Production of glycosylphosphatidylinositol-anchored proteins for vaccines and directed binding of immunoliposomes to specific cell types

Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist. Methods: We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested. Results: Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions: Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.(AU)

Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity.

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4 degrees C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4 degrees C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-beta-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites.

Screening for glycosylphosphatidylinositol-anchored proteins in the Paracoccidioides brasiliensis transcriptome.

Open reading frames in the transcriptome of Paracoccidioides brasiliensis were screened for potential glycosylphosphatidylinositol (GPI)-anchored proteins, which are a functionally and structurally diverse family of post-translationally modified molecules found in a variety of eukaryotic cells. Numerous studies have demonstrated that various GPI anchor sequences can affect the localization of these proteins in the plasma membrane or the cell wall. The GPI anchor core is produced in the endoplasmic reticulum by sequential addition of monosaccharides and phospho-ethanolamine to phosphatidylinositol. The complete GPI anchor is post-translationally attached to the protein carboxyl-terminus by GPI transamidases. Removal of this GPI lipid moiety by phospholipases generates a soluble form of the protein. The identification of putative GPI-attached proteins in the P. brasiliensis transcriptome was based on the following criteria: the presence of an N-terminal signal peptide for secretion and a hydrophobic region in the C-terminus presenting the GPI-attachment site. The proteins that were identified were in several functional categories: i) eight proteins were predicted to be enzymes (Gel1, Gel2, Gel3, alpha-amylase, aspartic proteinase, Cu-Zn SOD, DFG5, PLB); ii) Ag2/PRA, ELI-Ag1 and Gel1 are probably surface antigens; iii) Crh-like and the GPI-anchored cell wall protein have a putative structural role; iv) ECM33 and Gels (1, 2 and 3) are possibly involved in cell wall biosynthesis, and v) extracellular matrix protein is considered to be an adhesion protein. In addition, eight deduced proteins were predicted to localize in the plasma membrane and six in the cell wall. We also identified proteins involved in the synthesis, attachment and cleaving of the GPI anchor in the P. brasiliensis transcriptome.

Screening for glycosylphosphatidylinositol-anchored proteins in the Paracoccidioides brasiliensis transcriptome

Open reading frames in the transcriptome of Paracoccidioides brasiliensis were screened for potential glycosylphosphatidylinositol (GPI)-anchored proteins, which are a functionally and structurally diverse family of post-translationally modified molecules found in a variety of eukaryotic cells. Numerous studies have demonstrated that various GPI anchor sequences can affect the localization of these proteins in the plasma membrane or the cell wall. The GPI anchor core is produced in the endoplasmic reticulum by sequential addition of monosaccharides and phospho-ethanolamine to phosphatidylinositol. The complete GPI anchor is post-translationally attached to the protein carboxyl-terminus by GPI transamidases. Removal of this GPI lipid moiety by phospholipases generates a soluble form of the protein. The identification of putative GPI-attached proteins in the P. brasiliensis transcriptome was based on the following criteria: the presence of an N-terminal signal peptide for secretion and a hydrophobic region in the C-terminus presenting the GPI-attachment site. The proteins that were identified were in several functional categories: i) eight proteins were predicted to be enzymes (Gel1, Gel2, Gel3, alpha-amylase, aspartic proteinase, Cu-Zn SOD, DFG5, PLB); ii) Ag2/PRA, ELI-Ag1 and Gel1 are probably surface antigens; iii) Crh-like and the GPI-anchored cell wall protein have a putative structural role; iv) ECM33 and Gels (1, 2 and 3) are possibly involved in cell wall biosynthesis, and v) extracellular matrix protein is considered to be an adhesion protein. In addition, eight deduced proteins were predicted to localize in the plasma membrane and six in the cell wall. We also identified proteins involved in the synthesis, attachment and cleaving of the GPI anchor in the P. brasiliensis transcriptome.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-gamma-primed-macrophages.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-gamma-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1alpha or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-gamma. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-gamma may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.

Structural variation in the glycoinositolphospholipids of different strains of Trypanosoma cruzi.

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasite Trypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man4(AEP)GlcN-Ins-PO4 core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors of T. cruzi. The core can be further substituted by terminal (1-3)-linked beta-galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of beta-galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection by T. cruzi.

Characterization of inositolphospholipids in Trypanosoma cruzi trypomastigote forms.

In vivo labeling experiments with [3H]palmitic acid, [3H]inositol, and [3H]glucose allowed the identification of two main classes of inositolphospholipids (IPLs) from the trypomastigote stage of Trypanosoma cruzi. Purification of these compounds was achieved by ion-exchange chromatography, high performance liquid chromatography and thin layer chromatography. Specific phosphatidyl-inositol phospholipase C digestion, dephosphorylation and acid methanolysis showed a ceramide structure for the lower migrating IPL1. Palmitoyldihydrosphingosine and palmitoylsphingosine were detected by reverse-phase thin-layer chromatography. On the other hand, IPL2 showed to be a mixture of diacylglycero- and alkylacylglycero-phospholipids in a 1:1 ratio. After PI-PLC digestion, the lipids were separated by preparative TLC and individually analysed. The diacylglycerol contained mainly C18:0 fatty acid together with a low amount of C16:0. Hexadecylglycerol esterified with the C18:0 fatty acid was the only alkylacylglycerol detected. The C18:2 and C18:1 fatty acids, preponderant in the PI molecules of epimastigote forms, were not detected in trypomastigote forms. This is the first report on inositol phospholipids, putative precursors of lipid anchors in the infective stage of T. cruzi.

Trypanosoma cruzi: the Tc-85 surface glycoprotein shed by trypomastigotes bears a modified glycosylphosphatidylinositol anchor.

Tc-85, an 85-kDa surface glycoprotein specific for the trypomastigote stage of Trypanosoma cruzi, has been implicated in the invasion of host cells by the parasite. Radioactive palmitic acid was incorporated into Tc-85 immunoprecipitated from the culture medium with the H1A10 monoclonal antibody, suggesting that shedding occurs with Tc-85 bearing its GPI anchor. In contrast to the glycoprotein remaining in the parasites, the glycosylphosphatidylinositol moiety in shed Tc-85 is resistant to phosphatidylinositol phospholipase C and becomes susceptible to the enzyme following alkali treatment. An alkylglycerol was identified by thin layer chromatography of an ether extract after the enzymatic reaction. Resistance to cleavage by phospholipase C is due to fatty acid esterification of the inositol residue in shed Tc-85. This is the first example of inositol modification in anchors from a glycoprotein of Trypanosoma cruzi.

The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting.

Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.

Functional studies and cellular distribution of the F3 GPI-anchored adhesion molecule.

Many adhesion molecules of the immunoglobulin superfamily expressed in the nervous system are attached to the neuronal membrane by a glycan-phosphatidylinositol. Using neuronal glycoprotein F3 as a model we will discuss how this lipid modification might confer on molecules specific properties which may be particularly well suited to a role in modulating neuronal interactions. In particular, the following data dealing with the question of how the glycosylphosphatidylinositol (GPI) anchor influences the function, transport and localization of this molecule will be presented. 1) When anchored to the plasma membrane, F3 fulfills the operational criteria of an adhesion molecule while its soluble form is able to stimulate neurite outgrowth of sensory neurons in culture. 2) In the hypothalamo-hypophyseal system, immunoblot analysis indicates that there is more F3 in the neurohypophysis where secretory axons terminate than in the hypothalamic nuclei where the molecule is synthesized. In addition, GPI-linked forms predominate in the nuclei while there are mainly soluble forms in the neurohypophysis, suggesting that there is conversion of the GPI-bearing form to the soluble form during axonal transport. 3) In the cerebellum, F3 is polarized to the tips of the axons of granule cells, the major neuronal population in this system, as an indication that indeed GPI might be a signal for targeting molecules to axons. However, some neurons such as Golgi cells express F3 over all their surface.