GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice.

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease.

The release of glycosylphosphatidylinositol-anchored proteins from the cell surface.

Starting with the first description of the anchorage of a subset of cell surface proteins in eukaryotic cells from yeast to mammals with the aid of a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein, experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released into the extracellular environment has been accumulating. GPI-AP are released as soluble monomers or multimers having lost their anchor or within hetero-/multimeric assemblies with their complete anchor remaining attached. The configurations reported so far for those assemblies encompass carrier protein-bound monomers, phospholipid- and cholesterol-harboring micelle-like complexes as well as membrane vesicles and particles. Each of these configurations prevents direct contact of the GPI anchor with the aqueous environment. Their structural diversity is reflected in the different molecular mechanisms underlying their release, which involve (i) proteolytic or lipolytic cleavage of the protein or GPI moiety, respectively, (ii) masking of the GPI anchor in the binding pocket of carrier proteins or in the phospholipid mono- or bilayers of particles or vesicles, respectively, and (iii) direct transfer of anchor-harboring GPI-AP from donor to acceptor cells through intimate contact of their plasma membranes. Release of GPI-AP may occur spontaneously or in response to certain endogenous or environmental stress signals and exert specific roles in the (patho)physiology of eukaryotic organisms which, however, are only incompletely understood so far.

Strain-Specific Antagonism of the Human H1N1 Influenza A Virus against Equine Tetherin.

Tetherin/BST-2/CD317 is an interferon-induced host restriction factor that can block the budding of enveloped viruses by tethering them to the cell surface. Many viruses use certain proteins to counteract restriction by tetherin from their natural hosts, but not from other species. The influenza A virus (FLUAV) has a wide range of subtypes with different host tropisms. Human tetherin (huTHN) has been reported to restrict only specific FLUAV strains and the viral hemagglutinin (HA) and neuraminidase (NA) genes determine the sensitivity to huTHN. Whether tetherins from other hosts can block human FLUAV is still unknown. Here, we evaluate the impact of equine tetherin (eqTHN) and huTHN on the replication of A/Sichuan/1/2009 (H1N1) and A/equine/Xinjiang/1/2007 (H3N8) strains. Our results show that eqTHN had higher restriction activity towards both viruses, and its shorter cytoplasmic tail contributed to that activity. We further demonstrated that HA and NA of A/Hamburg/4/2009 (H1N1) could counteract eqTHN. Notably, our results indicate that four amino acids, 13T and 49L of HA and 32T and 80V of NA, were involved in blocking the restriction activity of eqTHN. These findings reveal interspecies restriction by eqTHN towards FLUAV, and the role of the HA and NA proteins in overcoming this restriction.

Molecular Insights into the Roles of Rab Proteins in Intracellular Dynamics and Neurodegenerative Diseases.

In eukaryotes, the cellular functions are segregated to membrane-bound organelles. This inherently requires sorting of metabolites to membrane-limited locations. Sorting the metabolites from ribosomes to various organelles along the intracellular trafficking pathways involves several integral cellular processes, including an energy-dependent step, in which the sorting of metabolites between organelles is catalyzed by membrane-anchoring protein Rab-GTPases (Rab). They contribute to relaying the switching of the secretory proteins between hydrophobic and hydrophilic environments. The intracellular trafficking routes include exocytic and endocytic pathways. In these pathways, numerous Rab-GTPases are participating in discrete shuttling of cargoes. Long-distance trafficking of cargoes is essential for neuronal functions, and Rabs are critical for these functions, including the transport of membranes and essential proteins for the development of axons and neurites. Rabs are also the key players in exocytosis of neurotransmitters and recycling of neurotransmitter receptors. Thus, Rabs are critical for maintaining neuronal communication, as well as for normal cellular physiology. Therefore, cellular defects of Rab components involved in neural functions, which severely affect normal brain functions, can produce neurological complications, including several neurodegenerative diseases. In this review, we provide a comprehensive overview of the current understanding of the molecular signaling pathways of Rab proteins and the impact of their defects on different neurodegenerative diseases. The insights gathered into the dynamics of Rabs that are described in this review provide new avenues for developing effective treatments for neurodegenerative diseases-associated with Rab defects.

Macrophage biology and their activation by protozoan-derived glycosylphosphatidylinositol anchors and hemozoin.

Despite recent advances in medical technology and a global effort to improve public health and hygiene, parasitic infections remain a major health and economic burden worldwide. The World Health Organization estimates that about 1/3 of the world's population is currently infected with a soil-transmitted helminth, and millions more suffer from diseases caused by protozoan parasites including Plasmodium, Trypanosoma, and Leishmania species. Due to the selective pressure applied by parasitic and other infections, animals have evolved an intricate immune system; however, the current worldwide prevalence of parasitic infections clearly indicates that these pathogens have adapted equally well. Thus, developing a better understanding of the host-parasite relationship, particularly by focusing on the host immune response and the mechanisms by which parasites evade this response, is a critical first step in mitigating the detrimental effects of parasitic diseases. Macrophages are critical contributors during the host response to protozoan parasites, and the success or failure of these cells often tips the balance in favor of the host or parasite. Herein, we briefly discuss macrophage biology and provide an update on our current understanding of how these cells recognize glycosylphosphatidylinositol anchors from protozoan parasites as well as malarial hemozoin.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Communicative functions of GPI-anchored surface proteins in unicellular eukaryotes.

Research on several unicellular eukaryotes has identified communicative surface proteins, which are anchored to the outer membrane by glycosylphosphatidylinositol (GPI). Surprisingly, these surface proteins are also released into the environment, raising questions regarding the underlying adaptive advantages and the physical mechanisms that allow for this shedding. This article reviews the current knowledge on several GPI-proteins of different protist species, assembles the puzzling data on the different functions of surface bound and released forms of these proteins, and summarizes their contribution to intra- and interspecific signaling. Recent advances in biochemistry and glycobiology indicate that the GPI-anchor is one of the prerequisites of protein function of membrane bound as well as of released proteins, and hence is a crucial invention for microbial molecular communication. The sensitivity of GPI-anchors (e.g. to phospholipase C) requires consideration of environmental lipase activity of different sources in microbial communities, as these may represent exogenous factors involved in surface protein release. We hypothesize a complex surface protein based communication network and discuss the known facts on protist GPIs in an evolutionary context.

Lipid storage in large and small rat adipocytes by vesicle-associated glycosylphosphatidylinositol-anchored proteins.

Adipose tissue mass in mammals expands by increasing the average cell volume and/or total number of the adipocytes. Upregulated lipid storage in fully differentiated adipocytes resulting in their enlargement is well documented and thought to be a critical mechanism for the expansion of adipose tissue depots during the growth of both lean and obese animals and human beings. A novel molecular mechanism for the regulation of lipid storage and cell size in rat adipocytes was recently elucidated for the physiological stimuli, palmitate and H(2)O(2), and the antidiabetic sulfonylurea drug, glimepiride. It encompasses (1) the release of small vesicles, so-called adiposomes, harboring the glycosylphosphatidylinositol -anchored (c)AMP-degrading phosphodiesterase Gce1 and 5'-nucleotidase CD73 from donor adipocytes, (2) the transfer of the adiposomes and their interaction with detergent-insoluble glycolipid-enriched microdomains of the plasma membrane of acceptor adipocytes, (3) the translocation of Gce1 and CD73 from the adiposomes to the intracellular lipid droplets of the acceptor adipocytes, and (4) the degradation of (c)AMP at the lipid droplet surface zone by Gce1 and CD73 in the acceptor adipocytes, leading to the upregulation of the esterification of fatty acids into triacylglycerol s and the downregulation of their release from triacylglycerols. This mechanism may provide novel strategies for the therapy of metabolic diseases, such as type 2 diabetes and obesity.

[Eculizumab in paroxysmal nocturnal hemoglobinuria]. / Anticorps monoclonal anti-C5 (éculizumab) dans l'hémoglobinurie paroxystique nocturne.

Paroxysmal nocturnal hemoglobinuria is a rare acquired clonal of the hematopoietic stem cell due to acquired mutation of the PIG-A gene. This results in the lack of two GPI-anchored membrane proteins involved in the inhibition of complement attack, thus explaining red cells hemolysis. The development of an anti-C5 monoclonal antibody (eculizumab) had profoundly modified the treatment of the the hemolytic form of the disease.

Inherited GPI deficiency: a disorder of histone hypoacetylation.

Co-operative interaction of transcription factors (TF) with epigenetic processes, such as chromatin remodeling and modification (acetylation or methylation), as well as DNA methylation, determine transcriptional activity, activation or repression of a given gene. Mutations disrupting binding of TF to their cognate DNA motifs would be expected to alter the epigenetic landscape of the promoter and selectively affect transcription of the given gene. We review here the transcriptional, epigenetic, biochemical, and clinical consequences of a constitutional mutation in the promoter of PIGM, a housekeeping gene that disrupts binding of the general TF, SP1, thus causing the autosomal recessive disease, inherited glycosylphosphatidylinositol (GPI) deficiency. We suggest that detailed dissection of the function of the mutated PIGM promoter provides important lessons pertinent to the transcriptional and epigenetic control of housekeeping genes as a whole and might have wider therapeutic implications.

The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro.

Transmissible spongiform encephalopathies are associated with an autocatalytic conversion of normal prion protein, PrP(C), to a protease-resistant form, PrPres. This autocatalytic reaction can be reproduced in vitro using a procedure called protein misfolding cyclic amplification (PMCA). Here we show that, unlike brain-derived PrP(C), bacterially-expressed recombinant prion protein (rPrP) is a poor substrate for PrPres amplification in a standard PMCA reaction. The differences between PrP(C) and rPrP appear to be due to the lack of the glycophosphatidylinositol anchor in the recombinant protein. These findings shed a new light on prion protein conversion process and have important implications for the efforts to generate synthetic prions for structural and biophysical studies.

Functional aspects of CD52 in reproduction.

CD52 is a GPI-anchored protein present in lymphocytes and male reproductive tissues (mrt) including mature sperm and seminal plasma. It has been shown that mrt-CD52 is synthesized in epithelial cells of the epididymis and vas deferens, but not in the testis. The mrt-CD52 is transported to mature sperm during sperm transition in the male reproductive tract. Lymphocyte CD52 functions to stimulate suppressor T cell induction, while mrt-CD52 is associated with seminogelin and involved in clot formation and liquefaction of semen. In a landmark study, a monoclonal antibody (Mab H6-3C4) from peripheral B lymphocytes of a patient with complement-dependent sperm-immobilizing antibody in the serum has been generated. Using Mab H6-3C4, the carbohydrate moiety of CD52 as an epitope for infertility-related antigen was identified. Mab H6-3C4 also shows strong complement-dependent sperm-immobilizing activity. This suggests CD52 may have a function in protecting sperm from complement activation. Indeed, purified mrt-CD52 from human sperm interferes with the classical pathway, but not lectin-binding or alternative pathways, of the complement systems. Recently, we found CD52 in ovulated cumulus cells from the female reproductive tissues (frt). The frt-CD52 is not recognized by Mab H6-3C4, suggesting that it harbors distinct carbohydrate antigenicity. Further studies are necessary to determine the molecular features and biological functions of CD52 in male and female reproductive tissues.

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals.

Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide) and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains). It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco) (sphingo)lipids of pathogenic/opportunistic fungi and trypanosomatids.

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals

Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide) and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains). It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco) (sphingo) lipids of pathogenic/opportunistic fungi and trypanosomatids.

Recentemente, glicoesfingolipídeos têm atraído atenção devido ao seu papel na biologia celular como segundo-mensageiro ou moduladores da transdução de sinal, afetando vários eventos, desde apoptose até a regulação do ciclo celular. Em fungos patogênicos, existem duas classes de glicolipídeos: monohexosil ceramidas neutras (glucosil-ou galactosilceramida) e glicosilinositol fosforilceramidas (os quais apresentam cadeias de carboidratos mais longas). É importante enfatizar que as monohexosil ceramidas exibem diferenças estruturais nas suas porções lipídicas quando comparadas às de mamíferos, enquanto que glicosilinositol fosforilceramidas exibem diferenças estruturais marcantes em suas porções carboidratos em comparação aos glicoesfingolipídeos de mamíferos. Observamos também que glicosilinositol fosforilceramidas são capazes de promover resposta imune em indíviduos infectados. Além do mais, inibição das vias biossintéticas de glicoesfingolipídeos de fungos acarreta a inibição da formação de colônias, germinação de esporos, ciclo celular, dimorfismo e crescimento de hifas. Outros patógenos, como os tripanosomatídeos, também apresentam glicolipídeos únicos, os quais apresentam um papel importante para o desenvolvimento do parasita e/ou para o estabelecimento da doença. Em relação à interação hospedeiro-patógeno, os "membrane rafts", estruturas da membrana plasmática enriquecidas em esfingolipídeos e esteróis, têm participação fundamental na infecção do parasita/fungo. Nesta revisão, discutimos os diferentes papéis biológicos dos (glico) (esfingo) lipídeos de fungos patogênicos/oportunistas e de tripanosomatídeos.

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.

Reverse signaling by glycosylphosphatidylinositol-linked Manduca ephrin requires a SRC family kinase to restrict neuronal migration in vivo.

Reverse signaling via glycosylphosphatidylinositol (GPI)-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), whereas adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways.

[Paroxysmal nocturnal hemoglobinuria (PNH). Pathogenesis, diagnosis and treatment]. / Paroxysmale nächtliche Hämoglobinurie (PNH). Pathogenese, Diagnostik und Therapie.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the classic clinical triad of corpuscular hemolytic anemia, thrombophilia and cytopenia. This is caused by an acquired mutation of the PIG(phosphatidylinositol glycan)-A gene of the pluripotent hematopoetic stem cell. This results in a deficiency of GPI(glycosylphosphatidylinositol)-anchors and GPI-anchored proteins on the surface of affected blood cells. Flow cytometry is the standard for diagnosis and measurement of type and size of the PNH clone. Treatment of PNH is mainly symptomatic. Allogeneic bone marrow transplantation is the only curative option in case of severe complications during the course of the diseases. A new targeted treatment strategy is the inhibition of the terminal complement cascade with a monoclonal antibody (eculizumab). As shown in clinical studies this is efficient to reduce complement mediated intravascular hemolysis, reduce the need for transfusions, improve the quality of life in patients with PNH and reduce the risk for thromboembolic complications, which are the main cause of mortality in PNH.

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.