Pro-inflammatory cytokines induced by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum contribute to malaria pathogenesis and hence, the naturally acquired anti-GPI antibody thought to provide protection against severe malaria (SM) by neutralizing the stimulatory activity of GPIs. In previous studies, the anti-GPI antibody levels increased with age in parallel with the development of acquired immunity, and high levels of anti-GPI antibodies were associated with mild malaria (MM) cases. In the present study, the relationship between the levels of pro-inflammatory cytokines and anti-GPI IgG antibody responses, parasitemia, and the clinical outcomes were evaluated in SM and mild malaria (MM) patients. Sera from a total of 110 SM and 72 MM cases after excluding of ineligible patients were analyzed for the levels of anti-GPI antibodies, IgG subclasses, and cytokine responses by ELISA. While the total anti-GPI antibody levels were similar in overall SM and MM groups, they were significantly higher in surviving SM patients than in fatal SM cases. In the case of cytokines, the TNF-α and IL-6 levels were significantly higher in SM compared to MM, whereas the IL-10 levels were similar in both groups. The data presented here demonstrate that high levels of the circulatory pro-inflammatory, TNF-α, and IL-6, are indicators of malaria severity, whereas anti-inflammatory cytokine IL-10 level does not differentiate SM and MM cases. Further, among SM patients, relatively low levels of anti-GPI antibodies are indicators of fatal outcomes compared to survivors, suggesting that anti-GPI antibodies provide some level of protection against SM fatality.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

Neosporosis due to Neospora caninum causes abortions in farm animals such as cattle. No treatment and vaccine exist to fight this disease, responsible for considerable economic losses. It is thus important to better understand the immune responses occurring during the pathogenesis to control them in a global strategy against the parasite. In this context, we studied the roles of N. caninum glycosylphosphatidylinositols (GPIs), glycolipids defined as toxins in the related parasite Plasmodium falciparum. We demonstrated for the first time that GPIs could be excreted in the supernatant of N. caninum culture and trigger cell signalling through the Toll-like receptors 2 and 4. In addition, antibodies specific to N. caninum GPIs were detected in the serum of infected mice. As shown for other protozoan diseases, they could play a role in neutralizing GPIs. N. caninum GPIs were able to induce the production of tumour necrosis factor-α, interleukin(IL)-1β and IL-12 cytokines by murine macrophages and dendritic cells. Furthermore, GPIs significantly reduced expression of major histocompatibility complex (MHC) molecules of class I on murine dendritic cells. In contrast to murine cells, bovine blood mononuclear cells produced increased levels of IFN-γ and IL-10, but reduced levels of IL-12p40 in response to GPIs. On these bovine cells, GPI had the tendency to up-regulate MHC class I, but to down-regulate MHC class II. Altogether, these results suggest that N. caninum GPIs might differentially participate in the responses of antigen presenting cells induced by the whole parasite in mouse models of neosporosis and in the natural cattle host.

Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane.

Dolichol-phosphate-mannose (DPM) synthase generates mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation. In Saccharomyces cerevisiae, this enzyme is encoded by DPM1. We reported previously that mammalian DPM synthase contains catalytic DPM1 and regulatory DPM2 subunits, and that DPM1 requires DPM2 for its stable expression in the endoplasmic reticulum. Here we report that human DPM synthase consists of three subunits. The third subunit, DPM3, comprises 92 amino acids associated with DPM1 via its C-terminal domain and with DPM2 via its N-terminal portion. The stability of DPM3 was dependent upon DPM2. However, overexpression of DPM3 in Lec15 cells, a null mutant of DPM2, restored the biosynthesis of DPM with an increase in DPM1, indicating that DPM3 directly stabilized DPM1. Therefore, DPM2 stabilizes DPM3 and DPM3 stabilizes DPM1. DPM synthase activity was 10 times higher in the presence of DPM2, indicating that DPM2 also plays a role in the enzymatic reaction. Schizosaccharomyces pombe has proteins that resemble three human subunits; S.pombe DPM3 restored biosynthesis of DPM in Lec15 cells, indicating its orthologous relationship to human DPM3.

Insulin regulation of energy metabolism is complex and involves numerous signaling cascades. Insulin has been suggested to stimulate a phospholipase that cleaves glycosylphosphatidylinositols resulting in the generation of an inositol glycan that serves as an insulin mediator. To determine if glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) may play a role in glucose metabolism, we examined the effect of overexpressing GPI-PLD using adenovirus-mediated gene transfer in C57BL/6 mice. Overexpressing GPI-PLD was associated with a decrease in fasting glucose as well as an improvement in glucose tolerance as determined by an intraperitoneal glucose tolerance test. This effect to improve glucose tolerance does not result from an increase in insulin sensitivity, as overexpressing GPI-PLD does not alter the response to insulin. In contrast, the insulin response during the glucose tolerance test in GPI-PLD-overexpressing mice was increased. Overexpressing GPI-PLD in an insulinoma cell line enhanced glucose-stimulated insulin secretion, suggesting that enhanced insulin secretion in vivo may have contributed to the improved glucose tolerance.

Biallelic pathogenic variants in the genes encoding the dolichol-phosphate mannose synthase subunits (DPM) which produce mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation, are rare causes of congenital disorders of glycosylation. Pathogenic variants in DPM1 and DPM2 are associated with muscle-eye-brain (MEB) disease, whereas DPM3 variants have mostly been reported in patients with isolated muscle disease-dystroglycanopathy. Thus far, only one affected individual with compound heterozygous DPM3 variants presenting with myopathy, mild intellectual disability, seizures, and nonspecific white matter abnormalities (WMA) around the lateral ventricles has been described. Here we present five affected individuals from four unrelated families with global developmental delay/intellectual disability ranging from mild to severe, microcephaly, seizures, WMA, muscle weakness and variable cardiomyopathy. Exome sequencing of the probands revealed an ultra-rare homozygous pathogenic missense DPM3 variant NM_018973.4:c.221A>G, p.(Tyr74Cys) which segregated with the phenotype in all families. Haplotype analysis indicated that the variant arose independently in three families. Functional analysis did not reveal any alteration in the N-glycosylation pathway caused by the variant; however, this does not exclude its pathogenicity in the function of the DPM complex and related cellular pathways. This report provides supporting evidence that, besides DPM1 and DPM2, defects in DPM3 can also lead to a muscle and brain phenotype.

Attachment of glycosylphosphatidylinositols (GPIs) to the C-termini of proteins is one of the most common posttranslational modifications in eukaryotic cells. GPI8/PIG-K is the catalytic subunit of the GPI transamidase complex catalyzing the transfer en bloc GPI to proteins. In this study, a T-DNA insertional mutant of rice with temperature-dependent drooping and fragile (df) shoots phenotype was isolated. The insertion site of the T-DNA fragment was 879 bp downstream of the stop codon of the OsGPI8 gene, which caused introns retention in the gene transcripts, especially at higher temperatures. A complementation test confirmed that this change in the OsGPI8 transcripts was responsible for the mutant phenotype. Compared to control plants, internodes of the df mutant showed a thinner shell with a reduced cell number in the transverse direction, and an inhomogeneous secondary wall layer in bundle sheath cells, while many sclerenchyma cells at the tops of the main veins of df leaves were shrunken and their walls were thinner. The df plants also displayed a major reduction in cellulose and lignin content in both culms and leaves. Our data indicate that GPI anchor proteins play important roles in biosynthesis and accumulation of cell wall material, cell shape, and cell division in rice.

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.

Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1-coding for a phospholipase required for the first step of the GPI lipid remodeling-was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the Δper1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the Δper1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway.

Glycosylphosphatidylinositols (GPIs) from several protozoan parasites are thought to elicit a detrimental stimulation of the host innate immune system aside their main function to anchor surface proteins. Here we analyzed the GPI biosynthesis of an avirulent Toxoplasma gondii type 2 strain (PTG) by metabolic radioactive labeling. We determined the biological function of individual GPI species in the PTG strain in comparison with previously characterized GPI-anchors of a virulent strain (RH). The GPI intermediates of both strains were structurally similar, however the abundance of two of six GPI intermediates was significantly reduced in the PTG strain. The side-by-side comparison of GPI-anchor content revealed that the PTG strain had only ∼ 34% of the protein-free GPIs as well as ∼ 70% of the GPI-anchored proteins with significantly lower rates of protein N-glycosylation compared to the RH strain. All mature GPIs from both strains induced comparable secretion levels of TNF-α and IL-12p40, and initiated TLR4/MyD88-dependent NF-κBp65 activation in macrophages. Taken together, these results demonstrate that PTG and RH strains differ in their GPI biosynthesis and possess significantly different GPI-anchor content, while individual GPI species of both strains induce similar biological functions in macrophages.

Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal blood disorder that manifests with hemolytic anemia, thrombosis, and peripheral blood cytopenias. The disease is caused by the deficiency of two glycosylphosphatidylinositols (GPI)-anchored proteins (CD55 and CD59) in the hemopoietic stem cells. The deficiency of GPI-anchored proteins has been associated with the somatic mutations in phosphatidylinositol glycan class A (PIGA). However, the mutations that do not cause PNH is associated with the multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2). To best of our knowledge, no computational study has been performed to explore at an atomistic level the impact of PIGA missense mutations on the structure and dynamics of the protein. Therefore, we focused our study to provide molecular insights into the changes in protein structural dynamics upon mutation. In the initial step, screening for the most pathogenic mutations from the pool of publicly available mutations was performed. Further, to get a better understanding, pathogenic mutations were mapped to the modeled structure and the resulting protein was subjected to 100 ns molecular dynamics simulation. The residues close to C- and N-terminal regions of the protein were found to exhibit greater flexibility upon mutation. Our study suggests that four mutations are highly effective in altering the structural conformation and stability of the PIGA protein. Among them, mutant G48D was found to alter protein's structural dynamics to the greatest extent, both on a local and a global scale.

Abnormalities of posttranslational protein modifications (PTMs) have recently been implicated in the pathophysiology of schizophrenia. Glycosylphosphatidylinositols (GPIs) are a class of complex glycolipids, which anchor surface proteins and glycoproteins to the cell membrane. GPI attachment to proteins represents one of the most common PTMs and GPI-associated proteins (GPI-APs) facilitate many cell surface processes, including synapse development and maintenance. Mutations in the GPI processing pathway are associated with intellectual disability, emphasizing the potential role of GPI-APs in cognition and schizophrenia-associated cognitive dysfunction. As initial endoplasmic reticulum (ER)-associated protein processing is essential for GPI-AP function, we measured protein expression of molecules involved in attachment (GPAA1), modification (PGAP1), and ER export (Tmp21) of GPI-APs, in homogenates and in an ER enriched fraction derived from dorsolateral prefrontal cortex (DLPFC) of 15 matched pairs of schizophrenia and comparison subjects. In total homogenate we found a significant decrease in transmembrane protein 21 (Tmp21) and in the ER-enriched fraction we found reduced expression of post-GPI attachment protein (PGAP1). PGAP1 modifies GPI-anchors through inositol deacylation, allowing it to be recognized by Tmp21. Tmp21 is a component of the p24 complex that recognizes GPI-anchored proteins, senses the status of the GPI-anchor, and regulates incorporation into COPII vesicles for export to the Golgi apparatus. Together, these proteins are the molecular mechanisms underlying GPI-AP quality control and ER export. To investigate the potential consequences of a deficit in export and/or quality control, we measured cell membrane-associated expression of known GPI-APs that have been previously implicated in schizophrenia, including GPC1, NCAM, MDGA2, and EPHA1, using Triton X-114 phase separation. Additionally, we tested the sensitivity of those candidate proteins to phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves GPI from GPI-APs. While we did not observe a difference in the amount of these GPI-APs in Triton X-114 phase separated membrane fractions, we found decreased NCAM and GPC1 within the PI-PLC sensitive fraction. These findings suggest dysregulation of ER-associated GPI-AP protein processing, with impacts on post-translational modifications of proteins previously implicated in schizophrenia such as NCAM and GPC1. These findings provide evidence for a deficit in ER protein processing pathways in this illness.