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Gp20 is a sialylglycoprotein of the human sperm surface related to maturation and capacitation and is homologous to CD52, a glycosyl- phosphatidyl-inositol (GPI)-anchored protein highly expressed in lymphocytes, monocytes, eosinophils, and epididymal cells, described by the monoclonal antibody family CAMPATH. The CAMPATH antigen is characterized by a very short peptide (12 amino acids) and an N-linked oligosaccharide chain bound to the asparagine located in the third position and a GPI anchor bound to the C-terminal serine. The CAMPATH epitope includes three amino acids at the C-terminus and part of the GPI anchor. It has been suggested that anti-gp20 interacts with the same peptide recognized by CAMPATH antibodies but with a different epitope, since it describes the corresponding antigen in a different way. For example, it localizes the corresponding antigen in the equatorial region of the sperm head when sperm are capacitated, whereas CAMPATH antibodies bind all over the sperm surface. Our results indicate that the anti-gp20 epitope does not include the peptide backbone, the GPI anchor, or the N-glycans but consists of O-linked oligosaccharide chains bound to a unique CD52 glycoform present both in sperm and leukocytes. This is suggested by results obtained using many different approaches, such as immunoblot analysis of gp20 after removal of N- and O-glycans and after jacalin ( Artocarpus integrifolia agglutinin)-affinity chromatography. [ABSTRACT FROM AUTHOR]

Extracellular signaling pathways regulating myoblast differentiation and cell-cycle withdrawal are not completely understood. Stem cell antigen-1 (Sca-1/Ly-6A/E) is a glycosylphosphatidylinositol-anchored membrane protein known for its role in T-cell activation, and recently described as a marker for regeneration-competent myoblasts. We previously determined that expression of Sca-1/Ly-6A is transiently upregulated during myocyte cell-cycle withdrawal; however, a specific function for Sca-1 in myogenesis has not been described. Here, we show that Sca-1 expression on the surface of a subpopulation of differentiating C2C12 myoblasts is maximal at the time of cell-cycle withdrawal, and that blocking Sca-1 with monoclonal antibodies or downregulating Sca-1 expression by antisense both promotes proliferation and inhibits myotube formation. Downregulating Sca-1 expression derepresses Fyn at the time of myoblast cell-cycle withdrawal, and dominant-negative and constitutively active Fyn mutants rescue and recapitulate the Sca-1 antisense phenotype, respectively. This suggests a Fyn-mediated mechanism for Sca-1 action. Thus, we demonstrate an unprecedented role for Sca-1 in early myogenesis in C2C12 cells, and propose a novel pathway from the myoblast cell surface to intracellular signaling networks controlling proliferation versus differentiation in mammalian muscle. These findings suggest that, beyond its role as a marker for muscle progenitors, Sca-1 may be an important therapeutic target for promoting muscle regeneration. [ABSTRACT FROM AUTHOR]

A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of bovine erythrocyte-ghost membrane. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition ofn-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model. [ABSTRACT FROM AUTHOR]

Bacterial phosphatidylinositol-specific phospholipases C have been shown not only to cause breakdown of phosphatidylinositol but atso to release GPI-anchored proteins from the plasma membranes of eucaryotes. Several enzymes in this group have been sequenced by cloning genomic DNA, and the enzymes of Bacillus cereus and Listeria monocytogenes were analyzed for their structures by X-ray crystallography. In the active sites of enzymes from Bacillus genera and L. monocytogenes, the roles of component amino acid residues in catalysis have been mostly clarified. The enzyme of Bacillus thuringiensis exhibited cytotoxicity against some cultivated cells. The enzyme of L monocytogenes was shown to contribute to listerial infection of epithelial cells and macrophages as a virulence factor cooperating with other factors such as listeriolysin O and phosphatidylcholine-preferring phosphotipase C. Recently, this enzyme proved to stimulate the signal-transduction system of host cells in listeriosis. The requirements for effective utilization of bacterial phosphatidylinositol-specific phospholipases C should be considered in research on GPI-anchored proteins, cellular transduction. and so forth, given the unique properties of these enzymes. [ABSTRACT FROM AUTHOR]

Our view of biological membranes has evolved dramatically over the last few decades. In the bilayer model from Singer & Nicholson (Science 1972;175:720–731), both proteins and lipids freely diffuse within the plane of the membrane. Currently, however, membranes are viewed as a mosaic of different compartments or domains maintained by an active cytoskeleton network (Ritchie et al. Mol Membr Biol 2003; 20:13–18). Due to interactions between membrane components, several types of subdomains can form with different characteristics and functions. Lipids are likely to play an important role in the formation of so-called lipid-enriched microdomains or lipid rafts, adding another order of complexity to the membrane model. Rafts represent a type of domain wherein lipids of specific chemistry may dynamically associate with each other, to form platforms important for membrane protein sorting and construction of signaling complexes (Simons & Toomre. Nat Rev Mol Cell Biol 2000;1:31–39). Currently, there are several hypotheses concerning the nature of rafts (reviewed in (Edidin. Annu Rev Biophys Biomol Struct 2003;32: 257–283; Zurzolo et al. EMBO Rep 2003;4:1117–1121)). The most commonly cited one, proposed by Kai Simons (Simons & Ikonen. Nature 1997;387:569–572; Pralle et al. J Cell Biol 2000;148:997–1008), suggests that rafts are relatively small structures (∼ 50 nm) enriched in cholesterol and sphingolipids within which associated proteins are likely to be concentrated. Another proposal (Anderson & Jacobson. Science 2002;296:1821–1825) suggests that rafts are constructed of lipid shells. These are small dynamic assemblies wherein ‘raft’ proteins are preferentially associated with certain types of lipids. These ‘shells’ are thermodynamically stable mobile entities in the plane of the membrane that are able to target the protein they encase to preexisting rafts/caveolae domains. In this review we summarize the data suggesting a specific role for lipid domains in intracellular trafficking and sorting and present a modification of the raft model that may help explain the observed phenomena. [ABSTRACT FROM AUTHOR]

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD  -  , and the isogenic cell line LD  -  PIGA  +  in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD  -  PIGA  +  cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab. [ABSTRACT FROM AUTHOR]

In this study we have optimised the enzyme immunoassay (ELISA) to quantify CD59 antigen in human serum or plasma. The glycosyl-phosphatidylinositol (GPI)-linked form of CD59 is known to complex with serum high-density lipoprotein. For ELISA optimisation, therefore, we investigated the effect of detergents, added to the sample diluent, on the determined values of CD59. Values obtained in the presence of octyl-glucoside (OG) for 20 adults aged 18–35 years and 17 children 1–5 years old were, respectively, 33–119 ng/ml (mean ± S.D.: 66±22 ng/ml) and 37–143 ng/ml (76±33 ng/ml). These results were higher than those measured without OG and were in contrast with published results showing absence, or eight to nine times lower levels, of the protein in serum. A known range for serum concentrations of CD59 in healthy individuals will establish an important reference point for clinical work and for the investigation of diseases involving the complement membrane attack complex (MAC) and its regulation. [Copyright &y& Elsevier]

Membrane domains are highly specialized parts of the cell plasma membrane, carrying on and augmenting the incoming signals. To study their structural and functional properties, it is crucial to find the least damaging mode of their isolation. Using two different cell lines, epithelial HEK cells (clone E2M11) and S49 lymphoma cells, three methods of membrane domain isolation (i.e., detergent extraction, alkaline treatment, and "drastic" homogenization) were tested for similarity and reproducibility by 2-D electrophoresis. Our data show that the protein composition of membrane domains obtained by different isolation methods is similar and that approximately 60% of the spots are present in all membrane domain preparations. Furthermore, the same degree of similarity of 2-D profiles of the most intensively silver stained spots found in membrane domains of the two cell lines derived from different tissues suggests that the composition of a large part of membrane domains proteins is conservative. We suggest that these proteins may either be involved in the organization of membrane domain structure or represent the conservative component of signal transduction machinery. [ABSTRACT FROM AUTHOR]

The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides. Manduca sexta, the tobacco hornworm, is the model insect for B. thuringiensis studies. Although brush border vesicles prepared from larval M. sexta midgut have been used in numerous mode-of-action studies of B. thuringiensis toxins, their protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome. Sub-proteomes were also established for B. thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins. Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches. Results generally did not produce matches to M. sexta proteins, but did match proteins of other Lepidoptera. Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N. Aminopeptidase N was the only GPI-anchored protein identified. Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots. [Copyright &y& Elsevier]

PrPC is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrPC remains unknown, a conformational isoform called PrPSc (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrPC to PrPSc conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrPC in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrPC is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrPC associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrPC to PrPSc . In order to discriminate whether detergent-resistant microdomains have a direct role in PrPSc conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrPC intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans -Golgi network sorting and transport of PrPC to... [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a highly specific enzyme whose only known substrate is the GPI anchor of cell surface proteins. GPI-PLD measurements, however, are technically difficult since the enzyme is expressed at low levels in cells and tissues, and serum contains large amounts of inactive, latent GPI-PLD interfering with protein-based assays. We have therefore developed a semi-quantitative RT-PCR method to measure mRNA expression of all known GPI-PLD isoforms in cells and tissues. In human ovarian cancer cell lines, GPI-PLD mRNA expression correlated with GPI-PLD enzyme activity and with the shedding of the GPI-anchored tumor and prognostic markers, urokinase receptor and CA125, from the cell surface. This supports a potential role for this enzyme in the generation of circulating prognostic markers in malignant tumors. Similarly, in human epithelial cells of the skin, GPI-PLD mRNA expression increased with tumor progression. Whereas normal keratinocytes did not express significant amounts of GPI-PLD mRNA, expression was dramatically induced by serum in immortalized HaCaT keratinocytes and constitutively high and independent of serum in tumorigenic A431 epidermoid carcinoma cells. In addition, GPI-PLD expression was significantly increased in highly malignant, H-ras-transfected murine bladder carcinoma cells as compared to the low malignant, non-transfected parental cells. The competitive RT-PCR described here represents the first quantitative assay specific for cellular GPI-PLD isoforms, and our in vitro analyses suggest that GPI-PLD expression might be associated with tumor malignancy. [ABSTRACT FROM AUTHOR]

Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediators of sorting and signaling functions of associated molecules. Apart from a limited number of biophysical studies in living cell membranes, raft-association has been defined by a simple biochemical criterion, namely the ability to partition with detergent-resistant membranes (DRMs). Here we examine the evidence for the specification of internalization mechanisms and endocytic pathways by rafts as defined by this criterion. We have surveyed the endocytic trafficking of a variety of molecules such as lipids, toxins, glycosylphosphatidylinositol (GPI)-anchored proteins, and DRM-associated transmembrane proteins. [Copyright &y& Elsevier]

Caveolae are flask-shaped invaginations present in the plasma membrane of many cell types. They have long been implicated in endocytosis, transcytosis, and cell signaling. Recent work has confirmed that caveolae are directly involved in the internalization of membrane components (glycosphingolipids and glycosylphosphatidylinositol-anchored proteins), extracellular ligands (folic acid, albumin, autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike clathrin-mediated endocytosis, internalization through caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized substances take are starting to emerge. [ABSTRACT FROM AUTHOR]

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations. [ABSTRACT FROM AUTHOR]

PNH is a disorder of the pluripotent stem cells resulting in a deficient expression of membrane-bound GPI-anchored proteins in different cell types. Several flow cytometric approaches are designed to detect this antigen deficiency. But they all require drawing and testing of normal samples as control. Therefore, in the present study two flow cytometric assays for the detection of CD55 and CD59 deficiency in erythrocytes (REDQUANT™ CD55/CD59) and granulocytes (CELLQUANT™ CD55/CD59) are proposed. Precalibrated beads are used to define the cut off between normal and deficient cell populations. The specificity of the tests has been evaluated in healthy blood donors (n=52) resulting in a clear and reproducible cut off (3%) for the normal percentage of GPI-deficient cells. This cut off has been confirmed in leukaemia and lymphoma patients not suspected for developing PNH. The sensitivity has been tested in patients suffering from known PNH (n=23). Both tests performed in combination allowed a reliable detection of PNH in all patients showing antigen deficiencies in both cell types in most patients (20/23). In contrast, the PNH clones in the investigated patients with MDS (4/19) or AA (4/22) were present in granulocytes or erythrocytes, only. This underlines the necessity of analysing erythrocytes as well as granulocytes. Preliminary data regarding a possible correlation between disease activity and percentage of antigen-deficient cells lead to the assumption that haemolytic crises can only be determined on granulocytes whereas deficient erythrocytes disappeared due to complement-mediated lysis of the PNH clone. In conclusion, the combination of the test kits enables the differential diagnosis of PNH clones in a standardized, simple and rapid approach which may have therapeutic consequences. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI‐anchored proteins (GPI‐APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI‐APs are delivered to endocytic organelles, devoid of markers of the clathrin‐mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate‐limiting step in the recycling of GPI‐APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid‐synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI‐APs to that of other membrane components. We have shown previously that endocytic retention of GPI‐APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI‐APs in the REC occurs via their association with sphingolipid and cholesterol‐enriched sorting platforms or 'rafts'. [ABSTRACT FROM AUTHOR]

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular haemolysis, venous thrombosis, marrow hypoplasia, frequent episodes of infection, and rarely leukaemic conversion. At the cellular level, PNH is characterized by the decrease or absence of glycosylphosphatidylinositol (GPI)-anchored molecules, such as CD55 and CD59, from the cell surface. PNH-like clones have been described in various haematological disorders. The link between PNH and aplastic anaemia (AA) has been established but the relationship of PNH with myelodysplastic syndromes (MDS) or myeloproliferative disorders (MPD) remains unclear. In this study, the presence of CD55 and/or CD59 defective (PNH-like) red cell populations was evaluated in 21 patients with AA, 133 with MDS, 197 with MPD, 7 with PNH and in 121 healthy blood donors using the Sephacryl Gel Test microtyping system. Red cell populations deficient in both molecules CD55 and CD59 were detected in 33.3% of AA patients, in 16.5% of MDS patients (50% with hypoplastic bone marrow), in 14.2% of MPD patients (more often in essential thrombocythemia, 21.2%) and in all PNH patients. CD55 deficient red cell populations were found in 14.2% of patients with AA, 12.7% of patients with MDS and 21.3% of patients with MPD. CD59 deficient populations were found in 9.5% of AA patients, 2.2% of MDS patients and 2% of MPD patients. These results indicate an association between PNH, AA and MDS or even between PNH and MPD. Further investigation is necessary to work out the mechanisms of this association, and to define classification criteria for borderline cases, where diagnosis is difficult. [ABSTRACT FROM AUTHOR]

In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The ‘rafts’ hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI-anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin–Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (∼30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X-100-insoluble microdomains, suggesting that at least in these cells ‘rafts’ are not involved in basolateral to apical transcytosis. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA. [ABSTRACT FROM AUTHOR]

The author discusses the impact of glycosyl-phosphatidyl-inositol (GPI) anchored major histocompatibility complex class I-related chain A (MICA) alleles on NKG2D-based therapies. Topics discussed include the association between immune regulation and human NKG2D-ligands, the role of viral immune evasion strategies in determining the heterogeneity in the ligands' cell biology, and the concept of personalized medicine.

Membrane proteins can be classified into two main categories—integral and peripheral membrane proteins—depending on the nature of their membrane interaction. Peripheral membrane proteins are highly unique amphipathic proteins that interact with the membrane indirectly, using electrostatic or hydrophobic interactions, or directly, using hydrophobic tails or GPI-anchors. The nature of this interaction not only influences the location of the protein in the cell, but also the function. In addition to their unique relationship with the cell membrane, peripheral membrane proteins often play a key role in the development of human diseases such as African sleeping sickness, cancer, and atherosclerosis. This review will discuss the membrane interaction and role of periplasmic nitrate reductase, CymA, cytochrome c, alkaline phosphatase, ecto-5'-nucleotidase, acetylcholinesterase, alternative oxidase, type-II NADH dehydrogenase, and dihydroorotate dehydrogenase in certain diseases. The study of these proteins will give new insights into their function and structure, and may ultimately lead to ground-breaking advances in the treatment of severe diseases. [ABSTRACT FROM AUTHOR]

Deficient expression of glycosylphosphatidyl inositol (GPI)-anchored proteins in aplastic anaemia (AA) patients has previously been reported to be associated with a poor response to immunosuppressive (IS) therapy. Here we report the response to IS therapy of 111 patients with AA and correlate this with GPI-anchored protein expression on peripheral blood cells by flow cytometry. A GPI-anchored protein deficient population was identified in 15% (17/111) of patients with AA who had a negative Ham's test and no laboratory evidence of haemolysis. Patients were treated with antilymphocyte globulin and/or cyclosporin A, or oxymetholone. Bone marrow transplantation was performed in 12 patients, seven of whom had not responded to IS therapy. In patients tested for GPI-anchored protein expression prior to IS therapy there was no difference in response rate to IS therapy between AA patients with a GPI-anchored protein deficiency and those with normal GPI-anchored protein expression (50% response rate versus 75%, respectively). Survival in these two groups was similar at 90% with follow-up over 140 months from diagnosis. Eight of the 17 AA patients who developed a GPI-anchored protein-deficient population later went on to develop a positive Ham's test. From this study we demonstrate a lower incidence of GPI-anchored protein deficiency in AA patients compared with previous reports. In addition we have shown that the presence of a GPI-anchored protein-deficient cell population in patients with AA who have a negative Ham's test is not a poor prognostic factor in terms of response and survival after IS therapy. [ABSTRACT FROM AUTHOR]

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract The proteins of flotillin-1 and flotillin-2 were originally discovered in axon regeneration of goldfish retinal ganglion cells. They are generally used as marker proteins of lipid rafts and considered to be scaffolding proteins of lipid microdomains. Although they are ubiquitously expressed and well-conserved from fly to man, their exact functions remain controversial. In this review, we summarize the structure of flotillins and some functions of them, such as regulating axon regeneration, endocytosis, T cell activation, insulin signaling, membrane protein recruitment, roles in the progression of some diseases and so on.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are membrane-conjugated cell-surface proteins with diverse structural, developmental, and signaling functions and clinical relevance. Typically, after biosynthesis and attachment to the preassembled GPI anchor, GPI-APs rapidly leave the endoplasmic reticulum (ER) and rely on post-ER quality control. Terminally misfolded GPI-APs end up inside the vacuole/lysosome for degradation, but their trafficking itinerary to this organelle and the processes linked to their uptake by the vacuole/lysosome remain uncharacterized. In a yeast mutant that is lacking Pep4, a key vacuolar protease, several misfolded model GPI-APs accumulated in the vacuolar membrane. In the same mutant, macroautophagy and the multi-vesicular body (MVB) pathway were intact, hinting at a hitherto-unknown trafficking pathway for the degradation of misfolded GPI-APs. To unravel it, we used a genome-wide screen coupled to high-throughput fluorescence microscopy and followed the fate of the misfolded GPI-AP: Gas1 ∗ . We found that components of the early secretory and endocytic pathways are involved in its targeting to the vacuole and that vacuolar transporter chaperones (VTCs), with roles in microautophagy, negatively affect the vacuolar uptake of Gas1 ∗ . In support, we demonstrate that Gas1 ∗ internalizes from vacuolar membranes into membrane-bound intravacuolar vesicles prior to degradation. Our data link post-ER degradation with microautophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The COBRA-like (COBL) gene family has been associated with the regulation of cell wall expansion and cellulose deposition. COBL mutants result in reduced levels and disorganized deposition of cellulose causing defects in the cell wall and inhibiting plant development. In this study, we report the identification of 24 COBL genes (GmCOBL) in the soybean genome. Phylogenetic analysis revealed that the COBL proteins are divided into two groups, which differ by about 170 amino acids in the N-terminal region. The GmCOBL genes were heterogeneously distributed in 14 of the 20 soybean chromosomes. This study showed that segmental duplication has contributed significantly to the expansion of the COBL family in soybean during all Glycine-specific whole-genome duplication events. The expression profile revealed that the expression of the paralogous genes is highly variable between organs and tissues of the plant. Only 20% of the paralogous gene pairs showed similar expression patterns. The high expression levels of some GmCOBLs suggest they are likely essential for regulating cell expansion during the whole soybean life cycle. Our comprehensive overview of the COBL gene family in soybean provides useful information for further understanding the evolution and diversification of COBL genes in soybean. [ABSTRACT FROM AUTHOR]

The plant defensin HsAFP1 is characterized by broad-spectrum antifungal activity and induces apoptosis in Candida albicans. In this study, we performed a transcriptome analysis on C. albicans cultures treated with HsAFP1 to gain further insight in the antifungal mode of action of HsAFP1. Various genes coding for cell surface proteins, like glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in cation homeostasis, autophagy and in cell cycle were differentially expressed upon HsAFP1 treatment. The biological validation of these findings was performed in the model yeast Saccharomyces cerevisiae. To discriminate between events linked to HsAFP1's antifungal activity and those that are not, we additionally used an inactive HsAFP1 mutant. We demonstrated that (i) HsAFP1-resistent S. cerevisiae mutants that are characterized by a defect in processing GPI-anchors are unable to internalize HsAFP1, and (ii) moderate doses (FC50, fungicidal concentration resulting in 50% killing) of HsAFP1 induce autophagy in S. cerevisiae , while high HsAFP1 doses result in vacuolar dysfunction. Vacuolar function is an important determinant of replicative lifespan (RLS) under dietary restriction (DR). In line, HsAFP1 specifically reduces RLS under DR. Lastly, (iii) HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. However, the latter HsAFP1-induced event is not linked to its antifungal activity, as the inactive HsAFP1 mutant also impairs the G2/M phase. In conclusion, we demonstrated that GPI-anchored proteins are involved in HsAFP1's internalization, and that HsAFP1 induces autophagy, vacuolar dysfunction and impairment of the cell cycle. Collectively, all these data provide novel insights in the mode of action of HsAFP1 as well as in S. cerevisiae tolerance mechanisms against this peptide. Unlabelled Image • GPI-anchored proteins mediate HsAFP1's internalization. • HsAFP1 induces autophagy and impairs vacuolar function. • HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. [ABSTRACT FROM AUTHOR]

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N -glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases.

Glycosylphosphatidylinositols (GPIs) anchored proteins are commonly localized onto lipid rafts. These extracellular proteins participate in a variety of cellular functions, including as receptors for viruses and toxins. Intracellular trafficking of World Health Organization recognized mosquito-larvicidal BinAB toxin is mediated via GPI-anchored Cqm1 receptor protein in Culex mosquitoes. We confirmed conformational change in Cqm1 dimer on interaction with BinA/BinB proteins by dynamic light scattering, modelling of hydrodynamic parameters using the atomic structures, and synchrotron Small Angle solution X-ray scattering (SAXS). A reliable model of the receptor-BinB complex was also constructed from joint SAXS/SANS refinement. We confirmed electrostatic interactions of the Cqm1 ectodomain with lipid rafts reconstituted in model membranes and report receptor-dependent impairment of model liposomes by BinA/B proteins. Liposomal disruption was toxin concentration-dependent as monitored by the release of encapsulated carboxyfluorescein dye. Interestingly, BinA alone, without BinB, showed efficient efflux of the fluorescent dye in agreement with the reported high larvicidal activity of BinA variants. The study provides insight into BinA/B toxin internalization mechanism in the membrane model that is toxin internalization is mediated via receptor-dependent pore formation mechanism. It also suggests a tangible and environmentally safe strategy for control of mosquito population.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens., (© 2019 Blackwell Verlag GmbH.)

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well-equilibrated two-dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans-bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano- and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein-protein, protein-lipid, and lipid-lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite., (© 2020 The Protein Society.)

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of β-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii , which had naturally more β-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and β-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells., (Copyright © 2020 Walker and Munro.)

Exosomes are 50-100 nm membranous vesicles actively released by cells which can be indicative of a diseased cell status. They contain various kinds of molecule - proteins, mRNA, miRNA, lipids - that are actively being studied as potential biomarkers. Hereafter I put forward several arguments in favor of the potential use of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as biomarkers especially of cancerous diseases. I will briefly update readers on the exosome field and review various features of GPI-APs, before further discussing the advantages of this class of proteins as potential exosomal biomarkers. I will finish with a few examples of exosomal GPI-APs that have already been demonstrated to be good prognostic markers, as well as innovative approaches developed to quantify these exosomal biomarkers., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)