GPI-anchored Proteins Research Articles

Homozygous PGAP2 mutation cause hyperphosphatasia with mental retardation syndrome-3 (HPMRS3): Genetic and clinical evaluation of the ultra rare inherited glycosylphosphatidylinositol (GPI) biosynthesis defect

Introduction: Inherited glycosylphosphatidylinositol biosynthesis defect is considered a subset of the congenital glycosylation disorder that result from mutations in the genes encoding proteins participating in glycosylphosphatidylinositol biosynthesis and modification. Glycosylphosphatidylinositol anchor proteins play important roles in numerous cellular processes including neurogenesis, cell adhesion, immune response and signalling. Hyperphosphatasia with mental retardation syndrome-3 is one of the Glycosylphosphatidylinositol anchor defects, characterized by moderate to severe intellectual disability, dysmorphic features, hypotonia, seizures and persistent hyperphosphatasia. The aim of this study was to investigating the clinical implications of the PGAP2 gene and identifying the severe phenotype. Case Presentation: A male patient with dysmorphic features, neurodevelopmental delay, seizures, hearing loss, Hirschsprung disease, central fever and elevated alkaline phosphatase was included in the study. The magnetic resonance imaging showed cerebral atrophy, corpus callosum hypoplasia. The whole exome sequencing analysis of the individual, and Sanger sequencing were performed for segregation. Additionally, next generation sequencing, whole transcriptome sequencing and homology modelling and analysis were performed. Whole exome sequencing revealed a homozygous c.651C>G (p.His217Gln) in the PGAP2 gene. The Sanger sequencing confirmed the parents were heterozygous. There is no splicing variant was detected by whole transcriptome sequencing. The AlphaFold model was interpreted hypothetically. It was observed the substitution of histidine, with glutamine and may affect the stability of protein. Discussion: Homozygous PGAP2 mutations in the patient we reported in our study resulted in a severe clinic including, severe developmental delay and intellectual disability, severe epilepsia, dysmorphic features, central fever, biochemical, hormonal and immunological abnormalities. This patient would be the youngest case published in the literatur. We showed that, the instability of mutant PGAP2 protein that cause Hyperphosphatasia with mental retardation syndrome-3 lead to more severe phenotypes.

Engineering Saccharomyces cerevisiae for the production of natural osmolyte glucosyl glycerol from sucrose and glycerol through Ccw12-based surface display of sucrose phosphorylase

BackgroundYeast Saccharomyces cerevisiae is widely recognised as a versatile chassis for constructing microbial cell factories. However, producing chemicals from toxic, highly concentrated, or cell-impermeable substrates, or chemicals dependent on enzymatic reactions incompatible with the yeast’s intracellular environment, remains challenging. One such chemical is 2-O-(α-D-glucopyranosyl)-sn-glycerol (glucosyl glycerol, αGG), a natural osmolyte used in the cosmetics and healthcare industries. This compound can be synthesised in a one-enzyme reaction from sucrose and glycerol by Leuconostoc mesenteroides sucrose phosphorylase (SucP), an enzyme which, in a low-water, glycerol-rich, phosphate-free environment, transfers the glucosyl moiety from sucrose to glycerol.ResultsIn this study, we engineered a yeast microbial cell factory for αGG production. For this purpose, we first focused on the abundant yeast GPI-anchored cell wall protein Ccw12 and used our insights to develop a miniature Ccw12-tag, which adds only 1.1 kDa to the enzyme of interest while enabling its covalent attachment to the cell wall. Next, we Ccw12-tagged SucP and expressed it in an invertase-negative strain of yeast S. cerevisiae from the PHO5 promoter, i.e., promoter strongly induced under phosphate-free conditions. Such SucP isoform, covalently C-terminally anchored to the outer cell surface, produced extracellularly 37.3 g l− 1 (146 mM) of αGG in five days, while the underlying chassis metabolised reaction by-products, thereby simplifying downstream processing.ConclusionsThe here-described S. cerevisiae strain, displaying C-terminally anchored sucrose phosphorylase on its cell surface, is the first eukaryotic microbial cell factory capable of a one-step αGG production from the readily available substrates sucrose and glycerol.

The concept of natural genome reconstruction. Part 1. Basic provisions of the “natural genome reconstruction” concept. Changing the genome of hematopoietic stem cells using several natural cellular mechanisms that are inherent in the hematopoietic cell and determine its biological status as “the source of the body’s reparative potential”

We present a series of articles proving the existence of a previously unknown mechanism of interaction between hematopoietic stem cells and extracellular double-stranded DNA (and, in particular, double-stranded DNA of the peripheral bloodstream), which explains the possibility of emergence and fixation of genetic information contained in double-stranded DNA of extracellular origin in hematopoietic stem cells. The concept of the possibility of stochastic or targeted changes in the genome of hematopoietic stem cells is formulated based on the discovery of new, previously unknown biological properties of poorly differentiated hematopoietic precursors. The main provisions of the concept are as follows. The hematopoietic stem cell takes up and internalizes fragments of extracellular double-stranded DNA via a natural mechanism. Specific groups of glycocalyx factors, including glycoproteins/proteoglycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors, take part in the internalization event. The binding sites for DNA fragments are heparin-binding domains and clusters of positively charged amino acid residues that are parts of protein molecules of these factors. Extracellular fragments delivered to the internal compartments of hematopoietic stem cells initiate terminal differentiation, colony formation, and proliferation of hematopoietic precursors. The molecular manifestation of these processes is the emergence and repair of pangenomic single-strand breaks. The occurrence of pangenomic single-strand breaks and restoration of genome (genomic DNA) integrity are associated with activation of a “recombinogenic situation” in the cell; during its active phase, stochastic homologous recombination or other recombination events between extracellular fragments localized in the nucleus and chromosomal DNA are possible. As a result, genetic material of initially extracellular localization either integrates into the recipient genome with the replacement of homologous chromosomal segments, or is transitively present in the nucleus and can manifest itself as a new genetic trait. It is assumed that as a result of stochastic acts of homologous exchange, chromosome loci are corrected in hematopoietic stem cells that have acquired mutations during the existence of the organism, which are the cause of clonal hematopoiesis associated with old age. In this regard, there is a fundamental possibility of changing the hematopoietic status of hematopoietic stem cells in the direction of polyclonality and the original diversity of clones. Such events can form the basis for the rejuvenation of the blood-forming cell system. The results of the laboratory’s work indicate that other stem cells in the body capture extracellular DNA fragments too. This fact creates a paradigm for the overall rejuvenation of the body.

Glycosylphosphatidylinositol-anchored proteins as non- DNA matter of inheritance: from molecular to cell to philosophical biology

Glycosylphosphatidylinositol-anchored proteins as non- DNA matter of inheritance: from molecular to cell to philosophical biology

A Thrilling Case of Anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare bone marrow failure disorder that presents with triad of peripheral blood cytopenias, hemolytic anemia and thrombosis. The absence of two glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that accounts for hemolysis and other PNH manifestations. GPI anchor protein deficiency is due to acquired somatic mutations in phosphatidylinositol glycan class A (PIGA) gene.Terminal complement inhibition with eculizumab and allogeneic bone marrow transplantation (BMT) are the only widely effective therapies for patients with classical PNH. Compared to eculizumab and ravulizumab that block the fifth component of complement (C5), pegcetacoplan blocks the third component of complement (C3).

Dampened α7 nAChR activity contributes to audiogenic seizures and hyperactivity in a mouse model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and often accompanied with debilitating pathologies including seizures and hyperactivity. FXS arises from a trinucleotide repeat expansion in the 5' UTR of the FMR1 gene that silences expression of the RNA-binding protein FMRP. Despite progress in understanding FMRP functions, the identification of effective therapeutic targets has lagged and at present there are no viable treatment options. Here we identify the α7 nicotinic acetylcholine receptor (nAChR) as candidate target for intervention in FXS. In the early postnatal hippocampus of Fmr1 knockout (KO) mice, an established pre-clinical model of FXS, the α7 nAChR accessory protein Ly6H is abnormally enriched at the neuronal surface and mislocalized in dendrites. Ly6H, a GPI-anchored protein, binds α7 nAChRs with high affinity and can limit α7 nAChR surface expression and signaling. We find that α7 nAChR-evoked Ca2+ responses are dampened in immature glutamatergic and GABAergic Fmr1 KO neurons compared to wild type. Knockdown of endogenous Ly6H in Fmr1 KO neurons is sufficient to rescue dampened α7 nAChR Ca2+ responses in vitro, providing evidence of a cell-autonomous role for Ly6H aberrant expression in α7 nAChR hypofunction. In line with intrinsic deficits in α7 nAChR activity in Fmr1 KO neurons, in vivo administration of the α7 nAChR-selective positive allosteric modulator PNU-120596 reduced hyperactivity and seizure severity in adolescent Fmr1 KO mice. Our mechanistic studies together with evidence of the in vivo efficacy of α7 nAChR augmentation implicate α7 nAChR hypofunction in FXS pathology.

Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance.

CD59, a GPI-anchored membrane protein, protects cancer cells from complement-dependent cytotoxicity (CDC) by inhibiting the formation of the membrane attack complex (MAC). It has been demonstrated to be overexpressed in most solid tumors, where it facilitates tumor cell escape from complement surveillance. The role of CD59 in cancer growth and interactions between CD59 and immune cells that modulate immune evasion has not been well explored. Using cancer patient database from The Cancer Genome Atlas (TCGA) and other public databases, we analyzed CD59 expression, its prognostic significance, and its association with immune cell infiltration in the tumor microenvironment, identifying associated genomic and functional networks and validating findings with invitro cell-line experimental data. This article describes the abundant expression of CD59 in multiple tumors such as cervical squamous cell carcinoma (CESC), kidney renal cell carcinoma (KIRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), and stomach adenocarcinoma (STAD), as well as in pan-cancer, using The Cancer Genome Atlas (TCGA) database and confirmed using multiple cancer cell lines. The expression of CD59 significantly alters the overall survival (OS) of patients with multiple malignancies such as CESC, GBM, HNSC, and STAD. Further, the correlation between CD59 and Treg and/or MDSC in the tumor microenvironment (TME) has shown to be strongly associated with poor outcomes in CESC, GBM, HNSC, and STAD as these tumors express high FOXP3 compared to KIRC. Moreover, unfavorable outcomes were strongly associated with the expression of CD59 and M2 tumor-associated macrophage infiltration in the TME via the IL10/pSTAT3 pathway in CESC and GBM but not in KIRC. In addition, TGFβ1-dominant cancers such as CESC, GBM, and HNSC showed a high correlation between CD59 and TGFβ1, leading to suppression of cytotoxic T cell activity. Overall, the correlation between CD59 and immune cells predicts its prognosis as unfavorable in CESC, GBM, HNSC, and STAD while being favorable in KIRC.

Deciphering the Cell Surface Sugar-Coating via Biochemical Pathways.

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i. e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol-anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and advancing glycan-targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid-based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications.

Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1.

The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis.

Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.

Investigation on the influence of GPI-AP for the production of malic acid in Aspergillus niger

Based on the potential of microbial fermentation in the sustainable and environmentally friendly production of L-malic acid (L-MA), this study aims to investigate the effect of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on enhancing the stability of Aspergillus niger (A. niger) cell wall to increase L-MA production. Through experiments, we discovered and screened GPI2, a GPI-anchored cell surface glycoprotein, and confirmed for the first time that it has a promoting effect on L-MA, a metabolite of A. niger. This recombinant strain RG0098 that overexpressed gpi2 showed an increase in cell wall thickness and robustness, a decrease in intracellular reactive oxygen species (ROS) levels, which improved the antioxidant capacity. After 108 h of fermentation, the production of L-MA reached 114.7 ± 2.12 g/L, and the productivity was 1.06 ± 0.025 g/L/h. The strain RG0098 has enhanced the productivity of L-MA by a notable margin of 49.7 %. This study provides a sustainable and environmentally friendly pathway for the production of L-MA through microbial fermentation.

A-152 Enhancing PNH Diagnosis with Multicolor Flow Cytometry Immunophenotyping Protocol in a Medical Center in Taiwan

Abstract Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from mutations in the X-linked PIG-A gene, leading to an inability to produce GPI-anchored proteins on RBCs and WBCs. PNH presents in three variants: classic (hemolytic), associated with specified bone marrow disorders, and subclinical. Flow cytometric analysis stands as the gold standard for PNH detection, crucial in diagnosis, monitoring, and clinical management. Recognizing potential variations in PNH testing among labs, it is essential to establish a precise and consistent protocol for accurate diagnosis and patient management. This report aims to demonstrate the effectiveness of a PNH immunophenotyping protocol adhering to ICCS/ESCCA PNH consensus guidelines. Methods A total of 501 samples were analyzed from Jan 1st, 2021 to Dec 31st, 2023. All the acquisitions were accomplished by a 3-laser and 10-PMT Navios EX (Beckman Coulter) flow cytometry. A typical combination of anti-CD235a-FITC (clone KC16) and anti-CD59-PE (clone MEM43) antibodies were adopted to identify the PNH clone size in the RBC tube. To achieve higher sensitivities in PNH granulocyte and PNH monocyte detection, a single tube, 6-color mixture of FLAER, monoclonal antibodies including CD14-APC, CD24-PE, CD33-APC-H7, CD15-PacB, and CD45-KrO, to immunostaining simultaneously was developed. An unstained control was run after the full-stained tube to determine the position of threshold for positivity, and to exam the potential carryover into the test sample. Results RBC and granulocyte assays can detect PNH phenotypes at the 0.009% level or higher with more than 50,000 cells of gated cells acquired, while the monocyte assay can detect at >=0.069% with at least 20,000 cells acquired. Analysis was conducted on five hundred and one patients to investigate potential PNH clones in their blood cells. Among them, 45 patients were identified as having classic PNH, while PNH clones were not detected in 168 cases, constituting 33.5% of total cases. Minor PNH clone (<1.0%) in single cell lineage were observed in 144 cases, making up 28.7% of all cases. Minor PNH clones found in more than one cell lineages were found in 155 cases (30.9% of 501). Additionally, minor PNH clones were identified across RBCs, granulocytes, and monocytes, with percentages of 32.9%, 23.8%, and 43.9%, respectively of all patients. Notably, monocyte clones exceeded granulocyte clones in 23.0% of cases with WBC PNH clones, irrespective of clone sizes. Furthermore, we investigated seven patients exhibiting persistent minor PNH clones in 2 to 3 cell lineages. Among them, five had aplastic anemia or myelodysplastic syndromes, while one underwent evaluation for young stroke and one was investigated for venous thromboembolism. Conclusions Flow cytometry labs aim not only to identify classic PNH patients but also to consistently detect minor or small clones in individuals with bone marrow disorders or subclinical conditions. Our findings are compatible with international reports, underscoring the importance of routinely examining patients with minor clones. Such assays serve as essential tools for ongoing monitoring, facilitating PNH diagnosis and management by physicians.

Enhancing the Cytotoxicity and Apoptotic Efficacy of Parasporin-2-Derived Variants (Mpp46Aa1) on Cancer Cell Lines.

Parasporin PS2Aa1, recently renamed Mpp46Aa1, is an anti-cancer protein known for its selectivity against various human cancer cell lines. We genetically modified native PS2Aa1 to create a library of approximately 100 mutants. From this library, we selected promising mutants based on their half-maximal inhibitory concentration (IC50) and sequence variations. In this study, Variant 3-35, with the G257V substitution, demonstrated increased cytotoxicity and selectivity against the colon cancer cell line SW480. Conversely, Variant N65, featuring substitutions N92D, K175R, and S218G, yielded the most favorable results against the cancer cell lines SW-620, MOLT-4, and Jurkat. The caspase 3/7 and 9, Annexin V-Cy3 and 6-GFDA activities, and, most notably, mitochondrial membrane permeabilization assays confirmed the apoptotic marker elevation. These findings indicate that residues 92, 175, 218, and 257 may play a critical role in the cytotoxic activity and selectivity. We successfully obtained genetically improved variants with substitutions at these key amino acid positions. Additionally, we conducted molecular dynamic simulations to explore the potential interactions between PS2Aa1 and the CD59 GPI-anchored protein. The simulation results revealed that residues 57, 92, and 101 were consistently present, suggesting their possible significance in the interactions between parasporin and the CD59 protein.

Glycosylphosphatidylinositol-anchored lipid transfer proteins influence root cap cuticle formation at primary root tips, promoting NaCl tolerance in Arabidopsis seedlings.

Root cap cuticles (RCCs), comprising mainly very-long-chain fatty acids (VLCFAs), promote salt tolerance by preventing ion influx. Glycosylphosphatidylinositol-anchored lipid transfer protein (LTPG)1 and LTPG2 participate in VLCFA deposition in the extracellular region, aiding RCC formation in the lateral roots. In this study, we investigated whether LTPG1 and LTPG2 have similar functions in the primary roots of young Arabidopsis thaliana. Phenotypic analyses, fluorescence microscopy, and quantitative real-time reverse transcription polymerase chain reaction confirmed that NaCl exposure induced LTPG1 and LTPG2 expression and promoted RCC formation in young primary roots. The loss of RCC in the ltpg1 and ltpg2 mutants resulted in increased NaCl sensitivity of root elongation. NaCl also upregulated the expression of several NaCl-responsive genes in ltpg1 and ltpg2. We conclude that RCC formation via LTPG function is pivotal in enhancing salt tolerance in young primary roots.

GPI anchoring controls cell wall integrity, immune evasion and surface localization of ChFEM1 for infection of Cochlibolus heterostrophus1

Glycosylphosphatidylinositol (GPI) anchoring is one of the common post-translational modifications in eukaryotic cells. In fungi, it exerts a wide range of biological functions by targeting proteins to the cell wall, but only few studies focus on the roles of GPI anchoring in plant pathogenic fungi. Here, we reveal a role of GPI anchoring in the maize fungal pathogen Cochlibolus heterostrophus. We found that GPI-anchored proteins were widely accumulated in hyphae, appressorium and infection hyphae of C. heterostrophus. Deletion of ChGPI7, which encodes a key enzyme involved in the biosynthesis of GPI anchors, resulted in significant reduction of vegetative growth and conidiation, as well as virulence due to impairment of appressorium formation and invasive growth. The ΔChgpi7 mutants also showed severe defects in cell wall integrity, resulting in a significant reduction of stress resistance. Deletion of ChGPI7 and hydrofluoric acid (HF) pyridine treatment both led to removal of cell wall GPI-anchored proteins and exposure of chitin, the results suggested that GPI anchored proteins could protect chitin from host immune recognition. A total of 124 proteins were predicted to be GPI anchored proteins in C. heterostrophus, including a putative cell wall glycoprotein ChFEM1. Deletion of ChFEM1 also resulted in significant reduction in virulence and defects in infection structures, as well as cell wall integrity. We further found that cell wall localization and protein abundance of ChFEM1 were affected by ChGPI7. Our results showed that GPI anchoring regulates cell wall integrity and immune evasion for infection of C. heterostrophus.

Semaphorin heterodimerization in cis regulates membrane targeting and neocortical wiring

Disruption of neocortical circuitry and architecture in humans causes numerous neurodevelopmental disorders. Neocortical cytoarchitecture is orchestrated by various transcription factors such as Satb2 that control target genes during strict time windows. In humans, mutations of SATB2 cause SATB2 Associated Syndrome (SAS), a multisymptomatic syndrome involving epilepsy, intellectual disability, speech delay, and craniofacial defects. Here we show that Satb2 controls neuronal migration and callosal axonal outgrowth during murine neocortical development by inducing the expression of the GPI-anchored protein, Semaphorin 7A (Sema7A). We find that Sema7A exerts this biological activity by heterodimerizing in cis with the transmembrane semaphorin, Sema4D. We could also observe that heterodimerization with Sema7A promotes targeting of Sema4D to the plasma membrane in vitro. Finally, we report an epilepsy-associated de novo mutation in Sema4D (Q497P) that inhibits normal glycosylation and plasma membrane localization of Sema4D-associated complexes. These results suggest that neuronal use of semaphorins during neocortical development is heteromeric, and a greater signaling complexity exists than was previously thought.

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

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

Identification of CNTN2 as a genetic modifier of PIGA-CDG through pedigree analysis of a family with incomplete penetrance and functional testing in Drosophila.

Loss of function mutations in the X-linked PIGA gene lead to PIGA-CDG, an ultra-rare congenital disorder of glycosylation (CDG), typically presenting with seizures, hypotonia, and neurodevelopmental delay. We identified two brothers (probands) with PIGA-CDG, presenting with epilepsy and mild developmental delay. Both probands carry PIGA S132C , an ultra-rare variant predicted to be damaging. Strikingly, the maternal grandfather and a great-uncle also carry PIGA S132C , but neither presents with symptoms associated with PIGA-CDG. We hypothesized genetic modifiers may contribute to this reduced penetrance. Using whole genome sequencing and pedigree analysis, we identified possible susceptibility variants found in the probands and not in carriers and possible protective variants found in the carriers and not in the probands. Candidate variants included heterozygous, damaging variants in three genes also involved directly in GPI-anchor biosynthesis and a few genes involved in other glycosylation pathways or encoding GPI-anchored proteins. We functionally tested the predicted modifiers using a Drosophila eye-based model of PIGA-CDG. We found that loss of CNTN2 , a predicted protective modifier, rescues loss of PIGA in Drosophila eye-based model, like what we predict in the family. Further testing found that loss of CNTN2 also rescues patient-relevant phenotypes, including seizures and climbing defects in Drosophila neurological models of PIGA-CDG. By using pedigree information, genome sequencing, and in vivo testing, we identified CNTN2 as a strong candidate modifier that could explain the incomplete penetrance in this family. Identifying and studying rare disease modifier genes in human pedigrees may lead to pathways and targets that may be developed into therapies.

Inherited glycosylphosphatidylinositol deficiency: a review from molecular and clinical perspectives.

Glycosylphosphatidylinositol (GPI) is a highly conserved post-translational modification in eukaryotes, which is essential for anchoring various proteins to the cell surface. Dysfunction of GPI biogenesis leads to human diseases, such as inherited GPI deficiency (IGD) caused by germline mutations in GPI-related genes. With accumulating reports on individuals with IGD, there has been increasing interest and studies on disease mechanism, diagnosis, and therapy. This review outlines the biosynthetic pathway of GPI-anchored proteins (GPI-APs) and summarizes clinical IGD cases from a molecular perspective. We also review current diagnostic and therapeutic approaches for IGD. Finally, we discuss future research directions to facilitate the understanding and treatment of GPI-related disorders.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.