Transbilayer mobility and distribution of red cell phospholipids during storage.

We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.

The influence of cellular ATP levels on dimyristoylphosphatidylcholine-induced release of vesicles from human erythrocytes.

Release of membrane vesicles from human erythrocytes was induced by modulation of red cell ATP levels, by incubation of erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) suspensions, or by a sequential combination of both procedures. When red blood cell ATP levels were decreased prior to incubation with DMPC, the lag-time between addition of the lipid and beginning of vesiculation was reduced. Furthermore, the rate of vesicle release itself was accelerated. Experiments carried out with a rapid ATP depletion technique showed that the onset of vesiculation and the release were most evidently accelerated in those cases where echinocytes had been formed prior to the addition of DMPC. The results suggest that red blood cells with reduced cellular ATP levels or an altered cell shape are more susceptible to a further perturbation of the membrane by addition of exogenous DMPC.

Modulation of red cell vesiculation by protease inhibitors.

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.

Modulation of erythrocyte vesiculation by amphiphilic drugs.

Release of acetylcholinesterase-containing vesicles from human erythrocyte membranes induced by dimyristoylphosphatidylcholine (DMPC) was inhibited by exposure of red cells to cationic amphiphilic drugs like tetracaine, chlorpromazine and primaquine which all are known to induce stomatocyte formation. On the other hand, the process was facilitated when red cells were exposed to crenators like the anionic drugs indomethacin and phenylbutazone or when DMPC was added to calcium-loaded red cells. The results suggest that agents which are known to modulate red cell shape do also influence the vesiculation behavior of the cells.

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.

Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells.

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells.

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.

Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins.

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.

'Crossed immunoblotting': identification of proteins after crossed immunoelectrophoresis and electrotransfer to nitrocellulose membranes.

Human serum proteins or membrane proteins from human erythrocytes were separated by crossed immunoelectrophoresis in agarose gels under non-denaturating and non-reducing conditions and precipitated by polyspecific antibodies against the respective protein mixtures. The separated proteins were subsequently transferred electrophoretically to nitrocellulose sheets and incubated with mouse monoclonal antibodies against individual protein species. Visualization of the complexes of antigen and monoclonal antibody on the nitrocellulose sheets was performed with peroxidase-conjugated second antibodies. The additional electroblotting step clearly improved the analytical resolution possibilities of crossed immunoelectrophoresis: crossed immunoblotting allows a straightforward identification of individual proteins in complex crossed immunoelectrophoresis patterns. At the same time separation and characterization of proteins is performed under non-denaturing conditions, which is not possible with blotting techniques based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Glycosyl inositolphospholipid-anchored structures in Herpetomonas davidi.

Glycosyl inositolphospholipid (GPI)-anchored structures in the monogenetic parasite Herpetomanas davidi, were labeled with [3H]glucosamine, and characterized by enzymatic and chemical treatments that are typical for the identification of GPI anchors. [3H]Myristate incorporated into two different pools of GPI-linked structures that could be separated by chromatography on octyl-Sepharose. One pool consisted of three GPI-anchored proteins with apparent molecular masses of 21,31 and 45 kDa, and the GPI lipid moieties were identified as alkyl-lysoglycerols. The label in the other pool associated with lipopeptidophosphoglycan (LPPG)-like structures of approximately 12-kDa molecular mass, containing ceramide-type GPI lipid anchors. While protein GPI anchors could also be labeled using [3H]glucosamine as radiolabeled GPI anchor precursor, hardly any radioactivity was incorporated into the LPPG-like structures. H. davidi is one of the few organisms identified to date that synthesizes two structurally different lipid moieties for GPI anchoring of membrane components.

Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei.

Trypanosoma brucei glycosylphosphatidylinositol phospholipase C (GPIPLC) is expressed in the bloodstream stage of the life cycle, but not in the procyclic form. It is capable of hydrolyzing GPI-anchored proteins and phosphatidylinositol (PI) in vitro. Several roles have been proposed for GPIPLC in vivo, in the release of variant surface glycoprotein during differentiation or in the regulation of GPI and PI levels, but none has been substantiated. To explore GPIPLC function in vivo, tetracycline-inducible GPIPLC gene (GPIPLC) conditional knock-out bloodstream form and tetracycline-inducible GPIPLC-expressing procyclic cell lines were constructed. We were unable to generate GPIPLC null mutants. Cleavage of GPI-anchored proteins was abolished in extracts from uninduced conditional knock-outs and was restored upon induction. Despite the barely detectable level of GPIPLC activity in uninduced conditional knock-out bloodstream forms, their growth was not affected. GPI-protein cleavage activity could be induced in procyclic cell extracts, up to wild-type bloodstream levels. Myo-[3H]inositol incorporation into [3H]inositol monophosphate was about 14-fold lower in GPIPLC conditional knock-out bloodstream forms than in the wild type. Procyclic cells expressing GPIPLC showed a 28-fold increase in myo-[3H]inositol incorporation into [3H]inositol monophosphate and a 1.5-fold increase in [3H]inositol trisphosphate levels, suggesting that GPIPLC may regulate levels of inositol phosphates, by cleavage of PI and phosphatidylinositol 4,5-bisphosphate.

Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms.

We present a quantitative description of the molecular species composition of the major phospholipid classes in bloodstream and procyclic forms of Trypanosoma brucei. Phospholipid classes were resolved by 2-dimensional thin-layer chromatography. Diradylglycerols were released from individual phospholipid classes by phospholipases C, converted into benzoate derivatives and separated into diacyl, alkylacyl and alk-1-enylacyl subclasses. Individual molecular species were quantitated and identified by HPLC and the assignments were confirmed by mass spectrometry. Comparison of the diacyl species of PC, PE and PI in bloodstream trypanosomes showed major differences in the relative amounts of individual molecular species between the different classes but not striking changes in the degree of saturation or overall chain length. In contrast, in procyclic trypanosomes the relative amounts of diacyl molecular species with polyunsaturated fatty acyl chains decreased in the order of PC > PE >> PI. Also, the alkylacyl and alk-1-enylacyl subclasses of PC and PE in bloodstream trypanosomes comprised a single molecular species, 18:0 18:2. Such exclusivity was not observed in procyclic trypanosomes among the same phospholipid subclasses, although 18:0 18:2 was the predominant species. Almost all the PI of bloodstream forms contained one 18:0 acyl species, which is consistent with the composition of the PI used for glycosylphosphatidylinositol synthesis.

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites.

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.

GPI anchor-hydrolyzing phospholipases.

Glycosylphosphatidylinositol(GPI) anchor-hydrolyzing phospholipases include C- and D-type phospholipases and have been described in a number of organisms including bacteria, protozoan parasites, plants, and mammals. Although these phospholipases efficiently cleave GPI structures in vitro, the physiological role of GPI hydrolysis by anchor-specific phospholipases is still unclear. In order to permit comparison of the known GPI anchor-hydrolyzing phospholipases, we studied the kinetic parameters of these enzymes and provide an overview of the currently available information.

Partial purification and characterization of a (glycosyl) inositol phospholipid-specific phospholipase C from peanut.

We have isolated a glycosyl inositol phospholipid (GIP) anchor-hydrolyzing activity from peanut seeds by a series of column chromatographic steps. The activity has a pH optimum below 6.0, requires calcium, and is inhibited by sulfhydryl reagents. It cleaves the GIP anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from Trypanosoma brucei. On the other hand, it does not act on membrane-bound GIP-anchored substrate or on inositol-acylated GIP anchor of human erythrocyte acetylcholinesterase. The only product released from [3H]myristate-labeled variant surface glycoprotein following treatment with the activity from peanut was 3H-labeled diacylglycerol. Together, these findings identify the activity from peanut seeds as a GIP anchor-hydrolyzing phospholipase C. The enzyme has been found to hydrolyze not only protein GIP anchors but also phosphatidylinositol, whereas it shows no activity against other phospholipids. The water-soluble products of phosphatidylinositol hydrolysis by peanut phospholipase C were characterized as a mixture of inositol 1,2-cyclic phosphate and inositol phosphate.

Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases.

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.

Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum.

It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.

Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein.

Glycosylphosphatidylinositol (GPI) membrane anchors are synthesized in the endoplasmic reticulum of eukaryotic cells. Synthesis of the core GPI structure is achieved by the sequential transfer of monosaccharides and phosphoethanolamine to phosphatidylinositol. The assembly process can be reproduced in vitro using membrane preparations supplemented with sugar nucleotides. With one exception, however, none of the biosynthetic enzymes involved have been isolated. One impediment to progress in the isolation of these enzymes is the nonavailability of adequate amounts of partially assembled GPI structures for use as assay substrates. In this paper we present procedures to prepare these structures from a GPI-anchored protein. The methods described include selective dephosphorylation of the GPI-anchored variant surface glycoprotein from Trypanosoma brucei variant 118 to generate Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6-myo-inositol-P-dimyristoylglycerol (Man3GlcN-PI), followed by exoglycosidase treatments and N-acetylation to produce Man2GlcN-PI, Man1GlcN-PI, GlcN-PI, and GlcNAc-PI. Procedures are also described for the stabilization and purification of these structures. It is anticipated that the convenient preparation of this range of partially assembled GPIs will be useful not only in developing assays for the eventual purification of the GPI biosynthetic enzymes but will also contribute to evaluating the specificity of the phospholipases that hydrolyze GPI anchors.