPI proteins (devoid of their lipid moiety), released from cilia. By labeling with 88[33P]phosphoric acid and [3H]ethanolamine, respectively, we identified membrane-bound GPI proteins, when anchored in the cilia membrane via the lipid of their GPI tail. We demonstrated that, in addition to the SAg (surface antigen) which is a high molecular weight protein implicated in the antigenic variation phenomenon, three other ciliary membrane proteins were also GPI-anchored. The membrane-bound and soluble forms of these GPI proteins had apparent molecular weights, in unreduced conditions, of 30,000 and 40 to 50,000, respectively. We named these surface GPI proteins SGPs, SGP1 to SGP3, SGP1 (surface GPI protein 1) and SGP2 (surface GPI protein 2) were the most abundantly expressed. Only SGP2 displayed a rapid turnover. By phosphatidylinositol-specific phospholipase C treatment of the membrane proteins recovered in the detergent phase, following partitioning with Triton X-114, we demonstrated that the SAg and SGPs are the major ciliary membrane proteins of P.primaurelia.

Glycolipids synthesized in a cell‐free system prepared from the free‐living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP‐[3H]mannose and UDP‐[3H]N‐acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol‐phosphoceramides (GIP‐ceramides). The ceramide‐based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP‐[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP‐ceramides were substituted with an acid‐labile modification identified as mannosyl phosphate. The modification of the glycosylinositol‐phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP‐ceramide intermediates in P.primaurelia was studied by a pulse‐chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP‐ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate‐6Man‐alpha 1–2Man‐alpha 1–6Man‐(mannosyl phosphate)‐alpha 1–4glucosamine‐inositol‐phosphoceramide.

Many eukaryotic membrane proteins have now been found to be anchored to the plasma membrane via a glycosylphosphatidylinositol membrane anchor (GPI-anchor). In Paramecium aurelia, a free-living ciliated protozoan, the major membrane protein, the surface antigen (SAg), is a GPI-anchored protein. This surface protein belongs to a multigene family, the expression and antigenic variation of which is controlled by environmental conditions. In order to screen for other Paramecium GPI-anchored proteins to identify those whose expression is also variable and influenced by external factors, we established a protocol permitting the rapid identification of GPI-proteins. The protocol is based upon the property of bacterial PI-PLCs to specifically release GPI-proteins. To overcome the resistance displayed by living paramecia to exogenous PI-PLCs, we used cilia purified from 35S-labeled cells obtained from various geographical strains grown under similar or different conditions. We observed a temperature-dependent variation in the electrophoretic patterns, as revealed by autoradiography of ciliary PI-PLC-releasable proteins from strain 513 of Paramecium primaurelia. In addition to a high molecular mass band corresponding to SAg molecules, three bands varying in apparent molecular mass from 30 to 50 kDa were observed at 23 degrees C. At 32 degrees C only one band of about 45 kDa was observed. Biosynthetic labeling experiments and the detection of the cross-reacting determinant after PI-PLC treatment (results reported elsewhere) provided definitive proof that these ciliary PI-PLC releasable proteins were actually GPI-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.

Glycosylphosphatidylinositol (GPI)-anchored cell surface proteins are widely expressed in tissues, including cells of immuno-hematopoietic origin. Cross-linking of GPI-linked proteins on T lymphocytes, such as Thy-1 (CD90), Ly-6 A/E, CD48, CD59 and others, induces T-cell mitogenesis. Similar to cross-linking with T-cell receptor (TcR)-specific antibodies, ligation of GPI-anchored proteins induces an intracellular flux of calcium, an up-regulation of activation-associated cell surface proteins and the elaboration of growth-promoting lymphokines. These events are dependent on p56lck-mediated tyrosine phosphorylation of substrates. GPI-linked proteins are constitutively clustered in sphingolipid-rich membrane domains. Actin-driven rearrangements of the cytoskeleton are probably responsible for the physical approximation of TcR and GPI-anchored proteins in mature immunological synapses. Functionally, GPI-linked proteins can supplant for signal 1 and productively collaborate with CD28 to fully activate T cells.

ABSTRACT In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface distribution of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly than the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidie nature of the SAg membrane anchor and to a possible role of an endogenous Paramecium phosphatidylinositol phospholipase C.

Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called ‘cross-reacting glycoproteins’ (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the ‘cross-reacting determinant’ (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called ‘det-2355’, is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs.

The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process.

ABSTRACT Paramecium aurelia can express a repertoire of surface antigens (SAgs) according to culture conditions. These high Mr proteins are anchored in the plasma membrane by a glycolipid, and they can be isolated in two different forms, an amphiphilic membrane-bound form (mSAg) and a hydrophilic soluble form (sSAg). Endogenous or exogenous phospholipase C can convert mSAg to sSAg with unmasking of a carbohydrate antigenic determinant similar to that found in the soluble form of Trypanosoma variant surface glycoproteins and called the cross-reacting determinant. By immunizing mice with cilia from strain 156 of P. primaurelia expressing the G SAg, we obtained six monoclonal antibodies against the 156G SAg, which could be classified into two groups. Y4 and Y8, representative of each group, have been characterized by checking their reactivity in situ and in vivo towards a series of allelic G and D SAgs in P. primaurelia and the 5IB SAg in P. tetraurelia. The monoclonal Y4 recognizes a conformational determinant, accessible in vivo and common to all the G SAgs. Thus, Y4 defines a G locus-specific epitope that corresponds to a conserved region inside a polymorphic domain. The monoclonal Y8 recognizes two homologous determinants whose detection depends on the presence or absence of the SAg membrane-anchor, and which are mutually exclusive: one is found in the reduced soluble form of all the SAgs and other surface proteins, the cross-reacting glycoproteins (CRGs); the other occurs in the unreduced membrane-bound form of the G SAgs. Thus, Y8 enables us to demonstrate that the membrane-anchor of Paramecium SAgs contains an additional hidden determinant close to the crossreacting determinant and to discriminate between the membrane-bound and soluble form of SAgs. The in situ organization of the 156G SAg molecules is also discussed on the basis of immunogold labelling obtained using Y4 and a polyclonal antiserum.

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci.