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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Glicosilfosfatidilinositoles , Retículo Endoplásmico/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Lipogénesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
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 (GPI-APs). Because the attachment of GPI is required for the cell-surface expression of GPI-APs, a thorough knowledge of the molecular basis of mammalian GPI-AP biosynthesis is important for understanding the basic biochemistry and biology of GPI-APs and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-APs and then examine new findings made since 2020.
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Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI and the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show that EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the Ect-5'-nucleotidase and Netrin G2. We find that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3, and with a small fraction in wild-type cells. Our findings modify the current view of GPI anchoring and provide a mechanistic basis for IGDs caused by PIGG mutations.
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Glicosilfosfatidilinositoles , Manosa , Animales , Etanolaminas/metabolismo , Proteínas Ligadas a GPI/genética , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Mamíferos/metabolismo , Manosa/metabolismo , FosfatosRESUMEN
Many eukaryotic proteins are attached to the cell surface via glycosylphosphatidylinositol (GPI) anchors. How GPI-anchored proteins (GPI-APs) are trafficked from the endoplasmic reticulum (ER) to the cell surface is poorly understood, but the GPI moiety has been postulated to function as a signal for sorting and transport. Here, we established mutant cells that were selectively defective in transport of GPI-APs from the ER to the Golgi. We identified a responsible gene, designated PGAP5 (post-GPI-attachment to proteins 5). PGAP5 belongs to a dimetal-containing phosphoesterase family and catalyzed the remodeling of the glycan moiety on GPI-APs. PGAP5 catalytic activity is a prerequisite for the efficient exit of GPI-APs from the ER. Our data demonstrate that GPI glycan acts as an ER-exit signal and suggest that glycan remodeling mediated by PGAP5 regulates GPI-AP transport in the early secretory pathway.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Polisacáridos/metabolismo , Animales , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Ratones , Hidrolasas Diéster Fosfóricas/genéticaRESUMEN
Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.
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Retículo Endoplásmico , Proteínas Ligadas a GPI , Glicosilfosfatidilinositoles , Señales de Clasificación de Proteína , Humanos , Retículo Endoplásmico/metabolismo , Glicosilación , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Priones/química , Priones/metabolismo , Transporte de ProteínasRESUMEN
Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.
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Flavina-Adenina Dinucleótido , Riboflavina , Humanos , Flavina-Adenina Dinucleótido/metabolismo , Fosfatasa Alcalina , 5'-Nucleotidasa/genética , Mononucleótido de Flavina/metabolismo , Hidrólisis , VitaminasRESUMEN
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.
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Acetilgalactosamina , Glicosilfosfatidilinositoles , Enfermedades por Prión , Priones , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animales , Encéfalo/metabolismo , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Ratones , Osteogénesis , Enfermedades por Prión/metabolismo , Priones/metabolismo , Relación Estructura-ActividadRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.
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Aciltransferasas/genética , Moléculas de Adhesión Celular/genética , Enfermedades Cerebelosas/genética , Epilepsia/genética , Variación Genética/genética , Hipotonía Muscular/genética , Trastornos del Neurodesarrollo/genética , Anomalías Múltiples/genética , Alelos , Femenino , Humanos , Discapacidad Intelectual/genética , Masculino , Malformaciones del Sistema Nervioso/genética , Linaje , SíndromeRESUMEN
Glycerophospholipids, sphingolipids and cholesterol assemble into lipid bilayers that form the scaffold of cellular membranes, in which proteins are embedded. Membrane composition and membrane protein profiles differ between plasma and intracellular membranes and between the two leaflets of a membrane. Lipid distributions between two leaflets are mediated by lipid translocases, including flippases and scramblases. Flippases use ATP to catalyze the inward movement of specific lipids between leaflets. In contrast, bidirectional flip-flop movements of lipids across the membrane are mediated by scramblases in an ATP-independent manner. Scramblases have been implicated in disrupting the lipid asymmetry of the plasma membrane, protein glycosylation, autophagosome biogenesis, lipoprotein secretion, lipid droplet formation and communications between organelles. Although scramblases in plasma membranes were identified over 10 years ago, most progress about scramblases localized in intracellular membranes has been made in the last few years. Herein, we review the role of scramblases in regulating lipid distributions in cellular membranes, focusing primarily on intracellular membrane-localized scramblases.
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Membranas Intracelulares , Membrana Dobles de Lípidos , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/metabolismo , Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
PURPOSE: Biallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported. METHODS: Genotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp). RESULTS: Biallelic-truncating variants were detected in 16 patients-10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased. Biallelic missense or mixed genotype were reported in the remaining 45 cases-32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon. For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period. CONCLUSION: We have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.
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Anomalías Múltiples , Trastornos Congénitos de Glicosilación , Epilepsia , Hernia Diafragmática , Embarazo , Femenino , Humanos , Hipotonía Muscular/genética , Epilepsia/genética , Anomalías Múltiples/genética , Hernia Diafragmática/genética , Convulsiones/genética , Fenotipo , Estudios de Asociación Genética , SíndromeRESUMEN
Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 proteins to the cell surface. Pathogenic variants in several genes that participate in GPI biosynthesis cause inherited GPI deficiency disorders. Here, we reported that homozygous null alleles of PIGG, a gene involved in GPI modification, are responsible for the rare Emm-negative blood phenotype. Using a panel of K562 cells defective in both the GPI-transamidase and GPI remodeling pathways, we show that the Emm antigen, whose molecular basis has remained unknown for decades, is carried only by free GPI and that its epitope is composed of the second and third ethanolamine of the GPI backbone. Importantly, we show that the decrease in Emm expression in several inherited GPI deficiency patients is indicative of GPI defects. Overall, our findings establish Emm as a novel blood group system, and they have important implications for understanding the biological function of human free GPI.
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Antígenos de Grupos Sanguíneos , Discapacidades del Desarrollo , Glicosilfosfatidilinositoles/deficiencia , Glicosilfosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Convulsiones , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Glicosilfosfatidilinositoles/genética , Humanos , Células K562 , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Convulsiones/enzimología , Convulsiones/genéticaRESUMEN
A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.
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Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Animales , Ratones , Humanos , Proteínas Priónicas/genética , Mutación Puntual , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Señales de Clasificación de Proteína/genética , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patología , Priones/genética , Priones/metabolismo , Mutación/genéticaRESUMEN
Glycosylphosphatidylinositol (GPI) functions to anchor certain proteins to the cell surface. Although defects in GPI biosynthesis can result in a wide range of phenotypes, most affected patients present with neurological abnormalities and their diseases are grouped as inherited-GPI deficiency disorders. We present two siblings with global developmental delay, brain anomalies, hypotonia, and contractures. Exome sequencing revealed a homozygous variant, NM_001035005.4:c.90dupC (p.Phe31Leufs*3) in C18orf32, a gene not previously associated with any disease in humans. The encoded protein is known to be important for GPI-inositol deacylation. Knockout of C18orf32 in HEK293 cells followed by a transfection rescue assay revealed that the PIPLC (Phosphatidylinositol-Specific Phospholipase C) sensitivity of GPI-APs (GPI-anchored proteins) was restored only by the wild type and not the mutant C18orf32. Immunofluorescence revealed that the mutant C18orf32 was localized to the endoplasmic reticulum and was also found as aggregates in the nucleus. In conclusion, we identified a pathogenic variant in C18orf32 as the cause of a novel autosomal recessive neurodevelopmental disorder with hypotonia and contractures. Our results demonstrate the importance of C18orf32 in the biosynthesis of GPI-anchors, the molecular impact of the variant on the protein function, and add a novel candidate gene to the existing repertoire of genes implicated in neurodevelopmental disorders.
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Contractura , Hipotonía Muscular , Malformaciones del Sistema Nervioso , Trastornos del Neurodesarrollo , Contractura/genética , Contractura/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Células HEK293 , Humanos , Hipotonía Muscular/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismoRESUMEN
The glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies.
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Aciltransferasas/genética , Encefalopatías/etiología , Epilepsia/etiología , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/deficiencia , Discapacidad Intelectual/etiología , Mutación , Convulsiones/patología , Adolescente , Adulto , Secuencia de Aminoácidos , Encefalopatías/patología , Niño , Preescolar , Epilepsia/patología , Femenino , Glicosilfosfatidilinositoles/genética , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/patología , Masculino , Linaje , Convulsiones/genética , Homología de Secuencia , Adulto JovenRESUMEN
Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.
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Anomalías Craneofaciales/etiología , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/deficiencia , Deformidades Congénitas de la Mano/etiología , Pérdida Auditiva Sensorineural/etiología , Discapacidad Intelectual/etiología , Manosiltransferasas/genética , Enfermedades Metabólicas/etiología , Mutación , Uñas Malformadas/etiología , Enfermedades del Sistema Nervioso Periférico/etiología , Convulsiones/patología , Adulto , Niño , Preescolar , Anomalías Craneofaciales/patología , Femenino , Glicosilfosfatidilinositoles/genética , Deformidades Congénitas de la Mano/patología , Pérdida Auditiva Sensorineural/patología , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/patología , Masculino , Enfermedades Metabólicas/patología , Uñas Malformadas/patología , Linaje , Enfermedades del Sistema Nervioso Periférico/patología , Convulsiones/genética , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK.Key words: Glycosylphosphatidylinositol, GPI transamidase complex, protein stability, transamidation, ERAD.
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Degradación Asociada con el Retículo Endoplásmico , Glicosilfosfatidilinositoles , Ubiquitina-Proteína Ligasas/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Moléculas de Adhesión Celular , MutaciónRESUMEN
The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.
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Calnexina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Oligosacáridos/metabolismo , Pliegue de Proteína , Calnexina/genética , Membrana Celular/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oligosacáridos/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that â¼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.