RESUMEN
Glycophosphatidylinositol (GPI) anchors are the predominant glycoconjugate in Plasmodium parasites, enabling modified proteins to associate with biological membranes. GPI biosynthesis commences with donation of a mannose residue held by dolichol-phosphate at the endoplasmic reticulum membrane. In Plasmodium dolichols are derived from isoprenoid precursors synthesised in the Plasmodium apicoplast, a relict plastid organelle of prokaryotic origin. We found that treatment of Plasmodium parasites with apicoplast inhibitors decreases the synthesis of isoprenoid and GPI intermediates resulting in GPI-anchored proteins becoming untethered from their normal membrane association. Even when other isoprenoids were chemically rescued, GPI depletion led to an arrest in schizont stage parasites, which had defects in segmentation and egress. In those daughter parasites (merozoites) that did form, proteins that would normally be GPI-anchored were mislocalised, and when these merozoites were artificially released they were able to attach to but not invade new red blood cells. Our data provides further evidence for the importance of GPI biosynthesis during the asexual cycle of P. falciparum, and indicates that GPI biosynthesis, and by extension egress and invasion, is dependent on isoprenoids synthesised in the apicoplast.
Asunto(s)
Apicoplastos , Glicosilfosfatidilinositoles , Plasmodium falciparum , Terpenos , Plasmodium falciparum/metabolismo , Apicoplastos/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Terpenos/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Eritrocitos/parasitología , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/metabolismo , Animales , Merozoítos/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. 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.
Asunto(s)
Glicosilfosfatidilinositoles , Humanos , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/química , Animales , Biosíntesis de ProteínasRESUMEN
Candida albicans is an opportunistic fungal pathogen that can switch between yeast and hyphal morphologies depending on the environmental cues it receives. The switch to hyphal form is crucial for the establishment of invasive infections. The hyphal form is also characterized by the cell surface expression of hyphae-specific proteins, many of which are GPI-anchored and important determinants of its virulence. The coordination between hyphal morphogenesis and the expression of GPI-anchored proteins is made possible by an interesting cross-talk between GPI biosynthesis and the cAMP-PKA signaling cascade in the fungus; a parallel interaction is not found in its human host. On the other hand, in the nonpathogenic yeast, Saccharomyces cerevisiae, GPI biosynthesis is shut down when filamentation is activated and vice versa. This too is achieved by a cross-talk between GPI biosynthesis and cAMP-PKA signaling. How are diametrically opposite effects obtained from the cross-talk between two reasonably well-conserved pathways present ubiquitously across eukarya? This Review attempts to provide a model to explain these differences. In order to do so, it first provides an overview of the two pathways for the interested reader, highlighting the similarities and differences that are observed in C. albicans versus the well-studied S. cerevisiae model, before going on to explain how the different mechanisms of regulation are effected. While commonalities enable the development of generalized theories, it is hoped that a more nuanced approach, that takes into consideration species-specific differences, will enable organism-specific understanding of these processes and contribute to the development of targeted therapies.
Asunto(s)
Candida albicans , Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Hifa , Saccharomyces cerevisiae , Transducción de Señal , Candida albicans/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hifa/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.
Asunto(s)
Diferenciación Celular , Glicosilfosfatidilinositoles , Placentación , Trofoblastos , Trofoblastos/metabolismo , Trofoblastos/citología , Femenino , Embarazo , Animales , Humanos , Ratones , Placentación/genética , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Placenta/metabolismo , Placenta/citología , Vía de Señalización Wnt , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Retículo Endoplásmico/metabolismo , Vías Biosintéticas/genética , Respuesta de Proteína Desplegada , Sistemas CRISPR-CasRESUMEN
GPI anchoring is an essential post-translational modification in eukaryotes that links proteins to the plasma membrane. In this issue, Liu et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208159) suggest, for the first time, a regulation on demand of the GPI glycolipid precursor biosynthesis.
Asunto(s)
Glicosilfosfatidilinositoles , Procesamiento Proteico-Postraduccional , Membrana Celular , Glucolípidos/biosíntesis , Glucolípidos/química , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/químicaRESUMEN
We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas Ligadas a GPI , Glicosilfosfatidilinositoles , Glicosilfosfatidilinositoles/biosíntesis , Proteínas Ligadas a GPI/metabolismo , Precursores de Proteínas/metabolismo , Señales de Clasificación de ProteínaRESUMEN
Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.
Asunto(s)
Proteínas Ligadas a GPI/fisiología , Glicosilfosfatidilinositoles/biosíntesis , Toxinas Bacterianas/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Manosiltransferasas/genética , Manosiltransferasas/fisiología , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMEN
The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1-6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1-6 GlcNAc-transferase complex.
Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Proteómica , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Electroforesis en Gel de Poliacrilamida Nativa , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.
Asunto(s)
Proteínas Ligadas a GPI/genética , Proteínas Recombinantes/genética , Selenocisteína , Animales , Línea Celular , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/genética , Humanos , Lipasa/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismoRESUMEN
Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g., PIG-L and PIG-O, cause reduced expression of surface GPI-anchored proteins. Here, we investigated the possibility that the loss of the GPI-anchor detected by the rat Pig-a assay also could be caused by mutation in other GPI-biosynthesis genes. 31 samples were obtained from 8 inbred and outbred rat strains commonly used for genetic toxicology assays. In order to investigate possible sources of variation in the Pig-a assay, variant DNA sequences were evaluated in Cd59 and 24 GPI-biosynthesis genes. In some genes, such as Pig-n and Pig-u, homozygous variations occurred in all animals, suggesting that these variations are due to deviations in the reference genome. Heterozygous Pig-s, Pig-w, Pig-o, Pig-c, Pgap1, Pgap2, Pig-k and Pig-t variations were found, however, indicating that these genes could serve as targets for mutation in the assay. Protein alignment for these altered genes was conducted with possible human, mouse and rat phenotypic mutants from the literature; this analysis demonstrated that many of the variations that we detected were in non-conserved sequences and that no phenotypes for any of these variants could be inferred from known mutants from the literature. All heterozygous variants were in outbred rats. Overall, the findings of this study cannot totally rule out the possibility that mutations in GPI-biosynthesis genes other than Pig-a are detected in the Pig-a assay, but suggest that if it occurs, it must occur only rarely and therefore mutations in genes other than Pig-a have little impact on rat-based experiments.
Asunto(s)
Vías Biosintéticas/genética , Glicosilfosfatidilinositoles/biosíntesis , Proteínas de la Membrana/genética , Mutación , Secuencia de Aminoácidos , Animales , Antígenos CD59/genética , Antígenos CD59/metabolismo , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas Endogámicas F344 , Ratas Long-Evans , Ratas Sprague-Dawley , Ratas Wistar , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.
Asunto(s)
Antifúngicos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Policétidos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Fermentación , Genes Reporteros , Glicosilfosfatidilinositoles/biosíntesis , Células HCT116 , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Células K562 , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos ICR , Mucorales , Familia de Multigenes , Rhizopus , Saccharomyces cerevisiaeRESUMEN
Through genome mining for fungal macrolide natural products, we discovered a characteristic family of putative macrolide biosynthetic gene clusters that contain a glycosylphosphatidylinositol-ethanolamine phosphate transferase (GPI-EPT) homologue. Through the heterologous expression of two clusters from Aspergillus kawachii and Colletotrichum incanum, new macrolides, including those with phosphoethanolamine or phosphocholine moieties, were formed. This study is the first demonstration of the tailoring steps catalyzed by GPI-EPT homologues in natural product biosynthesis, and it uncovers a new gene resource for phospholipid-resembling fungal macrolides.
Asunto(s)
Aspergillus/química , Colletotrichum/química , Etanolaminas/química , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Macrólidos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicosilfosfatidilinositoles/química , Macrólidos/química , Estructura Molecular , Familia de Multigenes , Biosíntesis de ProteínasRESUMEN
Vertical nystagmus is a known clinical feature, is however rarely observed in a specific neurodevelopmental disorder. Based on our experience with Polish patients with glycosylphosphatidylinositol biosynthesis defects (GPIBD) due to PIGN variants, supported by literature review, we have verified the clinical significance of this feature in PIGN-related disorder. We hope to underline the clinical implication of vertical nystagmus in the evaluation of patients with developmental encephalopathy with epilepsy, which may accelerate the neurological diagnosis process by orientating it towards PIGN-GPIBD.
Asunto(s)
Encefalopatías Metabólicas/genética , Nistagmo Patológico/genética , Fosfotransferasas/genética , Niño , Preescolar , Epilepsia/genética , Femenino , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/genética , Humanos , Masculino , MutaciónRESUMEN
Glycosylphosphatidylinositol (GPI) is a glycolipid added to the C-terminus of a large variety of proteins in eukaryotes, thereby anchoring these proteins to the cell surface. More than 150 different human proteins are modified with GPI, and GPI-anchored proteins (GPI-APs) play critical roles in embryogenesis, neurogenesis, immunity, and fertilization. GPI-APs are biosynthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the Golgi apparatus. During transport, GPI-APs undergo structural remodeling that is important for the efficient folding and sorting of GPI-APs. Asparagine-linked glycan-dependent folding and deacylation by PGAP1 work together to ensure that correctly folded GPI-APs are transported from the ER to the Golgi. Remodeling of the GPI lipid moiety is critical for the association of GPI-APs with lipid rafts. On the cell surface, certain GPI-APs are cleaved by GPI cleavage enzymes and released from the membrane, a key event in processes such as spermatogenesis and neurogenesis. In this review, we discuss the enzymes involved in GPI-AP biosynthesis and the fate of GPI-APs in mammalian cells, with a focus on the assembly, folding, degradation, and cleavage of GPI-APs.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/enzimología , Glicosilfosfatidilinositoles/biosíntesis , Aparato de Golgi/enzimología , Animales , Humanos , Masculino , Microdominios de Membrana/enzimología , Proteínas de la Membrana/metabolismo , Neurogénesis , Dominios Proteicos , Pliegue de Proteína , Transporte de Proteínas , EspermatogénesisRESUMEN
Deficiency of the endoplasmic reticulum transmembrane protein ARV1 leads to epileptic encephalopathy in humans and in mice. ARV1 is highly conserved, but its function in human cells is unknown. Studies of yeast arv1 null mutants indicate that it is involved in a number of biochemical processes including the synthesis of sphingolipids and glycosylphosphatidylinositol (GPI), a glycolipid anchor that is attached to the C-termini of many membrane bound proteins. GPI anchors are post-translational modifications, enabling proteins to travel from the endoplasmic reticulum (ER) through the Golgi and to attach to plasma membranes. We identified a homozygous pathogenic mutation in ARV1, p.Gly189Arg, in two brothers with infantile encephalopathy, and characterized the biochemical defect caused by this mutation. In addition to reduced expression of ARV1 transcript and protein in patients' fibroblasts, complementation tests in yeast showed that the ARV1 p.Gly189Arg mutation leads to deficient maturation of Gas1, a GPI-anchored protein, but does not affect sphingolipid synthesis. Our results suggest, that similar to mutations in other proteins in the GPI-anchoring pathway, including PIGM, PIGA, and PIGQ, ARV1 p.Gly189Arg causes a GPI anchoring defect and leads to early onset epileptic encephalopathy.
Asunto(s)
Encefalopatías/genética , Proteínas Portadoras/genética , Glicosilfosfatidilinositoles/biosíntesis , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Convulsiones/genética , Adolescente , Niño , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Homocigoto , Humanos , Lípidos/química , Masculino , Manosiltransferasas/genética , Mutación , Linaje , Dominios Proteicos , TemperaturaRESUMEN
BACKGROUND: Mutations 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. METHODS: Mutations 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. RESULTS: Here 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. CONCLUSION: Loss 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.
Asunto(s)
Anomalías Múltiples/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Discapacidades del Desarrollo/genética , Epilepsia/genética , Glicosilfosfatidilinositoles/deficiencia , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Anomalías Múltiples/fisiopatología , Adolescente , Empalme Alternativo/genética , Preescolar , Discapacidades del Desarrollo/fisiopatología , Epilepsia/fisiopatología , Femenino , Fibroblastos/metabolismo , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Homocigoto , Humanos , Lactante , Recién Nacido , Masculino , Mutación , Neutrófilos/metabolismo , Linaje , Secuenciación del ExomaRESUMEN
Inherited glycosylphosphatidylinositol (GPI) deficiencies are a group of clinically and genetically heterogeneous conditions belonging to the congenital disorders of glycosylation. PIGW is involved in GPI biosynthesis and modification, and biallelic pathogenic variants in this gene cause autosomal recessive GPI biosynthesis defect 11. Only five patients and two fetuses have been reported in the literature thus far. Here we describe a new patient with a novel homozygous missense variant in PIGW, who presented with hypotonia, severe intellectual disability, early-onset epileptic seizures, brain abnormalities, nystagmus, hand stereotypies, recurrent respiratory infections, distinctive facial features, and hyperphosphatasia. Our report expands the phenotype of GPI biosynthesis defect 11 to include stereotypies and recurrent respiratory infections. A detailed and long-term analysis of the electroclinical characteristics and review of the literature suggest that early-onset epileptic seizures are a key manifestation of GPI biosynthesis defect 11. West syndrome and focal-onset epileptic seizures are the most common seizure types, and the fronto-temporal regions may be the most frequently involved areas in these patients.
Asunto(s)
Aciltransferasas/genética , Glicosilfosfatidilinositoles/deficiencia , Glicosilfosfatidilinositoles/genética , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Convulsiones/genética , Encéfalo/anomalías , Encéfalo/patología , Niño , Preescolar , Femenino , Glicosilfosfatidilinositoles/biosíntesis , Humanos , Lactante , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/patología , Masculino , Hipotonía Muscular/complicaciones , Hipotonía Muscular/genética , Hipotonía Muscular/patología , Mutación Missense/genética , Convulsiones/complicaciones , Convulsiones/patología , Convulsiones/fisiopatologíaRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that ß1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Glicoesfingolípidos/biosíntesis , Glicosilfosfatidilinositoles/biosíntesis , Aciltransferasas/deficiencia , Aciltransferasas/genética , Aciltransferasas/metabolismo , Sistemas CRISPR-Cas , Degradación Asociada con el Retículo Endoplásmico/genética , Galactosiltransferasas/deficiencia , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Técnicas de Inactivación de Genes , Glicoesfingolípidos/genética , Glicosilfosfatidilinositoles/genética , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
ScGpi12 is a 304 amino residue long endoplasmic reticulum membrane protein, which participates in the de-N-acetylation of N-acetylglucosaminyl phosphatidylinositol to produce glucosaminyl phosphatidylinositol in the second step of GPI anchor biosynthesis pathway in Saccharomyces cerevisiae. ScGpi12 was cloned in a pMAL-c2x vector and expressed heterologously in Rosetta-gami (DE3) strain of E. coli. Affinity purification of the protein yielded low amounts of the MBP-tagged enzyme, which was active. To the best of our knowledge, this is the first successful purification of full-length Gpi12 enzyme, without the accompanying GroEL that was seen in other studies. The presence of the tag did not greatly alter the activity of the enzyme. ScGpi12 was optimally active in the pH range of 6.5-8.5 and at 30 °C. It was not sensitive to treatment with EDTA but was stimulated by multiple divalent cations. The divalent cation did not alter the pH profile of the enzyme, suggesting no role of the divalent metal in creating a nucleophile for catalysis. Divalent cations did, however, enhance the turnover number of the enzyme for its substrate, suggesting that they are probably required for the production of a catalytically competent active site by bringing the active site residues within optimum distance of the substrate for catalysis.
Asunto(s)
Acetilesterasa/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Acetilesterasa/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Vías Biosintéticas , Catálisis , Clonación Molecular , Retículo Endoplásmico/enzimología , Escherichia coli/genética , Cinética , Fosfatidilinositoles/metabolismo , Especificidad por SustratoRESUMEN
Manogepix (MGX) targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway. The prodrug fosmanogepix is currently in clinical development for the treatment of invasive fungal infections. We determined that the median frequencies of spontaneous mutations conferring reduced susceptibility to MGX in Candida albicans, C. glabrata, and C. parapsilosis ranged from 3 × 10-8 to <1.85 × 10-8 Serial passage on agar identified mutants of C. albicans and C. parapsilosis with reduced susceptibility to MGX; however, this methodology did not result in C. glabrata mutants with reduced susceptibility. Similarly, serial passage in broth resulted in ≤2-fold changes in population MIC values for C. tropicalis, C. auris, and C. glabrata A spontaneous V163A mutation in the Gwt1 protein of C. glabrata and a corresponding C. albicans heterozygous V162A mutant were obtained. A C. glabrata V163A Gwt1 mutant generated using CRISPR, along with V162A and V168A mutants expressed in C. albicans and Saccharomyces cerevisiae Gwt1, respectively, all demonstrated reduced susceptibility to MGX versus control strains, suggesting the importance of this valine residue to MGX binding across different species. Cross-resistance to the three major classes of antifungals was evaluated, but no changes in susceptibility to amphotericin B or caspofungin were observed in any mutant. No change was observed in fluconazole susceptibility, with the exception of a single non-Gwt1 mutant, where a 4-fold increase in the fluconazole MIC was observed. MGX demonstrated a relatively low potential for resistance development, consistent with other approved antifungal agents and those in clinical development.