IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

Immunoglobulin-like cell adhesion molecule (IgLON4) is a glycosylphosphatidylinositol-anchored membrane protein that has been associated with neuronal growth and connectivity, and its deficiency has been linked to increased fat mass and low muscle mass. Adequate information on IgLON4 is lacking, especially in the context of skeletal muscle. In this study, we report that IgLON4 is profusely expressed in mouse muscles and is intensely localized on the cell membrane. IgLON4 expression was elevated in CTX-injected mouse muscles, which confirmed its role during muscle regeneration, and was abundantly expressed at high concentrations at cell-to-cell adhesion and interaction sites during muscle differentiation. IgLON4 inhibition profoundly affected myotube alignment, and directional analysis confirmed this effect. Additionally, results demonstrating a link between IgLON4 and lipid rafts during myogenic differentiation suggest that IgLON4 promotes differentiation by increasing lipid raft accumulation. These findings support the notion that a well-aligned environment promotes myoblast differentiation. Collectively, IgLON4 plays a novel role in myogenesis and regeneration, facilitates myotube orientation, and is involved in lipid raft accumulation.

Emerging roles for the GPI-anchored tumor suppressor OPCML in cancers.

OPCML is a highly conserved glycosyl phosphatidylinositol (GPI)-anchored protein belonging to the IgLON family of cell adhesion molecules. OPCML functions as a tumor suppressor and is silenced in over 80% of ovarian cancers by loss of heterozygosity and by epigenetic mechanisms. OPCML inactivation is also observed in many other cancers suggesting a conservation of tumor suppressor function. Although epigenetic silencing and subsequent loss of OPCML expression correlate with poor progression-free and overall patient survival, its mechanism of action is only starting to be fully elucidated. Recent discoveries have demonstrated that OPCML exerts its tumor suppressor effect by inhibiting several cancer hallmark phenotypes in vitro and abrogating tumorigenesis in vivo, by downregulating/inactivating a specific spectrum of Receptor Tyrosine Kinases (RTKs), including EphA2, FGFR1, FGFR3, HER2, HER4, and AXL. This modulation of RTKs can also sensitize ovarian and breast cancers to lapatinib, erlotinib, and anti-AXL therapies. Furthermore, OPCML has also been shown to function in synergy with the tumor suppressor phosphatase PTPRG to inactivate pro-metastatic RTKs such as AXL. Recently, the identification of inactivating point mutations and the elucidation of the crystal structure of OPCML have provided valuable insights into its structure-function relationships, giving rise to its potential as an anti-cancer therapeutic.

A Membrane-Anchored Short-Peptide Fusion Inhibitor Fully Protects Target Cells from Infections of Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus.

Emerging studies demonstrate that the antiviral activity of viral fusion inhibitor peptides can be dramatically improved when being chemically or genetically anchored to the cell membrane, where viral entry occurs. We previously reported that the short-peptide fusion inhibitor 2P23 and its lipid derivative possess highly potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). To develop a sterilizing or functional-cure strategy, here we genetically linked 2P23 and two control peptides (HIV-1 fusion inhibitor C34 and hepatitis B virus [HBV] entry inhibitor 4B10) with a glycosylphosphatidylinositol (GPI) attachment signal. As expected, GPI-anchored inhibitors were efficiently expressed on the plasma membrane of transduced TZM-bl cells and primarily directed to the lipid raft site without interfering with the expression of CD4, CCR5, and CXCR4. GPI-anchored 2P23 (GPI-2P23) completely protected TZM-bl cells from infections of divergent HIV-1, HIV-2, and SIV isolates as well as a panel of enfuvirtide (T20)-resistant mutants. GPI-2P23 also rendered the cells resistant to viral envelope-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission. Moreover, GPI-2P23-modified human CD4+ T cells (CEMss-CCR5) fully blocked both R5- and X4-tropic HIV-1 isolates and displayed a robust survival advantage over unmodified cells during HIV-1 infection. In contrast, it was found that GPI-anchored C34 was much less effective in inhibiting HIV-2, SIV, and T20-resistant HIV-1 mutants. Therefore, our studies have demonstrated that genetically anchoring a short-peptide fusion inhibitor to the target cell membrane is a viable strategy for gene therapy of both HIV-1 and HIV-2 infections.IMPORTANCE Antiretroviral therapy with multiple drugs in combination can efficiently suppress HIV replication and dramatically reduce the morbidity and mortality associated with AIDS-related illness; however, antiretroviral therapy cannot eradiate the HIV reservoirs, and lifelong treatment is required, which often results in cumulative toxicities, drug resistance, and a multitude of complications, thus necessitating the development of sterilizing-cure or functional-cure strategies. Here, we report that genetically anchoring the short-peptide fusion inhibitor 2P23 to the cell membrane can fully prevent infections from divergent HIV-1, HIV-2, and SIV isolates as well as a panel of enfuvirtide-resistant mutants. Membrane-bound 2P23 also effectively blocks HIV-1 Env-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission, renders CD4+ T cells nonpermissive to infection, and confers a robust survival advantage over unmodified cells. Thus, our studies verify a powerful strategy to generate resistant cells for gene therapy of both the HIV-1 and HIV-2 infections.

Protozoan Parasites Glycosylphosphatidylinositol Anchors: Structures, Functions and Trends for Drug Discovery.

BACKGROUND: Glycosylphosphatidylinositol (GPI) anchors are molecules located on cell membranes of all eukaryotic organisms. Proteins, enzymes, and other macromolecules which are anchored by GPIs are essential elements for interaction between cells, and are widely used by protozoan parasites when compared to higher eukaryotes. METHODS: More than one hundred references were collected to obtain broad information about mammalian and protozoan parasites' GPI structures, biosynthetic pathways, functions and attempts to use these molecules as drug targets against parasitic diseases. Differences between GPI among species were compared and highlighted. Strategies for drug discovery and development against protozoan GPI anchors were discussed based on what has been reported on literature. RESULTS: There are many evidences that GPI anchors are crucial for parasite's survival and interaction with hosts' cells. Despite all GPI anchors contain a conserved glycan core, they present variations regarding structural features and biosynthetic pathways between organisms, which could offer adequate selectivity to validate GPI anchors as drug targets. Discussion was developed with focus on the following parasites: Trypanosoma brucei, Trypanosoma cruzi, Leishmania, Plasmodium falciparum and Toxoplasma gondii, causative agents of tropical neglected diseases. CONCLUSION: This review debates the main variances between parasitic and mammalian GPI anchor biosynthesis and structures, as well as clues for strategic development for new anti-parasitic therapies based on GPI anchors.

Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins.

Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.

Glycosyl Phosphatidylinositol-Anchored C34 Peptide Derived From Human Immunodeficiency Virus Type 1 Gp41 Is a Potent Entry Inhibitor.

Lipid rafts of the plasma membrane have been shown to be gateways for HIV-1 budding and entry. In nature, many glycosyl-phosphatidylinositol (GPI) anchored proteins are targeted to the lipid rafts. In the present study we constructed two fusion genes, in which C34 peptide or AVF peptide control was genetically linked with a GPI-attachment signal. Recombinant lentiviruses expressing the fusion genes were used to transduce TZM.bl and CEMss-CCR5 cells. Here, we show that with a GPI attachment signal both C34 and AVF are targeted to the lipid rafts through a GPI anchor. GPI-C34, but not GPI-AVF, in transduced TZM.bl cells efficiently blocks the infection of diverse HIV-1 strains of various subtypes. GPI-C34-transduced CEMss-CCR5 cells are totally resistant to HIV-1 infection. Importantly, maximum percentage of inhibition (MPI) by GPI-C34 is comparable to, if not higher than, a very high concentration of soluble C34. Potent blocking by GPI-C34 is likely due to its high local concentration, which allows GPI-C34 to efficiently bind to the prehairpin intermediate and prevent its transition to six helical bundle, thereby interfering with membrane fusion and virus entry. Our findings should have important implications in GPI-anchor-based therapy against HIV-1.

Synthesis of non-hydrolysable mimics of glycosylphosphatidylinositol (GPI) anchors.

Synthesis of first generation non-hydrolysable C-phosphonate GPI analogs, viz., 6-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol-1-O-(sn-3,4-bis(palmitoyloxy)butyl-1-phosphonate) and 6-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol-1-O-(sn-2,3-bis(palmitoyloxy)propyl-1-phosphonate) 23b, is reported. The target compounds were synthesized by the coupling of α-pseudodisaccharide 21 with phosphonic acids 18a and 18b respectively in quantitative yield followed by de-protection. These synthetic C-phosphonate GPI-probes were resistant to phosphatidylinositol specific phospholipase C (PI-PLC) and also showed moderate inhibition of the enzyme activity.

Recombinant TIMP-1-GPI inhibits growth of fibrosarcoma and enhances tumor sensitivity to doxorubicin.

Fibrosarcomas show a high incidence of recurrence and general resistance to apoptosis. Limiting tumor regrowth and increasing their sensitivity to chemotherapy and apoptosis represent key issues in developing more effective treatments of these tumors. Tissue inhibitor of metalloproteinase 1 (TIMP-1) broadly blocks matrix metalloproteinase (MMP) activity and can moderate tumor growth and metastasis. We previously described generation of a recombinant fusion protein linking TIMP-1 to glycosylphophatidylinositol (GPI) anchor (TIMP-1-GPI) that efficiently directs the inhibitor to cell surfaces. In the present report, we examined the effect of TIMP-1-GPI treatment on fibrosarcoma biology. Exogenously applied TIMP-1-GPI efficiently incorporated into surface membranes of human HT1080 fibrosarcoma cells. It inhibited their proliferation, migration, suppressed cancer cell clone formation, and enhanced apoptosis. Doxorubicin, the standard chemotherapeutic drug for fibrosarcoma, was tested alone or in combination with TIMP-1-GPI. In parallel, the influence of treatment on HT1080 side population cells (exhibiting tumor stem cell-like characteristics) was investigated using Hoechst 33342 staining. The sequential combination of TIMP-1-GPI and doxorubicin showed more than additive effects on apoptosis, while TIMP-1-GPI treatment alone effectively decreased "stem-cell like" side population cells of HT1080. TIMP-1-GPI treatment was validated using HT1080 fibrosarcoma murine xenografts. Growing tumors treated with repeated local injections of TIMP-1-GPI showed dramatically inhibited fibrosarcoma growth and reduced angiogenesis. Intraoperative peritumoral application of GPI-anchored TIMP-1 as an adjuvant to surgery may help maintain tumor control by targeting microscopic residual fibrosarcoma cells and increasing their sensitivity to chemotherapy.

Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria.

The mechanism of bone marrow failure (BMF) in paroxysmal nocturnal hemoglobinuria (PNH) is not yet known. Because in PNH the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI) is disrupted in hematopoietic stem and progenitor cells by a somatic mutation in the PIG-A gene, BMF might result from an autoimmune attack, whereby T cells target GPI in normal cells, whereas PIG-A mutant GPI-negative cells are spared. In a deliberate test of this hypothesis, we have demonstrated in PNH patients the presence of CD8(+) T cells reactive against antigen-presenting cells (APCs) loaded with GPI. These T cells were significantly more abundant in PNH patients than in healthy controls; their reactivity depended on CD1d expression and they increased upon coculture with CD1d-expressing, GPI-positive APCs. In GPI-specific T cells captured by CD1d dimer technology, we identified, through global T-cell receptor α (TCRα) analysis, an invariant TCRVα21 sequence, which was then found at frequencies higher than background in the TCR repertoire of 6 of 11 PNH patients. Thus, a novel, autoreactive, CD1d-restricted, GPI-specific T-cell population, enriched in an invariant TCRα chain, is expanded in PNH patients and may be responsible for BMF in PNH.

Trypomastigotes and amastigotes of Trypanosoma cruzi induce apoptosis and STAT3 activation in cardiomyocytes in vitro.

The haemoflagellate Trypanosoma cruzi is the causative agent of Chagas' disease that occurs in approximately 8 million people in Latin America. Patients infected with T. cruzi frequently suffer of cardiomegaly and may die of myocardial failure. Here we show that T. cruzi trypomastigotes (extracellular form) increased in vitro apoptosis of rat cardiomyocytes. Additionally, we demonstrated that amastigotes (intracellular form), for which a method for purification was established, were also able to induce cardiomyocyte apoptosis. Increase of apoptosis was associated with up-regulation of the apoptotic gene bax by trypomastigotes, while expression of the anti-apoptotic gene bcl-2 was down-regulated by amastigotes. The transcription factor STAT3 but not STAT1 was activated in cardiomyocytes by trypomastigotes. In addition, tlr7 gene expression was up-regulated in cardiomyocytes incubated with trypomastigotes, suggesting that this Toll-like receptor is involved in the intracellular recognition after host cell invasion by T. cruzi. Glycosylphosphatidylinositols purified from trypomastigotes did not induce cardiomyocyte apoptosis and STAT activation but down-regulated tlr7 gene expression. In conclusion, cardiomyopathy observed in Chagas' disease might be in part due to apoptosis of cardiomyocytes induced directly by the parasite.

Synthetic inositol phosphoglycans related to GPI lack insulin-mimetic activity.

Insulin signaling has been suggested, at least in part, to be affected by an insulin-mimetic species of low molecular weight. These inositol phosphoglycans (IPGs) are generated upon growth hormone/cytokine stimulation and control the activity of a multitude of insulin effector enzymes. The minimal structural requirements of IPGs for insulin-mimetic action have been debated. Two types of IPGs were suggested, and the IPG-A type resembles the core glycan of glycosylphosphatidylinositol (GPI)-anchors. In fact, purified GPI-anchors of lower eukaryotic origin have been shown to influence glucose homeostasis. To elucidate active IPGs, a collection of synthetic IPGs designed on the basis of previous reports of activity were tested for their insulin-mimetic activity. In vitro and ex vivo assays in rodent adipose tissue as well as in vivo analyses in mice were employed to test the synthetic IPGs. None of the IPGs we tested mimic insulin actions as determined by PKB/Akt phosphorylation and quantification of glucose transport and lipogenesis. Furthermore, none of the IPGs had any effect in in vivo insulin tolerance assays. In stark contrast to previous claims, we conclude that neither of the compounds tested is insulin-mimetic.

DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

MAPK-activated protein kinase 2 differentially regulates plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

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

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

Disruption of JNK2 decreases the cytokine response to Plasmodium falciparum glycosylphosphatidylinositol in vitro and confers protection in a cerebral malaria model.

Host inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in the pathophysiology of severe malaria. However, relatively little is known about the signal transduction pathways involved in pfGPI-stimulated inflammatory response and its potential contribution to severe malaria syndromes. In this study, we investigated the role of MAPK activation in pfGPI-induced cytokine secretion and examined the role of selected MAPKs in a model of cerebral malaria in vivo. We demonstrate that ERK1/2, JNK, p38, c-Jun, and activating transcription factor-2 became phosphorylated in pfGPI-stimulated macrophages. A JNK inhibitor (1,9-pyrazoloanthrone) inhibited pfGPI-induced phosphorylation of JNK, c-Jun, and activating transcription factor-2 and significantly decreased pfGPI-induced TNF-alpha secretion. pfGPI-stimulated JNK and c-Jun phosphorylation was absent in Jnk2(-/-) macrophages but unchanged in Jnk1(-/-) and Jnk3(-/-) macrophages compared with wild-type macrophages. Jnk2(-/-) macrophages secreted significantly less TNF-alpha in response to pfGPI than macrophages from Jnk1(-/-), Jnk3(-/-), and wild-type counterparts. Furthermore, we demonstrate a role for JNK2 in mediating inflammatory responses and severe malaria in vivo. In contrast to wild-type or Jnk1(-/-) mice, Jnk2(-/-) mice had lower levels of TNF-alpha in vivo and exhibited significantly higher survival rates when challenged with Plasmodium berghei ANKA. These results provide direct evidence that pfGPI induces TNF-alpha secretion through activation of MAPK pathways, including JNK2. These results suggest that JNK2 is a potential target for therapeutic interventions in severe malaria.

Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface.

Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.

Ethanolaminephosphate side chain added to glycosylphosphatidylinositol (GPI) anchor by mcd4p is required for ceramide remodeling and forward transport of GPI proteins from endoplasmic reticulum to Golgi.

Glycosylphosphatidylinositol (GPI) anchors of mammals as well as yeast contain ethanolaminephosphate side chains on the alpha1-4- and the alpha1-6-linked mannoses of the anchor core structure (protein-CO-NH-(CH(2))(2)-PO(4)-6Manalpha1-2Manalpha1-6Manalpha1-4GlcNH(2)-inositol-PO(4)-lipid). In yeast, the ethanolaminephosphate on the alpha1-4-linked mannose is added during the biosynthesis of the GPI lipid by Mcd4p. MCD4 is essential because Gpi10p, the mannosyltransferase adding the subsequent alpha1-2-linked mannose, requires substrates with an ethanolaminephosphate on the alpha1-4-linked mannose. The Gpi10p ortholog of Trypanosoma brucei has no such requirement. Here we show that the overexpression of this ortholog rescues mcd4Delta cells. Phenotypic analysis of the rescued mcd4Delta cells leads to the conclusion that the ethanolaminephosphate on the alpha1-4-linked mannose, beyond being an essential determinant for Gpi10p, is necessary for an efficient recognition of GPI lipids and GPI proteins by the GPI transamidase for the efficient transport of GPI-anchored proteins from the endoplasmic reticulum to Golgi and for the physiological incorporation of ceramides into GPI anchors by lipid remodeling. Furthermore, mcd4Delta cells have a marked defect in axial bud site selection, whereas this process is normal in gpi7Delta and gpi1. This also suggests that axial bud site selection specifically depends on the presence of the ethanolaminephosphate on the alpha1-4-linked mannose.

Effect of AgK114 on picryl chloride-induced chronic contact hypersensitivity responses.

BACKGROUND: Mouse AgK114 (a glycosylphosphatidylinositol (GPI) anchored membrane-associated protein) expression is found in somatotrophs of the pituitary gland in correlation with the expression of growth hormone. In this study, the effects of AgK114 on the systemic immune response were examined in contact hypersensitivity (CHS) model mice. MATERIALS AND METHODS: AgK114 was intraperitoneally injected into BALB/c mice that were sensitized and challenged with picryl chloride (PiCl). Serum IgE levels and the antigen-specific cytokine production by lymph node (LN) cells were examined. RESULTS: The serum IgE levels in the CHS mice treated with 10 microg/head of AgK114 during the repeated challenge with PiCl were significantly decreased compared with those of the control mice. Moreover, IL-4 production by LN cells in response to 2,4,6-trinitrobenzene-sulfonic acid sodium salt-treated splenocytes was decreased in the AgK114-treated CHS mice compared with that of the control mice. CONCLUSION: Our results suggest that systemic administration of AgK114 exerted immunoregulatory functions on the allergic responses, resulting in the inhibition of IgE production.

mAgK114 suppresses lymphocyte infiltration into epidermis in the picryl chloride-induced atopic dermatitis-like skin lesions of NC/Nga mice.

BACKGROUND: We have previously shown that when mouse AgK114 (mAgK114, a glycosylphosphatidylinositol anchored membrane-associated protein) is applied to the wound area, the inflammatory responses in the early recovery phase of damaged tissue are enhanced and wound closure is accelerated. This suggests that mAgK114 has an important effect on skin wound repairing. MATERIALS AND METHODS: Whether mAgK114 supresses the development, in NC/Nga mice, of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (picryl chloride, PiCl) was examined under specific pathogen-free conditions. RESULTS: Histopathologically, the application of mAgK114-ointment to the PiCl-treated NC/Nga mice remarkably suppressed severe lymphocytic infiltration into the epidermis, although total skin severity scores, histological changes in hypertrophy, erosion and infiltration of inflammatory cells into the corium and subcutaneous tissues were comparable between the mAgK114-treated group of mice and the control group. CONCLUSION: Our results suggest that mAgK114 would be beneficial for the treatment of atopic dermatitis by suppressing severe lymphocytic infiltration into the epidermis.

Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness.

It has been suggested that compounds affecting glycosylphosphatidylinositol (GPI) biosynthesis in bloodstream form Trypanosoma brucei should be trypanocidal. We describe cell-permeable analogues of a GPI intermediate that are toxic to this parasite but not to human cells. These analogues are metabolized by the T. brucei GPI pathway, but not by the human pathway. Closely related nonmetabolizable analogues have no trypanocidal activity. This represents the first direct chemical validation of the GPI biosynthetic pathway as a drug target against African human sleeping sickness. The results should stimulate further inhibitor design and synthesis and encourage the search for inhibitors in natural product and synthetic compound libraries.