Glycosylphosphatidylinositol Anchor Research Articles

Synthesis of glycosylphosphatidylinositol analogues with an unnatural β-D-glucosamine-(1→6)-myo-inositol motif

Glycosylphosphatidylinositol (GPI) anchors contain a unique α-D-glucosamine-(1→6)-myo-inositol [αGlcN(1,6)Ins] motif in their conserved core structure. To facilitate investigations of the functional roles of this structural motif, two GPI analogues containing unnatural βGlcN(1,6)Ins, instead of αGlcN(1,6)Ins, and an alkyne group at different positions of the GPI core were designed and synthesized. To this end, an orthogonally protected pseudopentasaccharide derivative of GPIs with the βGlcN(1,6)Ins motif was convergently constructed via [3 + 2] glycosylation and used as the common intermediate to prepare both GPI analogues by streamlined synthetic protocols. The pseudopentasaccharide intermediate and developed protocols can be widely applicable to access various GPI analogues with the βGlcN(1,6)Ins motif. The target GPI analogues contain an alkyne, which allows their further modification to introduce various molecular labels via click chemistry, making them useful probes for the study of GPI anchorage. The differences in reactivity and NMR behavior of the two GPI analogues, as well as the differences of these analogues from previously reported GPI derivatives of similar structure containing an αGlcN(1,6)Ins motif, suggest that the 2-O-phosphoethanolamine moiety on mannose-I and the linkage form of GlcN in GPIs can have a decisive impact on the structure, which is likely relevant to biology.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

PIGA mutations and glycosylphosphatidylinositol anchor dysregulation in polyposis-associated duodenal tumorigenesis.

The pathogenesis of duodenal tumours in the inherited tumour syndromes Familial Adenomatous Polyposis (FAP) and MUTYH-associated Polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumours and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of PIGA in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyses the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. Implications: PIGA somatic mutation in duodenal tumours from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

The clinical and genetic spectrum of inherited glycosylphosphatidylinositol deficiency disorders.

Inherited glycosylphosphatidylinositol deficiency disorders (IGDs) are a group of rare multisystem disorders arising from pathogenic variants in glycosylphosphatidylinositol anchor pathway (GPI-AP) genes. Despite associating 24 of at least 31 GPI-AP genes with human neurogenetic disease, prior reports are limited to single genes without consideration of the GPI-AP as a whole and with limited natural history data. In this multinational retrospective observational study, we systematically analyse the molecular spectrum, phenotypic characteristics, and natural history of 83 individuals from 75 unique families with IGDs, including 70 newly reported individuals: the largest single cohort to date. Core clinical features were developmental delay or intellectual disability (DD/ID, 90%), seizures (83%), hypotonia (72%), and motor symptoms (64%). Prognostic and biologically significant neuroimaging features included cerebral atrophy (75%), cerebellar atrophy (60%), callosal anomalies (57%), and symmetric restricted diffusion of the central tegmental tracts (60%). Sixty-one individuals had multisystem involvement including gastrointestinal (66%), cardiac (19%), and renal (14%) anomalies. Though dysmorphic features were appreciated in 82%, no single dysmorphic feature had a prevalence >30%, indicating substantial phenotypic heterogeneity. Follow-up data were available for all individuals, 15 of whom were deceased at the time of writing. Median age at seizure onset was 6 months. Individuals with variants in synthesis stage genes of the GPI-AP exhibited a significantly shorter time to seizure onset than individuals with variants in transamidase and remodelling stage genes of the GPI-AP (P=0.046). Forty individuals had intractable epilepsy. The majority of individuals experienced delayed or absent speech (95%); motor delay with non-ambulance (64%); and severe-to-profound DD/ID (59%). Individuals with a developmental epileptic encephalopathy (51%) were at greater risk of intractable epilepsy (P=0.003), non-ambulance (P=0.035), ongoing enteral feeds (P<0.001), and cortical visual impairment (P=0.007). Serial neuroimaging showed progressive cerebral volume loss in 87.5% and progressive cerebellar atrophy in 70.8%, indicating a neurodegenerative process. Genetic analyses identified 93 unique variants (106 total), including 22 novel variants. Exploratory analyses of genotype-phenotype correlations using unsupervised hierarchical clustering identified novel genotypic predictors of clinical phenotype and long-term outcome with meaningful implications for management. In summary, we expand both the mild and severe phenotypic extremities of the IGDs; provide insights into their neurological basis; and, vitally, enable meaningful genetic counselling for affected individuals and their families.

Transfer of membrane(s) matter(s)-non-genetic inheritance of (metabolic) phenotypes?

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer phospholipid layer of eukaryotic plasma membranes exclusively by a glycolipid. GPI-APs are not only released into extracellular compartments by lipolytic cleavage. In addition, certain GPI-APs with the glycosylphosphatidylinositol anchor including their fatty acids remaining coupled to the carboxy-terminus of their protein components are also detectable in body fluids, in response to certain stimuli, such as oxidative stress, radicals or high-fat diet. As a consequence, the fatty acid moieties of GPI-APs must be shielded from access of the aqueous environment by incorporation into membranes of extracellular vesicles or into micelle-like complexes together with (lyso)phospholipids and cholesterol. The GPI-APs released from somatic cells and tissues are transferred via those complexes or EVs to somatic as well as pluripotent stem cells with metabolic consequences, such as upregulation of glycogen and lipid synthesis. From these and additional findings, the following hypotheses are developed: i) Transfer of GPI-APs via EVs or micelle-like complexes leads to the induction of new phenotypes in the daughter cells or zygotes, which are presumably not restricted to metabolism. ii) The membrane topographies transferred by the concerted action of GPI-APs and interacting components are replicated by self-organization and self-templation and remain accessible to structural changes by environmental factors. iii) Transfer from mother cells and gametes to their daughter cells and zygotes, respectively, is not restricted to DNA and genes, but also encompasses non-genetic matter, such as GPI-APs and specific membrane constituents. iv) The intergenerational transfer of membrane matter between mammalian organisms is understood as an epigenetic mechanism for phenotypic plasticity, which does not rely on modifications of DNA and histones, but is regarded as molecular mechanism for the inheritance of acquired traits, such as complex metabolic diseases. v) The missing interest in research of non-genetic matter of inheritance, which may be interpreted in the sense of Darwin's "Gemmules" or Galton's "Stirps", should be addressed in future investigations of the philosophy of science and sociology of media.

Hedgehog on the Move: Glypican-Regulated Transport and Gradient Formation in Drosophila.

Glypicans (Glps) are a family of heparan sulphate proteoglycans that are attached to the outer plasma membrane leaflet of the producing cell by a glycosylphosphatidylinositol anchor. Glps are involved in the regulation of many signalling pathways, including those that regulate the activities of Wnts, Hedgehog (Hh), Fibroblast Growth Factors (FGFs), and Bone Morphogenetic Proteins (BMPs), among others. In the Hh-signalling pathway, Glps have been shown to be essential for ligand transport and the formation of Hh gradients over long distances, for the maintenance of Hh levels in the extracellular matrix, and for unimpaired ligand reception in distant recipient cells. Recently, two mechanistic models have been proposed to explain how Hh can form the signalling gradient and how Glps may contribute to it. In this review, we describe the structure, biochemistry, and metabolism of Glps and their interactions with different components of the Hh-signalling pathway that are important for the release, transport, and reception of Hh.

MDGA2 Constrains Glutamatergic Inputs Selectively onto CA1 Pyramidal Neurons to Optimize Neural Circuits for Plasticity, Memory, and Social Behavior.

Synapse organizers are essential for the development, transmission, and plasticity of synapses. Acting as rare synapse suppressors, the MAM domain containing glycosylphosphatidylinositol anchor (MDGA) proteins contributes to synapse organization by inhibiting the formation of the synaptogenic neuroligin-neurexin complex. A previous analysis of MDGA2 mice lacking a single copy of Mdga2 revealed upregulated glutamatergic synapses and behaviors consistent with autism. However, MDGA2 is expressed in diverse cell types and is localized to both excitatory and inhibitory synapses. Differentiating the network versus cell-specific effects of MDGA2 loss-of-function requires a cell-type and brain region-selective strategy. To address this, we generated mice harboring a conditional knockout of Mdga2 restricted to CA1 pyramidal neurons. Here we report that MDGA2 suppresses the density and function of excitatory synapses selectively on pyramidal neurons in the mature hippocampus. Conditional deletion of Mdga2 in CA1 pyramidal neurons of adult mice upregulated miniature and spontaneous excitatory postsynaptic potentials, vesicular glutamate transporter 1 intensity, and neuronal excitability. These effects were limited to glutamatergic synapses as no changes were detected in miniature and spontaneous inhibitory postsynaptic potential properties or vesicular GABA transporter intensity. Functionally, evoked basal synaptic transmission and AMPAR receptor currents were enhanced at glutamatergic inputs. At a behavioral level, memory appeared to be compromised in Mdga2 cKO mice as both novel object recognition and contextual fear conditioning performance were impaired, consistent with deficits in long-term potentiation in the CA3-CA1 pathway. Social affiliation, a behavioral analog of social deficits in autism, was similarly compromised. These results demonstrate that MDGA2 confines the properties of excitatory synapses to CA1 neurons in mature hippocampal circuits, thereby optimizing this network for plasticity, cognition, and social behaviors.

JAGGER localization and function are dependent on GPI anchor addition.

GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

GPI-anchored Gas1 protein regulates cytosolic proteostasis in budding yeast.

The decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation through the "mitochondrial as guardian in cytosol" (MAGIC) pathway. Here, we report an unexpected role of Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase in the budding yeast, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of GAS1 inhibits MAGIC but elevates protein ubiquitination and UPS-mediated protein degradation. Interestingly, we found that the Gas1 protein exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins through MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1-E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

Deletion of the auxiliary α2δ1 voltage sensitive calcium channel subunit in osteocytes and late-stage osteoblasts impairs femur strength and load-induced bone formation in male mice.

Osteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel's α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol anchor and regulates the channel's calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (P < .05) by DXA, reduced relative osteoid surface (P < .05), and altered osteoblast and osteocyte regulatory gene expression (P < .01). Cacna2d1f/f, Cre + male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo micro-computed tomography measurements at the proximal tibia (P < .01) compared to controls, whereas Cacna2d1f/f, Cre + female mice showed impaired 20-wk cancellous and cortical bone ex vivo micro-computed tomography measurements (P < .05) vs controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.

Neuronal Wiring Receptors Dprs and DIPs Are GPI Anchored and This Modification Contributes to Their Cell Surface Organization.

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that most Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr and DIP localization, we replaced it with a transmembrane domain and observed perturbation of subcellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane coreceptors.

Folate Receptor Alpha-A Novel Approach to Cancer Therapy.

Folate receptor α (FR) was discovered many decades ago, along with drugs that target intracellular folate metabolism, such as pemetrexed and methotrexate. Folate is taken up by the cell via this receptor, which also targeted by many cancer agents due to the over-expression of the receptor by cancer cells. FR is a membrane-bound glycosyl-phosphatidylinositol (GPI) anchor glycoprotein encoded by the folate receptor 1 (FOLR1) gene. FR plays a significant role in DNA synthesis, cell proliferation, DNA repair, and intracellular signaling, all of which are essential for tumorigenesis. FR is more prevalent in cancer cells compared to normal tissues, which makes it an excellent target for oncologic therapeutics. FRα is found in many cancer types, including ovarian cancer, non-small-cell lung cancer (NSCLC), and colon cancer. FR is widely used in antibody drug conjugates, small-molecule-drug conjugates, and chimeric antigen-receptor T cells. Current oncolytic therapeutics include mirvetuximab soravtansine, and ongoing clinical trials are underway to investigate chimeric antigen receptor T cells (CAR-T cells) and vaccines. Additionally, FRα has been used in a myriad of other applications, including as a tool in the identification of tumor types, and as a prognostic marker, as a surrogate of chemotherapy resistance. As such, FRα identification has become an essential part of precision medicine.

Macrocephaly and Digital Anomalies Expand the Phenotypic Spectrum of PGAP2 Variants in Hyperphosphatasia with Impaired Intellectual Development Syndrome 3 (HPMRS3)

Glycosylphosphatidylinositols (GPIs) anchor over 150 proteins as GPI-anchored proteins (GPI-APs) with crucial roles in diverse biological processes. The highly conserved biosynthesis of GPI-APs involves precise steps with at least 21 genes, categorized as PIG and PGAP genes. Pathogenic variants in these genes are linked to human diseases, highlighting the importance of each biosynthesis step. PGAP2 stands out among these genes due to its association with an expanded clinical spectrum of neurodevelopmental disorder (NDD) phenotypes with biallelic pathogenic variants. We present four patients from two families, one consanguineous and the other nonconsanguineous, each displaying distinct clinical presentations, including intellectual disability, hyperphosphatasia, hearing impairment, and epilepsy, as well as craniofacial and digital anomalies. Genetic analyses revealed homozygous and novel compound heterozygous missense variants in PGAP2 in four affected individuals, confirming the molecular diagnosis of hyperphosphatasia with impaired intellectual development syndrome 3 (HPMRS3). Importantly, the three amino acids affected by missense variants exhibit complete conservation in 10 vertebrate species, illuminating their crucial role in the gene’s functionality. Protein modeling provided additional evidence for the pathogenicity of the three substitutions, demonstrating their detrimental impact on protein folding and putative protein-protein interactions, ultimately leading to impaired protein function. The four patients in our study displayed common phenotypic features, such as brachydactyly, camptodactyly, and syndactyly, which have not been previously documented in individuals with PGAP2 variants. Notably, the occurrence of macrocephaly in two affected brothers from a consanguineous Pakistani family represents a novel finding. These previously unreported digital anomalies, along with macrocephaly and the identification of novel compound heterozygous variants, contribute to the expansion of the phenotypic and genotypic spectrum of HPMRS3 associated with PGAP2 variants.

Drosophila models of phosphatidylinositol glycan biosynthesis class A congenital disorder of glycosylation (PIGA-CDG) mirror patient phenotypes.

Mutations in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene cause a rare, X-linked recessive congenital disorder of glycosylation. Phosphatidylinositol glycan biosynthesis class A congenital disorder of glycosylation (PIGA-CDG) is characterized by seizures, intellectual and developmental delay, and congenital malformations. The PIGA gene encodes an enzyme involved in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis. There are over 100 GPI-anchored proteins that attach to the cell surface and are involved in cell signaling, immunity, and adhesion. Little is known about the pathophysiology of PIGA-CDG. Here, we describe the first Drosophila model of PIGA-CDG and demonstrate that loss of PIG-A function in Drosophila accurately models the human disease. As expected, complete loss of PIG-A function is larval lethal. Heterozygous null animals appear healthy but, when challenged, have a seizure phenotype similar to what is observed in patients. To identify the cell-type specific contributions to disease, we generated neuron- and glia-specific knockdown of PIG-A. Neuron-specific knockdown resulted in reduced lifespan and a number of neurological phenotypes but no seizure phenotype. Glia-knockdown also reduced lifespan and, notably, resulted in a very strong seizure phenotype. RNA sequencing analyses demonstrated that there are fundamentally different molecular processes that are disrupted when PIG-A function is eliminated in different cell types. In particular, loss of PIG-A in neurons resulted in upregulation of glycolysis, but loss of PIG-A in glia resulted in upregulation of protein translation machinery. Here, we demonstrate that Drosophila is a good model of PIGA-CDG and provide new data resources for future study of PIGA-CDG and other GPI anchor disorders.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Lipid metabolism of plasma-derived small extracellular vesicles in COVID-19 convalescent patients

The coronavirus disease 2019 (COVID-19), which affects multiple organs, is causing an unprecedented global public health crisis. Most COVID-19 patients recover gradually upon appropriate interventions. Viruses were reported to utilize the small extracellular vesicles (sEVs), containing a cell-specific cargo of proteins, lipids, and nucleic acids, to escape the attack from the host’s immune system. This study aimed to examine the sEVs lipid profile of plasma of recovered COVID-19 patients (RCs). Plasma sEVs were separated from 83 RCs 3 months after discharge without underlying diseases, including 18 recovered asymptomatic patients (RAs), 32 recovered moderate patients (RMs), and 33 recovered severe and critical patients (RSs), and 19 healthy controls (HCs) by Total Exosome Isolation Kit. Lipids were extracted from sEVs and then subjected to targeted liquid chromatography-mass spectrometry. The size, concentration, and distribution of sEVs did not differ in RCs and HCs as validated by transmission electron microscopy, nanoparticle tracking analysis, and immunoblot analysis. Fifteen subclasses of 508 lipids were detected in plasma sEVs from HCs, RAs, RMs, and RSs, such as phosphatidylcholines (PCs) and diacylglycerols (DAGs), etc. Total lipid intensity displayed downregulation in RCs compared with HCs. The relative abundance of DAGs gradually dropped, whereas PCs, lysophosphatidylcholines, and sphingomyelins were higher in RCs relative to HCs, especially in RSs. 88 lipids out of 241 in sEVs of RCs were significantly different and a conspicuous increase was revealed with disease status. The sEVs lipids alternations were found to be significantly correlated with the clinical indices in RCs and HCs, suggesting that the impact of COVID-19 on lipid metabolism lingered for a long time. The lipid abnormalities bore an intimate link with glycerophospholipid metabolism and glycosylphosphatidylinositol anchor biosynthesis. Furthermore, the lipidomic analysis showed that RCs were at higher risk of developing diabetes and sustaining hepatic impairment. The abnormality of immunomodulation in RCs might still exist. The study may offer new insights into the mechanism of organ dysfunction and help identify novel therapeutic targets in the RCs.

The PIG-A gene mutation assay in human biomonitoring and disease.

The blood cell phosphatidylinositol glycan class A (PIG-A) gene mutation assay has been extensively researched in rodents for in vivo mutagenicity testing and is now being investigated in humans. The PIG-A gene is involved in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis. A single mutation in this X-linked gene can lead to loss of membrane-bound GPI anchors, which can be enumerated via corresponding GPI-anchored proteins (e.g., CD55) using flow cytometry. The studies published to date by different research groups demonstrate a remarkable consistency in PIG-A mutant frequencies. Moreover, with the low background level of mutant erythrocytes in healthy subjects (2.9-5.56 × 10-6 mutants), induction of mutation post genotoxic exposure can be detected. Cigarette smoking, radiotherapy, and occupational exposures, including lead, have been shown to increase mutant levels. Future applications of this test include identifying new harmful agents and establishing new exposure limits. This mutational monitoring approach may also identify individuals at higher risk of cancer development. In addition, identifying protective agents that could mitigate these effects may reduce baseline somatic mutation levels and such behaviors can be encouraged. Further technological progress is required including establishing underlying mechanisms of GPI anchor loss, protocol standardization, and the development of cryopreservation methods to improve GPI-anchor stability over time. If successful, this assay has the potential be widely employed, for example, in rural and low-income countries. Here, we review the current literature on PIG-A mutation in humans and discuss the potential role of this assay in human biomonitoring and disease detection.