Profiling Glycosylphosphatidylinositol (GPI)-Interacting Proteins in the Cell Membrane Using a Bifunctional GPI Analogue as the Probe.

Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.

Free, unlinked glycosylphosphatidylinositols on mammalian cell surfaces revisited.

Glycosylphosphatidylinositols (GPIs) are linked to many cell-surface proteins, anchor these proteins in the membrane, and are well characterized. However, GPIs that exist in the free form on the mammalian cell surface remain largely unexplored. To investigate free GPIs in cultured cell lines and mouse tissues, here we used the T5-4E10 mAb (T5 mAb), which recognizes unlinked GPIs having an N-acetylgalactosamine (GalNAc) side chain linked to the first mannose at the nonreducing terminus. We detected free GPIs bearing the GalNAc side chain on the surface of Neuro2a and CHO, but not of HEK293, K562, and C2C12 cells. Furthermore, free GPIs were present in mouse pons, medulla oblongata, spinal cord, testis, epididymis, and kidney. Using a panel of Chinese hamster ovary cells defective in both GPI-transamidase and GPI remodeling pathway, we demonstrate that free GPIs follow the same structural remodeling pathway during passage from the endoplasmic reticulum to the plasma membrane as do protein-linked GPI. Specifically, free GPIs underwent post-GPI attachment to protein 1 (PGAP1)-mediated inositol deacylation, PGAP5-mediated removal of the ethanolamine phosphate from the second mannose, and PGAP3- and PGAP2-mediated fatty acid remodeling. Moreover, T5 mAb recognized free GPIs even if the inositol-linked acyl chain or ethanolamine-phosphate side chain linked to the second mannose is not removed. In contrast, addition of a fourth mannose by phosphatidylinositol glycan anchor biosynthesis class Z (PIGZ) inhibited T5 mAb-mediated detection of free GPIs. Our results indicate that free GPIs are normal components of the plasma membrane in some tissues and further characterize free GPIs in mammalian cells.

Rapid glycosyl-inositol-phospho-ceramide fingerprint from filamentous fungal pathogens using the MALDI Biotyper Sirius system.

RATIONALE: Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis. METHODS: Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000. RESULTS: MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis. CONCLUSIONS: We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii.

Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.

Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

A GPI anchor explains the unique biological features of the common NKG2D-ligand allele MICA*008.

The human MICA (MHC I-related chain A) gene, encoding a ligand for the NKG2D (NKG2-D type II integral membrane protein) receptor, is highly polymorphic. A group of MICA alleles, named MICA 5.1 (prototype, MICA*008), produce a truncated protein due to a nucleotide insertion in the transmembrane domain. These alleles are very frequent in all of the human populations studied and they have different biological properties, compared with full-length alleles, e.g. recruitment into exosomes, which makes them very potent for down-modulating the NKG2D receptor in effector immune cells. Moreover, MICA*008 is not affected by viral immune evasion mechanisms that target other MICA alleles. In the present study, we demonstrate that MICA*008 acquires a GPI (glycosylphosphatidylinositol) anchor and that this modification is responsible for many of the distinct biological features of the truncated MICA alleles, including recruitment of the protein to exosomes. MICA*008 processing is also unusual as it is observed in the endoplasmic reticulum as a Triton™ X-114 soluble protein, partially undergoing GPI modification while the rest is exocytosed, suggesting a new model for MICA*008 release. This is the first report of a GPI-anchored MICA allele. The finding that this modification occurs in both families of human NKG2D ligands, as well as in the murine system, suggests positive pressure to maintain this biochemical feature.

Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by ß-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method.

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins I: Localization at Plasma Membranes and Extracellular Compartments.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).

Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS-PAGE shotgun proteomics.

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI.

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.

Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells.

HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane. While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood. Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes. Our data strengthen evidence that Gag-Env interaction is important in envelope association with lipid rafts containing GPI-anchored proteins for efficient assembly onto mature virions resulting in productive infection of primary CD4+ T cells.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway.

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

CDG biochemical screening: Where do we stand?

BACKGROUND: Glycosylation is one of the most complex post-translational modifications of proteins and lipids, notably requiring many glycosyltransferases, glycosidases and sugar transporters encoded by about 1-2% of all human genes. Deleterious variants in any of them may result in improper protein or lipid glycosylation, thus yielding the so-called 'congenital disorders of glycosylation' or CDG. SCOPE OF REVIEW: We first review the current state of knowledge on the common blood and cellular glycoproteins used in the biochemical screening of CDG, as well as the emerging ones for an improved diagnosis. We then provide an overview of the current state-of-the-art methodologies ranging from gel electrophoresis to mass spectrometry to measure improper glycosylation. Finally, we discuss how additional tools such as metabolomics and microfluidics can be added to the current toolbox to better diagnose and delineate CDG. MAJOR CONCLUSIONS: Combining several biochemical indicators and related methods is often required to cope with the large clinical heterogeneity of CDG and establish a definitive diagnosis. GENERAL SIGNIFICANCE: This review aims to critically present current available CDG biochemical biomarkers and dedicated methods in the context of highly diverse glycosylation pathways and related inherited diseases.

Proteomic scale high-sensitivity analyses of GPI membrane anchors.

Glycosylphosphatidylinositol (GPI) anchored proteins are ubiquitous in eukaryotic cells. Earlier analysis methods required large amounts of purified protein to elucidate the structure of the GPI. This paper describes methods for analyzing GPIs on a 'proteomic' scale. Partially purified proteins may be run on sodium dodecyl sulphate polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride (PVDF) membrane. Following identification of the protein the piece of PVDF may be subjected to various chemical treatments, which are specific for GPI structures. The first method uses gas chromatography-mass spectrometry and it enables the presence of a GPI anchor to be confirmed. The second method depends on the cleavage of phosphate bonds and permits the carbohydrate structure to be elucidated by electrospray or matrix assisted laser desorption ionization-time of flight mass spectrometry. The final method described uses deamination of the glucosamine residue to release the lipid moiety for analysis by mass spectrometry.

Ponticulin is an atypical membrane protein.

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are sorted to the apical surface of many epithelial cell types. To better understand the mechanism for apical segregation of these proteins, we analyzed the lateral mobility and molecular associations of a model GPI-anchored protein, herpes simplex virus gD1 fused to human decay accelerating factor (gD1-DAF) (Lisanti, M. P., I. W. Caras, M. A. Davitz, and E. Rodriguez-Boulan. 1989. J. Cell Biol. 109:2145-2156) shortly after arrival and after long-term residence at the surface of confluent, polarized MDCK cells. FRAP measurements of lateral diffusion showed that the mobile fraction of newly arrived gD1-DAF molecules was much less than the mobile fraction of long-term resident molecules (40 vs. 80-90%). Fluorescence resonance energy transfer measurements showed that the newly arrived molecules were clustered, while resident molecules were not. Newly delivered gD1-DAF molecules were clustered but not immobilized in mutant, Concanavalin A-resistant MDCK cells that failed to sort gD1-DAF. Our results indicate that GPI-anchored proteins in MDCK cells are clustered before delivery to the surface. However, clustering alone does not target molecules for apical delivery. The immobilization observed when gD1-DAF is correctly sorted suggests that the clusters must associate some component of the cell's cytoplasm.

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site.

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae.

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.