Expression Of GPI-anchored Proteins Research Articles

Towards a thorough understanding of mammalian glycosylphosphatidylinositol-anchored protein biosynthesis.

Glycosylphosphatidylinositols (GPIs) are glycolipids found ubiquitously in eukaryotes. They consist of a glycan and an inositol phospholipid, and act as membrane anchors of many cell-surface proteins by covalently linking to their C-termini. GPIs also exist as unlinked, free glycolipids on the cell surface. In human cells, at least 160 proteins with various functions are GPI-anchored proteins. Because the attachment of GPI is required for the cell-surface expression of GPI-anchored proteins, a thorough knowledge of the molecular basis of mammalian GPI-anchored protein biosynthesis is important for understanding the basic biochemistry and biology of GPI-anchored proteins and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-anchored proteins and then examine new findings made since 2020.

Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 2 Caused by a Novel PIGA Variant Not Associated with a Skewed X-Inactivation Pattern

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Damaging novel mutations in PIGN cause developmental epileptic-dyskinetic encephalopathy: a case report

BackgroundMutations in PIGN, resulting in a glycosylphosphatidylinositol (GPI) anchor deficiency, typically leads to multiple congenital anomalies-hypotonia-seizures syndrome. However, the link between PIGN and epilepsy or paroxysmal non-kinesigenic dyskinesia (PNKD) is not well-described. This study reported a patient with PIGN mutation leading to developmental and epileptic encephalopathy and PNKD, to expand upon the genotype–phenotype correlation of PIGN.Case presentationDuring the first 10 days of life, a girl exhibited paroxysmal staring episodes with durations that ranged from several minutes to hours. These episodes occurred 2–5 times daily and always occurred during wakefulness. Ictal electroencephalography revealed no abnormalities, and PNKD was diagnosed. The patient also exhibited severely delayed psychomotor development and generalized seizures at the age of 4 months. Results of brain magnetic resonance imaging and metabolic screenings were normal, but trio-based whole-exome sequencing identified two novel compound heterozygous PIGN mutations (NM_176787; c.163C > T [p.R55 > X] and c.283C > T [p.R95W]). Flow cytometry analysis of the patient’s granulocytes revealed dramatically reduced expression of GPI-anchored proteins. This indicated that the mutations compromised GPI functions. The patient got seizure-free for 1 year, and her dyskinesia episodes reduced significantly (1–2 times/month) after treatment with levetiracetam (600 mg/day) and clonazepam (1.5 mg/day). No progress was observed with respect to psychomotor development; however, no craniofacial dysmorphic features, cleft lip/palate, brachytelephalangy with nail hypoplasia, and internal malformations have been observed until now (6 years of age).ConclusionThis is the first study to document developmental and epileptic encephalopathy with PNKD in a human with PIGN mutations. This report expanded our understanding of the genotype–phenotype correlation of PIGN, and PIGN may be considered a potentially relevant gene when investigating cases of epilepsy or PNKD.

Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive β-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support β-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

Effects of Eculizumab Treatment on the Quality of Life in Patients with Paroxysmal Nocturnal Hemoglobinuria Treated in Japan

Introduction: PNH is a hematopoietic stem cell disorder with the expansion of a PIGA mutated clone(s). PNH clones are deficient in the membrane expression of GPI-anchored proteins including CD55 and CD59, which leave erythrocytes susceptible to complement attack, resulting in intravascular hemolysis. Many symptoms experienced by PNH patients such as fatigue, dyspnea, and stomachache have been found to be associated with serum free hemoglobin (Hb) released from erythrocytes by intravascular hemolysis (JAMA. 2005;293(13):1653-1662). Eculizumab (Soliris®), a humanized monoclonal antibody against Complement Factor C5, has shown dramatic effect in blocking intravascular hemolysis. Previous reports have revealed that eculizumab has not only improved anemia but also patients' quality of life (QoL) (N Engl J Med. 2006; 355: 1233-1243, Blood. 2008; 111: 1840-1847, Int J Hematol. 2011; 93: 36-46). However, factors which may be associated with improvement of QoL have yet to be fully understood. To clarify the clinical significance of QoL improvement in PNH patients treated with eculizumab, we analyzed the change in QoL in 54 PNH patients treated with eculizumab based on the data from the post-marketing surveillance (PMS) of eculizumab in Japan.Methods: All PNH patients treated with eculizumab have been registered in PMS based on the revised Pharmaceutical Affairs Law in Japan since June 2010. Of 491 patients registered as at March 2017, QoL questionnaires were obtained from 225 patients. 54 patients who received both baseline and one-year assessment of QoL were analyzed in this study. QoL was assessed using Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. Summary statistics were obtained from the figures of QoL scoring systems and the lab values, and paired Student's t-test was used for the change of the values in one year from the baseline. Canonical correlation analysis was performed among QoL variates (consisted of 16 items) and lab data variates (LDH, Hb, and platelets).Results: There was no bias in the baseline characteristics between the total (491) and the analyzed (54) patients. One-year administration of eculizumab improved serum LDH level (mean: from 1435.3 to 289.5 U/L), Hb level (mean: from 8.22 to 9.46 g/dL), and QoL in most of their items in comparison with the baseline. In particular, significant improvement of EORTC QLQ-C30 was shown in the items of fatigue, dyspnea, physical function, and global health status. Canonical correlation analysis revealed a high correlation between QoL variates and lab data variates (Canonical R=0.8393, p=0.004). Of the lab data variates, LDH (Canonical correlation coefficient: -0.91) and Hb (0.76) showed strong correlations with QoL improvement as expected. Interestingly, QoL improvement was independent of patient's baseline characteristics such as the coexistence of aplastic anemia and/or myelodysplastic syndrome and the degree of lactate dehydrogenase (LDH) (Fig. 1, 2).Conclusion: In this study, QoL improvement following treatment with eculizumab was statistically strongly correlated to the degree of hemolysis, which is consistent with the previous reports. Herein we also demonstrated, for the first time, that the degree of improvement was independent of the baseline serum LDH level before eculizumab treatment and of the presence or absence of bone marrow failure. Those PNH patients without eculizumab treatment due to mild hemolysis might benefit from eculizumab treatment regardless of the presence of bone marrow failure syndromes. [Display omitted] DisclosuresUeda:Alexion Pharma GK: Honoraria, Research Funding. Obara:Alexion Pharma GK: Honoraria, Research Funding. Yonemura:Alexion Pharma GK: Honoraria, Research Funding. Noji:Alexion Pharma GK: Honoraria, Research Funding. Matsuda:Alexion Pharma G.K.: Employment. Akiyama:Alexion Pharma G.K.: Employment. Chiba:Nippon Shinyaku: Honoraria, Research Funding. Kawaguchi:Alexion Pharma G.K.: Honoraria; Novartis,Pfizer: Consultancy, Honoraria. Shichishima:Alexion Pharma GK: Honoraria. Okamoto:Bristol-Myers K.K: Honoraria; Pfizer Japan: Speakers Bureau; Alexion Pharma G.K.: Honoraria; Astellas: Honoraria; Pfizer: Research Funding; Chugai Pharmaceutical Co.: Research Funding; Novartis: Research Funding; Bristol-Myers K.K: Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Kanakura:Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Nippon Shinyaku: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Toyama Chemical: Research Funding. Ninomiya:Alexion Pharma GK: Honoraria.

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number–neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

SELENOI is important for maintenance of phosphatidylethanolamine and glycosylphosphatidylinositol anchor protein during T cell activation and immune response

Abstract Selenoproteins are important for both innate and adaptive immune responses. As one of the selenoprotein members, SELENOI is characterized by its ethanolamine phosphotransferase activity in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis and PE is also the donor of phosphoethanolamine moiety in glycosylphosphatidylinositol anchor protein (GPI-AP). To explore how SELENOI is involved in the immune response, we generated three mouse models with different SELENOI expression levels, including defined selenium diets, doxycycline inducible SELENOI knockdown (KD) and T cell specific SELENOI knockout (KO) mice models. We analyzed both the quantity of PE species on activated CD3 T cell membrane by LCMS and GPI-AP expression on activated T cell surface by FACS in all these three models. The SELENOI KO T cells exhibit a lower level of PE species on T cell membrane compared to WT T cells, and reduced expression of GPI-anchored proteins including CD52, CD73, CD48, CD109 and CD44 on cell surface upon activation as well. We also detected the same pattern of reduced PE species and GPI-anchor proteins on defined selenium diets and doxycycline-inducible SELENOI KD mouse models. Furthermore, the SELENOI KO T cells exhibited a reduced proliferation rate upon CD3/CD28 co-stimulation. Vaccination with LCMV peptides in SELENOI deficient mice results in less CD8+ and CD4+ LCMV specific T cells and reduced IFN-γ production, indicating an impaired immune response when SELENOI is absent. These results indicate that SELENOI is crucial for both PE and GPI-AP synthesis during T cell activation and proliferation due to the importance of PE during the activation process.

Homozygous splice-variants in human ARV1 cause GPI-anchor synthesis deficiency

BackgroundMutations in the ARV1 Homolog, Fatty Acid Homeostasis Modulator (ARV1), have recently been described in association with early infantile epileptic encephalopathy 38. Affected individuals presented with epilepsy, ataxia, profound intellectual disability, visual impairment, and central hypotonia. In S. cerevisiae, Arv1 is thought to be involved in sphingolipid metabolism and glycophosphatidylinositol (GPI)-anchor synthesis. The function of ARV1 in human cells, however, has not been elucidated. MethodsMutations were discovered through whole exome sequencing and alternate splicing was validated on the cDNA level. Expression of the variants was determined by qPCR and Western blot. Expression of GPI-anchored proteins on neutrophils and fibroblasts was analyzed by FACS and immunofluorescence microscopy, respectively. ResultsHere we describe seven patients from two unrelated families with biallelic splice mutations in ARV1. The patients presented with early onset epilepsy, global developmental delays, profound hypotonia, delayed speech development, cortical visual impairment, and severe generalized cerebral and cerebellar atrophy. The splice variants resulted in decreased ARV1 expression and significant decreases in GPI-anchored protein on the membranes of neutrophils and fibroblasts, indicating that the loss of ARV1 results in impaired GPI-anchor synthesis. ConclusionLoss of GPI-anchored proteins on our patients' cells confirms that the yeast Arv1 function of GPI-anchor synthesis is conserved in humans. Overlap between the phenotypes in our patients and those reported for other GPI-anchor disorders suggests that ARV1-deficiency is a GPI-anchor synthesis disorder.

Current Status and Optimal Management of Eculizumab Poor-Responders Due to C5 Polymorphisms

(Introduction) Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare but life-threatening stem cell disease caused by expansion of PIGA mutated clone(s). PIGA mutation abolishes the expression of GPI-anchored proteins on the cell surface including CD55 and CD59 which protect red blood cells from complement attack, resulting in complement-mediated destruction of PNH erythrocytes. Eculizumab (Ecu) effectively ameliorates the intravascular hemolysis in PNH patients by blocking complement C5 in the terminal pathway. We previously reported that 3-4 % of the Japanese patients did not respond to Ecu due to C5 polymorphism c.2654G>A (predicting p.Arg885His) and one Argentina patient with similar but different polymorphism c.2653C>T (predicting p.Arg885Cys) (N Engl J Med 370:632, 2014). Since then, we have received various consults regarding poor response cases and requests for analysis from around the world. Here, we summarize the latest series of analyses in C5 polymorphisms and propose optimal management based on these findings. (Method) Once poor response to Ecu, defined as sustained high serum LDH, was suspected with sustained high serum LDH level, peripheral blood samples with clinical data were sent to our institute after obtaining patients' informed consent. DNA was extracted from the samples, and the hot spot of C5 polymorphisms at exon 21 was sequenced by Sanger method. If no polymorphism was identified, all 41 exons of C5 were sequenced. (Results) At the initial publication (2014), 11 cases of c.2654G>A were identified out of 345 PNH patients (3.2%). As of July, 2018, a total of 22 patients were identified among roughly 600 patients treated with Ecu in Japan (3.7%). To determine the distribution of the polymorphism, a DNA panel containing 120 Han Chinese persons were previously screened, and one had the polymorphism. The same C5 polymorphism was newly identified in one among 89 patients treated with Ecu in Korea. A similar polymorphism c.2653C>T (predicting p.Arg885Cys) was also previously identified in an Argentina patient. Another polymorphism c.2653C>A (predicting p.Arg885Ser) was identified in a Dutch patient. Furthermore, a novel mutation c.2422 G>A (predicting p.Val 808 Ile) was found in a poor responder in Israel, and currently under functional analyses. (Discussion) In PNH patients treated with Ecu, serum LDH level usually drops importantly after the first loading dose, and mostly comes to upper limit of normal range after second or third loading dose. If the serum LDH level remains high, poor response should be considered. CH50 is usually not detected in Ecu responsive patients, so monitoring of CH50 level is critical to evaluate the responsiveness to Ecu. Once poor response is suspected, hot spot of exon 21 should initially be sequenced, and then the whole 41 exons of C5 may need to be sequenced. c.2654G>A was found among Han Chinese in our previous study, so it is reasonable that a Korean case had the same polymorphism considering the geography. Surprisingly, a Caucasian case with the same polymorphism has been reported (Blood Advances 1:1254, 2017) in addition to the Dutch case, underlining the importance of poor responsiveness due to polymorphisms (p.Arg885) even outside of Asia. We previously reported that Coversin, a C5 inhibitor derived from tick saliva protein, blocked hemolysis in vitro using the serum from the patients with c.2654G>A, and it was reported that post-transplant thrombotic microangiopathy was successfully treated with Coversin for the patient with the polymorphism (Blood Advances 1:1254, 2017). C5 inhibitors targeting a different epitope or having different mechanisms from Ecu as well as other upcoming complement inhibitors targeting Factor D or C3 are expected to benefit patients with C5 polymorphism and resistance to Ecu. Further analyses and clinical trials may pave the way to a second generation anti-complement drug to treat PNH patients. [Display omitted] DisclosuresUeda:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Muus:Akari Therapeutics: Consultancy. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Nishimura:Chugai Pharmaceuticals: Consultancy, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

A PIGH mutation leading to GPI deficiency is associated with developmental delay and autism.

We identified an individual with a homozygous missense variant (p.Ser103Pro) in a conserved residue of the glycosylphosphatidylinositol (GPI) biosynthesis gene PIGH. This gene encodes an essential component of the phosphatidylinositol N-acetylglucosaminyltransferase complex, in the first step of the biosynthesis of GPI, a glycolipid anchor added to more than one hundred human proteins, several being critical for embryogenesis and neurological functions. The affected individual had hypotonia, moderate developmental delay, and autism. Unlike other reported individuals with GPI deficiency, the proband did not have epilepsy; however, he did have two episodes of febrile seizures. He had normal alkaline phosphatase and no brachytelephalangy. Upon analysis of the surface expression of GPI-anchored proteins on granulocytes, he was demonstrated to have GPI deficiency. This suggests that PIGH mutations may cause a syndrome with developmental delay and autism, but without an epileptic encephalopathy, and should increase the awareness of the potentially deleterious nature of biallelic variants in this gene.

F202. ABNORMAL REMODELING PROCESSING IN NEURAL GPI-APS SECRETORY PATHWAY IN SCHIZOPHRENIA

BackgroundAbnormalities in post translational modifications (PTMs) such as glycosylation have become targets of schizophrenia (SCZ) research and are implicated in the neuropathophysiology of this illness. Glycosylphosphatidylinositol (GPI) attachment to proteins and glycoproteins events; proteins essential to cellular function, including neurotransmitter rreceptors, adhesion molecules, and enzymes, are all modified by GPI. Biosynthesis of GPI -APs occurs in the endoplasmic reticulum (ER). Once GPI-APs are synthesized, they are transported from the ER to the cell surface through the Golgi apparatus. Inositol deacylation of the GPI-APs by is common PTMs. The GPI-anchored proteins (GPI-APs) play an essential role in many biological PGAP1 is required for efficient export from the ER and acts a molecular mechanism for quality control of GPI-APs. The p24 complex binds specifically to GPI-APs and plays a role in their selective trafficking by sensing the status of the GPI anchor and to promote efficient ER exit of remodeled GPI-APs. In this study, we identified abnormalities of proteins associated with the ER exit of GPI-APs in SCZ. To address mechanisms of GPI-APs ER exit, we measured expression of proteins of the GPI-APs ER exit and targeting pathway. We also measured expression of GPI-APs which have been previously implicated in SCZ, including GPC1, NCAM, MDGA2 and EPHA1.MethodsWe assessed the total expression and subcellular localization of proteins involved in ER export processing of GPI-APs from the DLPFC of 15 matched pairs of SCZ and comparison subjects. Specifically, we measured levels of PGAP1 and Tmp21 (p24). Additionally, we performed a Triton X-114 phase separation to distinguish between membrane-associated and cytosolic forms of protein substrates. We confirmed the sensitivity of each target GPI-AP to phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves GPI from GPI-APs.ResultsWe found a significant decrease in p24 in total tissue homogenates and PGAP1 in an ER enriched fraction from subjects with SCZ. We also identified diminished sensitivity of the GPI-APs, GPC1 and NCAM, to PI-PLC treatment in SCZ.DiscussionDecreased PGAP1 in an ER enriched fraction in consistent with reduced inositol deacylation and potential dysfunction as the gatekeeper of GPI-AP ER exit in SCZ. This also suggests that the GPI-anchor is not correctly modified. Decreased p24 levels suggest downregulation of transport between the Golgi and the ER in SCZ. Additionally, we observed unchanged total level of GPI-APs in Triton X-114 phase separation, but a significant decrease in the amount of NCAM and GPC1 that was sensitive to PI-PLC in SCZ. This finding may be consistent with abnormal GPI modification of these two candidate proteins. Together, these findings suggest dysregulation of the GPI-APs remodeling system in SCZ, which may impact the structure of the GPI-anchor for SCZ-relevant proteins like NCAM and GPC1.

Reduced cell surface levels of GPI-linked markers in a new case with PIGG loss of function.

Glycosylphosphatidylinositol (GPI) is a glycolipid that tethers more than 150 different proteins to the cell surface. Aberrations in biosynthesis of GPI anchors cause congenital disorders of glycosylation with clinical features including intellectual disability (ID), seizures, and facial dysmorphism. Here, we present two siblings with ID, cerebellar hypoplasia, cerebellar ataxia, early‐onset seizures, and minor facial dysmorphology. Using exome sequencing, we identified a homozygous nonsense variant (NM_001127178.1:c.1640G>A, p.Trp547*) in the gene Phosphatidylinositol Glycan Anchor Biosynthesis, Class G (PIGG) in both the patients. Variants in several other GPI anchor synthesis genes lead to a reduced expression of GPI‐anchored proteins (GPI‐APs) that can be measured by flow cytometry. No significant differences in GPI‐APs could be detected in patient granulocytes, consistent with recent findings. However, fibroblasts showed a reduced global level of GPI anchors and of specific GPI‐linked markers. These findings suggest that fibroblasts might be more sensitive to pathogenic variants in GPI synthesis pathway and are well suited to screen for GPI‐anchor deficiencies. Based on genetic and functional evidence, we confirm that pathogenic variants in PIGG cause an ID syndrome, and we find that loss of function of PIGG is associated with GPI deficiency.

A novel mutation in PGAP2 gene causes developmental delay, intellectual disability, epilepsy and microcephaly in consanguineous Saudi family

PGAP2 (Post-GPI Attachment to Proteins 2) gene is involved in lipid remodeling steps of Glycosylphosphatidylinositol (GPI)-anchor maturation. At the surface of the cell this gene is required for proper expression of GPI-anchored proteins. Hyperphosphatasia with mental retardation syndrome-3 is an autosomal recessive disorder usually characterized by severe mental retardation. Mutations in the PGAP2 gene cause hyperphosphatasia mental retardation syndrome-3. We have identified a large consanguineous family from Saudi origin segregating developmental delay, intellectual disability, epilepsy and microcephaly. Whole exome sequencing with 100× coverage was performed on two affected siblings of the family. Data analysis in the patient revealed a novel missense mutation c.191C>T in PGAP2 gene resulting in Alanine to Valine substitution (Ala64Val). The mutation was reconfirmed and validated by subsequent Sanger sequencing method. The mutation was ruled out in 100 unrelated healthy controls. We suggest that this pathogenic mutation disrupts the proper function of the gene proteins resulting in the disease state.

Pathogenic Variants in PIGG Cause Intellectual Disability with Seizures and Hypotonia

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 various proteins to the cell surface. At least 27 genes are involved in biosynthesis and transport of GPI-anchored proteins (GPI-APs). To date, mutations in 13 of these genes are known to cause inherited GPI deficiencies (IGDs), and all are inherited as recessive traits. IGDs mainly manifest as intellectual disability, epilepsy, coarse facial features, and multiple organ anomalies. These symptoms are caused by the decreased surface expression of GPI-APs or by structural abnormalities of GPI. Here, we present five affected individuals (from two consanguineous families from Egypt and Pakistan and one non-consanguineous family from Japan) who show intellectual disability, hypotonia, and early-onset seizures. We identified pathogenic variants in PIGG, a gene in the GPI pathway. In the consanguineous families, homozygous variants c.928C>T (p.Gln310(∗)) and c.2261+1G>C were found, whereas the Japanese individual was compound heterozygous for c.2005C>T (p.Arg669Cys) and a 2.4 Mb deletion involving PIGG. PIGG is the enzyme that modifies the second mannose with ethanolamine phosphate, which is removed soon after GPI is attached to the protein. Physiological significance of this transient modification has been unclear. Using B lymphoblasts from affected individuals of the Egyptian and Japanese families, we revealed that PIGG activity was almost completely abolished; however, the GPI-APs had normal surface levels and normal structure, indicating that the pathogenesis of PIGG deficiency is not yet fully understood. The discovery of pathogenic variants in PIGG expands the spectrum of IGDs and further enhances our understanding of this etiopathogenic class of intellectual disability.

Coversin Blocked in Vitro Hemolysis in an Eculizumab-Resistant PNH Patient with the C5 Polymorphism (c.2654G>A)

Background:Paroxysmal Nocturnal Hemoglobinurea (PNH) is a rare stem cell disease caused by the expansion of PIGA mutated clone(s). PNH-type cells are deficient in the expression of GPI-anchored proteins including DAF and CD59, which protect red blood cells (RBC) from complement-mediated intravascular hemolysis. Eculizumab (Soliris®, Alexion Pharmaceuticals) is a humanized monoclonal antibody against C5 which efficiently inhibits hemolysis by blocking the terminal complement cascade. Eculizumab dramatically ameliorates several clinical symptoms, and improves the prognosis in PNH patients. However, among 345 Japanese PNH patients who were treated with eculizumab, 11 patients showed poor response. All the poor responders had a single missense C5 heterozygous mutation, c.2654G>A, which predicts the polymorphism p.Arg885His (Nishimura et al, NEJM. 2014 13;370(7):632-9). Two of those patients have already passed away due to severe complications related to PNH, and the rest of them are still suffered from various clinical symptoms including hemolytic episodes and RBC transfusion. In these circumstances, multiple new anti-complement drugs are under development in Japan. Coversin (Volution Immuno Pharmaceuticals) is a recombinant protein (16,740 Da) derived from a secreted protein in the saliva of the Ornithodoros moubata tick, and it blocks complement-mediated hemolysis at C5 level. In this study, we examined this new anti-complement agent to a PNH patient with C5 polymorphism c.2654G>A, as well as those without the polymorphism.Materials:Peripheral blood samples were collected from a poor responder to eculizumab and hemolytic PNH patients with written informed consent as approved by the Institutional Review Board of Osaka University Hospital.In vitro hemolytic assay:RBC from ABO-matched PNH patients off eculizumab treatment were washed 3 times in saline, and subsequently incubated with Mg2+ supplemented serum of the poor responder in the presence or absence of an anti-complement agent. Alternative pathway was activated by adding HCl (22:1 of 0.4M HCl) to the serum. Heat-inactivated (56°C for 30min) serum was used as a negative control. After a 24-hour incubation at 37°C, hemolysis was quantified by measuring the optical density at 405nm (OD405). The hemolytic activity was normalized against maximum hemolysis as induced by HCl (100%) and minimum hemolysis with inactivated acidified serum (0%).Results:A 41-year-old male with fatigue was diagnosed as aplastic anemia with PNH in 2008, and cyclosporine (CyA) was initiated at the dose of 150mg/day. The PNH clone expanded from 30.6% to 70.2% in granulocytes from 2008 to 2011 with elevated LDH (700 U/L) and the patient was referred to our hospital to undergo eculizumab treatment. CyA was reduced to 100mg/day and eculizumab was initiated in May 2012. Eculizumab treatment did not change the serum LDH level without any improvement of the symptoms: fatigue, abdominal pain, and periodical hemoglobinurea. A heterozygous mutation c.2654G>A was identified as the cause of the failure to eculizumab treatment, and he is still suffered from continuous intravascular hemolysis (LDH > 1400 U/L) with periodical acute hemolytic episodes, requiring frequent RBC transfusion.In the hemolytic assay, Coversin completely blocked hemolysis at the concentration of 10ug/ml, similar to the effective inhibition with hemolytic PNH patients without the polymorphism.Discussion:Eculizumab has dramatically improved the quality-of-life in the majority of the PNH patients by blocking intravascular hemolysis, but there are still some concerns; poor response due to C5 polymorphism, C3b deposition on the RBC, high cost and burden for scheduled infusion. Blocking the complement cascade at C5 level has shown to be relatively safe if meningococcal vaccination is properly performed, but still an extravascular hemolysis remains problematic at least in some cases. Inhibiting C3 amplification would resolve both intra and extravascular hemolysis, but susceptibility to infections remains a major concern.Our study showed that Coversin efficiently blocked in vitro hemolysis in the eculizumab resistant patient with C5 heterozygous mutation, c.2654G>A. Coversin might be a therapeutic option for the population of C5 polymorphism c.2654G>A in PNH patients. Our results warrant further investigation to explore new anti-complement agents for hemolytic PNH patients. DisclosuresUeda:Alexion Pharma: Research Funding. Osato:Alexion Pharma: Research Funding. Weston-Davies:Volution Immuno Pharmaceuticals (UK) Ltd: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Hayashi:Alexion Pharma: Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

Stable cell surface expression of GPI-anchored proteins, but not intracellular transport, depends on their fatty acid structure.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI-APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi-to-plasma membrane transport or selective endocytosis via GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI-APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI-APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI-APs particularly under potential membrane lipid perturbation.

PIGA mutations cause early-onset epileptic encephalopathies and distinctive features

To investigate the clinical spectrum caused by mutations in PIGA at Xp22.2, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, among patients with early-onset epileptic encephalopathies (EOEEs). Whole-exome sequencing was performed as a comprehensive genetic analysis for a cohort of 172 patients with EOEEs including early myoclonic encephalopathy, Ohtahara syndrome, and West syndrome, and PIGA mutations were carefully investigated. We identified 4 PIGA mutations in probands showing early myoclonic encephalopathy, West syndrome, or unclassified EOEE. Flow cytometry of blood granulocytes from patients demonstrated reduced expression of GPI-anchored proteins. Expression of GPI-anchored proteins in PIGA-deficient JY5 cells was only partially or hardly restored by transient expression of PIGA mutants with a weak TATA box promoter, indicating a variable loss of PIGA activity. The phenotypic consequences of PIGA mutations can be classified into 2 types, severe and less severe, which correlate with the degree of PIGA activity reduction caused by the mutations. Severe forms involved myoclonus and asymmetrical suppression bursts on EEG, multiple anomalies with a dysmorphic face, and delayed myelination with restricted diffusion patterns in specific areas. The less severe form presented with intellectual disability and treatable seizures without facial dysmorphism. Our study confirmed that PIGA mutations are one genetic cause of EOEE, suggesting that GPI-anchor deficiencies may be an underlying cause of EOEE.

Parasporin-2 requires GPI-anchored proteins for the efficient cytocidal action to human hepatoma cells

Parasporin-2 (PS2) is a Bacillus thuringiensis inclusion protein that reacts intensively with human hepatoma cells. This antitumour toxin oligomerizes at the cell surface via binding to lipid rafts, leading to the cell lysis with typical blebs around peripheral cells. We find here that glycosylphosphatidylinositol (GPI)-anchored proteins are involved in the cytocidal actions. Depletion of the cellular cholesterol and loss of sphingolipid in lipid rafts slightly decreased cytolysis by PS2. Beyond those, the cells temporally resisted PS2 with reduction of the toxin binding after GPI-anchored proteins were cleaved off by phosphatidylinositol-specific phospholipase C. PS2 and aerolysin showed individual cytocidal specificity while aerolysin's receptor is GPI-anchored proteins. When we confirmed expression of GPI-anchored proteins on four cell lines, showing different cytotoxicity by PS2, GPI-anchored proteins were evenly expressed on the cells. Therefore, PS2 requires a kind of GPI-anchored proteins for the effective cytolysis.