The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid

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.     

A class of membrane proteins with a C-terminal anchor

Integral membrane proteins are generally targeted to translocation-competent membranes by virtue of signal sequences located close to the N-terminus of the polypeptide chain. Membrane anchoring is caused by the signal sequence or other hydrophobic segments located after it in the amino acid sequence. However, some integral membrane proteins do not follow these rules. The members of one class of nonconformist membrane proteins have no signal sequence, but instead possess a hydrophobic segment near the C-terminus that orients them with their N-termini in the cytoplasm. Members of this class are found in many organelles and are probably inserted into membranes by an unusual mechanism.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.     

Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.     

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.     

Cell-free processing of nascent proteins destined to be linked to the plasma membrane by a phosphatidylinositol-glycan anchor.

Certain proteins are anchored to the outer plasma membrane by a phosphatidylinositol-glycan (PI-G) linker. Nascent forms of PI-G anchored proteins contain both NH2- and COOH-terminal signal peptides. The function and structural requirements of the COOH-terminal signal peptide as discussed and some studies on the cell-free processing of a nascent protein to its mature PI-G tailed form are presented.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.     

Prenyl proteins in eukaryotic cells: a new type of membrane anchor

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.     

Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins

Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.     

Membrane phospholipid dynamics during cytokinesis: regulation of actin filament assembly by redistribution of membrane surface phospholipid

To study molecular motion and function of membrane phospholipids, we have developed various probes which bind specifically to certain phospholipids. Using a novel peptide probe, RoO9-0198, which binds specifically to phosphatidylethanolamine (PE) in biological membranes, we have analyzed the cell surface movement of PE in dividing CHO cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membranebound peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced transbilayer movement of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding of the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex bound specifically to cleavage furrow and inhibited both actin filament disassembly at cleavage furrow and subsequent plasma membrane fusion. Binding of the peptide complex to interphase cells also induced immediate disassembly of stress fibers followed by assembly of cortical actin filaments to the local area of plasma membrane where the peptide complex bound. The cytoskeletal reorganizations caused by the peptide complex were fully reversible; removal of the surface-bound peptide complex by incubating with PE-containing liposome caused gradual disassembly of the cortical actin filaments and subsequent formation of stress fibers. These observations suggest that the redistribution of plasma membrane phospholipids act as a regulator of actin cytoskeleton organization and may play a crucial role in mediating a coordinate movement between plasma membrane and actin cytoskeleton to achieve successful cell division.     

Biogenesis of plasma membrane glycoproteins. Tracer kinetic study of two rat liver plasma membrane glycoproteins in vivo

Antibodies to purified nucleotide pyrophosphatase (NPPase) and dipeptidyl peptidase IV (DPP IV) were used to study the biogenesis of these rat liver plasma membrane glycoproteins in vivo. Following injection of tritiated leucine, the radioactivity in NPPase and DPP IV decayed at markedly different rates in the plasma membrane, with apparent half-lives of about 1 and 5 days, respectively. In short term experiments, labeling of total plasma membrane proteins was rapid and insensitive to colchicine, while labeling of both NPPase and DPP IV showed a lag of about 15 min, followed by colchcine-sensitive/cycloheximide-insensitive increases to half-maximal and maximal values at about 1 and 2 h, respectively. A peak of labeled DPP IV in rough microsomes at 15 min showed increased mobility on polyacrylamide gels and was largely inaccessible to antibodies in intact microsomes, consistent with its being an underglycosylated precursor, exposed on the cisternal side of the rough endoplasmic reticulum. In contrast, the behavior of unlabeled DPP IV in preparations of rough microsomes and Golgi was consistent with its being contributed by contaminating right-side-out plasma membrane vesicles. This conclusion was also necessary to fit the tracer kinetic data to a simple membrane-flow model, which gave precursor pools (1 microgram/g of liver) and fluxes (1 microgram/h/g of liver) for both DPP IV and NPPase which were about 3 orders of magnitude less than those for the synthesis of rat serum albumin. Thus, unlike hepatoma tissue culture cells (Doyle, D., Baumann, H., England, B., Friedman, E., Hou, E., and Tweto, J. (1978) J. Biol. Chem. 253, 967-973), normal rat liver does not contain large amounts of preformed intracellular plasma membrane precursors.     

Characterization of the cross-reacting determinant (CRD) of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein

The cross-reacting determinant (CRD epitope) of the glycosyl-phosphatidylinositol (GPI) membrane anchor of Trypanosoma brucei variant surface glycoprotein has been analysed by selective chemical and enzymic modification of the isolated GPI structure combined with the use of a competitive ELISA inhibition assay for the detection of CRD epitopes. The data show that the CRD consists of at least three overlapping epitopes involving different regions of the molecule including the inositol 1,2-cyclic phosphate, the non-N-acetylated-glucosamine residue and the galactose branch. Although the presence of all three of these structural features is required for quantitative binding of anti-CRD antibodies in ELISA and Western blotting, the Western blot reaction obtained in the presence of any one epitope is still significant. The use of anti-CRD antibodies for the detection of GPI anchors is discussed.     

The variant surface glycoproteins of Trypanosoma equiperdum. Identification of a phosphorylated glycopeptide as the cross-reacting antigenic determinant

The cross-reacting antigenic determinant in the variant surface glycoproteins (VSGs) of Trypanosoma equiperdum was studied by testing the ability of VSG glycopeptides to bind heterologous anti-VSG sera. VSG glycopeptide purification revealed the presence of 3 oligosaccharide sidechains on the mature VSG. These consist of two sidechains containing only mannose and glucosamine and a third containing galactose and mannose (in a 5:1 ratio) as well as phosphorous and ethanolamine. This phosphorylated fragment completely blocked the binding of VSG to heterologous anti-VSG and therefore contained the cross-reacting determinants.     

Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.     

Detection of glycoproteins in the Acanthamoeba plasma membrane

In the present study we have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB3H4 and galactose oxidase/NaB3H4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with Mr of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [35S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.     

Structure and expression of genes for surface proteins in Paramecium.

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.     

Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK _m value of 2.56 · 10^–4 m and an apparentV _max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface. Abbreviations: PLC, phospholipase C; PIPLC, phospholipase C acting selectively on phosphatidylinositol; NeuAc,N-acetylneuraminic acid; MU, 4-methylumbelliferone; PBS, Dulbecco's phosphate buffer saline solution. Gangliosides were coded according to Svennerholm [42] and the IUPAC-IUB recommendations [43].