Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Boron (B) is essential for plant cell-wall structure and membrane functions. Compared with its role in cross-linking the pectic domain rhamnogalacturonan II (RG-II), little information is known about the biological role of B in membranes. Here, we investigated the involvement of glycosylinositol phosphorylceramides (GIPCs), major components of lipid rafts, in the membrane requirement for B. Using thin-layer chromatography and mass spectrometry, we first characterized GIPCs from Rosa cell culture. The major GIPC has one hexose residue, one hexuronic acid residue, inositol phosphate, and a ceramide moiety with a C18 trihydroxylated mono-unsaturated long-chain base and a C24 monohydroxylated saturated fatty acid. Disrupting B bridging (by B starvation in vivo or by treatment with cold dilute HCl or with excess borate in vitro) enhanced the GIPCs' extractability. As RG-II is the main B-binding site in plants, we investigated whether it could form a B-centred complex with GIPCs. Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex. Last, using polyacrylamide gel electrophoresis, we showed that added GIPCs facilitate RG-II dimerization in vitro. We conclude that B plays a structural role in the plasma membrane. The disruption of membrane components by high borate may account for the phytotoxicity of excess B. Moreover, the in-vitro formation of a GIPC-B-RG-II complex gives the first molecular explanation of the wall-membrane attachment sites observed in vivo. Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy.

BACKGROUND: Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. METHODOLOGY/PRINCIPAL FINDINGS: A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. CONCLUSIONS: The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.

Intraspecies variation in Trypanosoma cruzi GPI-mucins: biological activities and differential expression of α-galactosyl residues.

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

[Identification of glycosylphosphatidylinositol-anchored protein from Schistosoma japonicum].

OBJECTIVE: To identify glycosylphosphatidylinositol (GPI) anchored protein of Schistosoma japonicum. METHODS: Based on the gene sequence of Schistosoma mansoni GPI anchored protein Sm200 (GenBank Assess No: XM_002569560.1), bioinformatics analysis was performed to find out its homologous gene sequence in S. japonicum, then a selected partial coding sequence (SjGPIs, about 933 bp) from the homologous gene sequence were amplified, and cloned into PET-28a(+) vector. The recombinant plasmid pET-28a(+)SjGPIs were transformed into E. coli Top10 cells and induced with IPTG for protein expression. The recombinant protein SjGPIs was purified with Ni-NTA resin, and the purified recombinant SjGPIs protein was used as antigen to prepare antiserum in New Zealand rabbit. The antiserum was used to detect S. japonicum GPI-anchored protein. To identify a GPI-anchored protein, the detected protein were identified by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. White blood cells from S. japonicum-infected mice was examined whether they endocytosed GPI-anchored proteins by Western blotting. RESULTS: The homologous gene sequence of S. mansoni GPI Sm200 gene was found in S. japonicum genome. A 3 495 bp coding sequence was obtained, containing the complete C-terminal sequence. The selected gene sequence (SjGPIs) were amplified and the recombinant plasmid pET-28a(+)-SjGPIs was established. According to the analysis of C-terminal sequence, Western blotting and enzyme digestion of PI-PLC, a GPI-anchored protein was present in S. japonicum tegument (about 1M(r)200000), named SjGPI200. The protein was detected in white blood cells of infected mice. CONCLUSION: SjGPI200 protein exists in S. japonicum, and anchored to parasite tegument via GPI.

Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds--applications in acidic modification-specific proteomics.

Characterization of the biomolecules involved in molecular processes occurring in biological systems such as the human cell remains central to biology, biotechnology, and medicine. One of the preferred methods of selectively purifying specific classes of biomolecules from complex biological matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field of phosphoproteomics. Additional studies have shown the potential of this sorbent in purification of other acidic post-translational modified peptides, such as sialylated glycopeptides, thereby targeting the sialiome, defined as the content of sialic acid containing glycoproteins of a given cell, body fluid or tissue. The development of TiO(2)-based chromatographic strategies for separation of various biomolecules from its introduction for small molecules more than 20 years ago until recent proteomics applications today will be reviewed here.

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro, such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens. Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlalpha1/beta2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I(max) of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlalpha1/beta2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlalpha1(Y151S) mutation was included (Nlalpha1(Y151S)/beta2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlalpha1/beta2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlalpha1(Y151S)/beta2 (3.25-fold and 2.86-fold) than in Nlalpha1/beta2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens. These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.

GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi.

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

Surface engineering of microparticles by novel protein transfer for targeted antigen/drug delivery.

Biodegradable microparticles can function as an adjuvant by targeting antigens to professional antigen presenting cells such as dendritic cells and macrophages. To enhance targeting of microparticles, we have developed a novel method of attaching immunostimulatory molecules such as B7-1 to the surface of albumin microparticles utilizing the glycosylphosphatidyl inositol (GPI) anchor. GPI-B7-1 attaches to the surface of albumin microparticles in a protein transfer mediated process and is functionally active. This protein transfer was dependent on the concentration of the GPI-anchored protein, and independent of temperature and incubation time. Results show that the binding of the GPI-anchored protein is specifically occurring through an interaction between the GPI-anchor and the albumin microparticle surface. Stability studies indicate that the GPI-anchored protein can remain attached to the surface of the microparticle up to 7 days, with storage at 4 degrees C providing the optimal stability. Finally, we were able to simultaneously attach two different GPI-anchored proteins, GPI-B7-1 and GPI-ICAM-1, to the microparticles, demonstrating the capability of attaching more than one GPI-anchored protein to the microparticle surface. This novel method of attaching proteins to the surface of microparticles has potential implications in using microparticles as an antigen delivery device in vaccines as well as in targeted drug delivery.

Toxoplasma gondii glycosylphosphatidylinositols up-regulate major histocompatibility complex (MHC) molecule expression on primary murine macrophages.

Toxoplasma gondii is an obligatory intracellular parasite able to block the IFN-gamma-induced up-regulation of major histocompatibility complex (MHC) class I and class II molecules. This facilitates parasite-mediated evasion of T-cell responses. Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and we investigated if GPIs are responsible for inhibition of MHC expression on macrophages. In contrast to the blockade observed in cells infected with viable tachyzoites, T. gondii GPIs up-regulated MHC class I and class II molecules on the surface of both unstimulated and IFN-gamma-stimulated primary murine macrophages. This effect was correlated to the ability of GPIs to increase the antigen presentation to CD8(+) lymphocytes. T. gondii GPIs did not activate STAT1, one of the factors involved in the transcription of MHC class I and class II genes. However, the GPI-induced MHC class I up-regulation was abrogated by SN50, a specific NF-KB inhibitor. Up-regulation of surface MHC molecules by GPIs may lead to the elimination of non-infected cells of the host immune system, contributing to the immune escape strategy of T. gondii.

Rat osseous plate alkaline phosphatase as Langmuir monolayer--an infrared study at the air-water interface.

A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase-OAP) was studied as monolayer (pure and mixed with lipids) at the air-water interface. Surface pressure and surface potential-area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m(-1). Isotherms for mixed dimyristoylphosphatidic acid (DMPA)-OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air-water interface as observed from in situ p-polarized light Fourier transform-infrared reflection-absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed.

FragAnchor: a large-scale predictor of glycosylphosphatidylinositol anchors in eukaryote protein sequences by qualitative scoring.

A glycosylphosphatidylinositol (GPI) anchor is a common but complex C-terminal post-translational modification of extracellular proteins in eukaryotes. Here we investigate the problem of correctly annotating GPI-anchored proteins for the growing number of sequences in public databases. We developed a computational system, called FragAnchor, based on the tandem use of a neural network (NN) and a hidden Markov model (HMM). Firstly, NN selects potential GPI-anchored proteins in a dataset, then HMM parses these potential GPI signals and refines the prediction by qualitative scoring. FragAnchor correctly predicted 91% of all the GPI-anchored proteins annotated in the Swiss-Prot database. In a large-scale analysis of 29 eukaryote proteomes, FragAnchor predicted that the percentage of highly probable GPI-anchored proteins is between 0.21% and 2.01%. The distinctive feature of FragAnchor, compared with other systems, is that it targets only the C-terminus of a protein, making it less sensitive to the background noise found in databases and possible incomplete protein sequences. Moreover, FragAnchor can be used to predict GPI-anchored proteins in all eukaryotes. Finally, by using qualitative scoring, the predictions combine both sensitivity and information content. The predictor is publicly available at [see text].

Plasmodium falciparum glycosylphosphatidylinositol induces limited apoptosis in liver and spleen mouse tissue.

Plasmodium falciparum malaria affects about 500 million people worldwide and is responsible for approximately 2.5 million deaths per year. Glycosylphosphatidylinositol (GPI) is the major anchor for membrane-associated proteins of P. falciparum and GPI plays a major role as a toxin in the pathology of malaria. Therefore, we tested the hypothesis that GPI, like LPS, induces apoptosis in vitro and in vital organs of mice. Our data does not provide evidence for direct cardiomyocyte apoptosis induced by GPI in vitro. However, in vivo injection of GPI induced limited apoptosis in mouse liver and spleen tissue. Apoptosis may be due to a direct GPI apoptotic effect or to an indirect effect via the induction of TNFalpha and nitric oxide production.

Supported planar bilayers for study of the immunological synapse.

Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC-peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two-dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid-anchored membrane proteins, 6-histidine-tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers. Reagents developed for the MHC-peptide tetramer staining method (UNIT 17.3) can readily be adapted to presentation on planar bilayers. The unique advantage of this approach is that the proteins presented on the surface of the supported bilayer are laterally mobile. This provides a more physiological presentation of cell-surface molecules and supports visualization of protein rearrangement on the bilayer by live cells.

MDGA1, an IgSF molecule containing a MAM domain, heterophilically associates with axon- and muscle-associated binding partners through distinct structural domains.

Molecules belonging to the immunoglobulin superfamily (IgSF) are reported to be involved in intercellular communication in the developing nervous system. We have identified a novel GPI-anchored IgSF molecule containing a MAM (meprin, A5 protein, PTPmu) domain, named MDGA1, by screening for genes that are expressed by subpopulations of cells in the embryonic chick spinal cord. MDGA1 is selectively expressed by brachial LMCm motor neurons, some populations of DRG neurons, and interneurons. We found that MDGA1 interacts heterophilically with axon-rich regions, mainly through its MAM domain. Interestingly, MDGA1 also interacts with differentiating muscle through its N-terminal region, which contains Ig domains. These results suggest that MDGA1 functions in MDGA1-expressing nerves en route to and at their target site.

Synthesis and structural characterization of a mimetic membrane-anchored prion protein.

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.

Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi.

GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed.

Removal of phospholipid contaminants through precipitation of glycosylphosphatidylinositols.

Parasitic glycosylphosphatidylinositols (GPIs) are thought to be involved in induced cell signaling that leads to proinflammatory responses. Increasing interest in elucidation of the mechanisms involved in signaling pathways drives the finding of rapid and reliable methods to purify GPIs. GPIs are usually extracted using mixtures of chloroform/methanol/water, followed by a phase partition between water and water-saturated n-butanol. GPIs recovered in the butanol phase are separated by thin-layer chromatography, scraped, eluted from the silica, and used for studying the structure-function relationship. The presence of phospholipid contaminants or other hydrophobic components in the samples cannot be excluded. Furthermore, the standard procedures to purify GPIs harbor several drawbacks, including the need to handle large amounts of culture, poor yields, time-consuming, and interfering contaminants. Here we report on the development of a simple and reliable method to isolate and purify both free and bound GPIs from one cell pellet. We exploited the low solubility of GPIs in water-saturated n-butanol to remove the phospholipid contaminants completely. After delipidation, GPI proteins were solubilized from the pellet using a mixture of organic solvent containing ethanol and water.

Identification of the inositol isomers present in Tetrahymena.

The inositol isomer composition of phosphoinositides, polyphosphoinositols, phosphatidylinositol-linked glycans, and glycosyl phosphatidylinositol-anchored proteins of logarithmic phase Tetrahymena vorax was determined by GC-MS analysis of trimethylsilylimadazole derivatives. The most abundant inositol found was the myo-isomer; however, appreciable percentages of scylloinositol were present in the free inositol pool, phosphatidylinositol-linked glycan fraction, and glycosyl phosphatidylinositol-anchored protein fraction. Trace quantities of chiro- and neo-inositols also were present.

GLUT4-containing vesicles are released from membranes by phospholipase D cleavage of a GPI anchor.

We have previously developed a cell-free assay from rat skeletal muscle that displayed in vitro glucose transporter 4 (GLUT4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT4 transfer assay, we have purified a glycosylphosphatidylinositol (GPI) phospholipase D (PLD) that induces transfer of GLUT4 from small to large membranes. The in vitro GLUT4 transfer was activated and inhibited by suramin and 1,10-phenanthroline (an activator and an inhibitor of GPI-PLD activity, respectively). Furthermore, upon purification of the GLUT4 transporter protein, the protein displayed an elution profile in which the molecular mass was related to the charge, suggesting the presence or absence of phosphate. Second, by photoaffinity labeling of the purified GLUT4 with 3-(trifluoromethyl)-3-(m-[(125)I]iodopenyl)diazirine, both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of Triton X-114, the purified GLUT4 was found to be partly detergent resistant, which is a known characteristic of GPI-linked proteins. Fourth, the purified GLUT4 protein was recognized by an antibody raised specifically against GPI links. In conclusion, GLUT4-containing vesicles may be released from a membrane compartment by action of a GPI-PLD.