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Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies usingiDaf1 siRNA/idemonstrated worsened lung fibrosis detected by higher mRNA levels ofiCol1a1/iand epithelial injury-relatediMuc1/iandiSnai1,/iwith exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungsivia/iDAF induction that will restrain complement dysregulation and lung injury.

Pattern recognition receptors (PRRs) sense ligands in pattern-triggered immunity (PTI). Plant PRRs include numerous receptor-like proteins (RLPs), but many RLPs remain functionally uncharacterized. Here, we examine an Arabidopsis thaliana RLP, RLP53, which positively regulates immune signaling. Our forward genetic screen for suppressors of enhanced disease resistance1 (edr1) identified a point mutation in RLP53 that fully suppresses disease resistance and mildew-induced cell death in edr1 mutants. The rlp53 mutants showed enhanced susceptibility to virulent pathogens, including fungi, oomycetes, and bacteria, indicating that RLP53 is important for plant immunity. The ectodomain of RLP53 contains leucine-rich repeat (LRR) motifs. RLP53 constitutively associates with the LRR receptor-like kinase SUPPRESSOR OF BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE (BAK1)-INTERACTING RECEPTOR KINASE1 (SOBIR1) and interacts with the co-receptor BAK1 in a pathogen-induced manner. The double mutation sobir1-12 bak1-5 suppresses edr1-mediated disease resistance, suggesting that EDR1 negatively regulates PTI modulated by the RLP53-SOBIR1-BAK1 complex. Moreover, the glycosylphosphatidylinositol (GPI)-anchored protein LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) interacts with RLP53 and mediates RLP53 accumulation in the plasma membrane. We thus uncovered the role of a novel RLP and its associated immune complex in plant defense responses and revealed a potential new mechanism underlying regulation of RLP immune function by a GPI-anchored protein.

A bifunctional derivative of the core structure of glycosylphosphatidylinositol (GPI) anchors having a clickable alkynyl group and a photoreactive diazirine group attached to the GPI glucosamine and lipid moieties, respectively, was synthesized from myo-inositol, D-glucosamine, and (R)-1,2-O-acetonized glycerol. The target molecule should be useful for the investigation of GPI-interacting components in the cell membrane that play a key role in the signal transduction and other biological functions of GPI-anchored proteins.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The

Many calcifying organisms utilize metabolic CO 2 to generate CaCO 3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO 2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H + -selective electrodes were used to demonstrate eCA-catalyzed CO 2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO 2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pH i regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO 3 − uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO 2 by Cara7 coupled to HCO 3 − uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO 2 to generate a versatile construction material.

Glycosphingolipids (GSLs) form GSL-enriched microdomains, together with sphingomyelin (SM), cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and membrane-associated signaling molecules. GSL-enriched microdomains mediate a variety of physiological functions, including innate immune responses. Innate immune responses are initialized by the binding of host pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) expressed in microorganisms. This binding triggers phagocytosis and leads to the formation of a phagosome-containing microorganism and the subsequent lysosomal fusion with a phagosome. To detect the molecular interaction between GSL-enriched microdomains, sphingolipids, and signaling molecules from the uptake of the microorganism until the phagosome-containing microorganism fuses with lysosomes, biochemical and microscopic approaches are indispensable. Here, we describe the detailed methods for isolating phagosomes and observing the molecular interaction using a superresolution microscope. Our methodology provides a strategy for exploring the molecular interaction between the host and pathogen and for developing new treatment approaches.

Glycosylphosphatidylinositol (GPI) anchoring is a common protein modification that targets proteins to the plasma membrane (PM). Knowledge about the GPI lipid tail, which guides the secretion of GPI-anchored proteins (GPI-APs), is limited in plants. Here, we report that rice (Oryza sativa) BRITTLE CULM16 (BC16), a membrane-bound O-acyltransferase (MBOAT) remodels GPI lipid tails and governs cell wall biomechanics. The bc16 mutant exhibits fragile internodes, resulting from reduced cell wall thickness and cellulose content. BC16 is the only MBOAT in rice and is located in the endoplasmic reticulum and Golgi apparatus. Yeast gup1Δ mutant restoring assay and GPI lipid composition analysis demonstrated BC16 as a GPI lipid remodelase. Loss of BC16 alters GPI lipid structure and disturbs the targeting of BC1, a GPI-AP for cellulose biosynthesis, to the PM lipid nanodomains. Atomic force microscopy revealed compromised deposition of cellulosic nanofibers in bc16, leading to an increased Young's modulus and abnormal mechanical properties. Therefore, BC16-mediated lipid remodeling directs the GPI-APs, such as BC1, to the cell surface to fulfill multiple functions, including cellulose organization. Our work unravels a mechanism by which GPI lipids are remodeled in plants and provides insights into the control of cell wall biomechanics, offering a tool for breeding elite crops with improved support strength.

The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.

Lily bulb and Rehmannia decoction (LBRD), consisting ofThis study was aimed to investigate the metabolic mechanism of LBRD in treating depression.Network pharmacology was conducted using SwissTargetPrediction, DisGeNET, DrugBank, Metascape, etc., to construct component-target-pathway networks. The depression-like model was induced by intraperitoneal injection with lipopolysaccharide (LPS) (0.3 mg/kg) for 14 consecutive days. After the administration of LBRD (90 g/kg) and fluoxetine (2 mg/kg) for 14 days, we assessed behaviour and the levels of neurotransmitter, inflammatory cytokine and circulating stress hormone. Prefrontal metabolites of rats were detected by using liquid chromatography-mass spectrometry metabolomics method.The results of network pharmacology showed that LBRD mainly acted on neurotransmitter and second messenger pathways. Compared to the model group, LBRD significantly ameliorated depressive phenotypes and increased the level of 5-HT (13.4%) and GABA (24.8%), as well as decreased IL-1β (30.7%), IL-6 (32.8%) and TNF-α (26.6%). Followed by LBRD treatment, the main metabolites in prefrontal tissue were contributed to retrograde endocannabinoid signalling, glycerophospholipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, autophagy signal pathway, etc.LBRD were effective at increasing neurotransmitter, attenuating proinflammatory cytokine and regulating glycerophospholipid metabolism and glutamatergic synapse, thereby ameliorating depressive phenotypes. This research will offer reference for elucidating the metabolomic mechanism underlying novel antidepressant agents contained LBRD formula.

CD160 is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein expressed on cytotoxic natural killer (NK) cells and T-cell subsets. It plays a crucial role in the activation of NK-cell cytotoxicity and cytokine production. It also modulates the immune system and is involved in some pathologies, such as cancer. CD160 is abnormally expressed in B-cell chronic lymphocytic leukemia (CLL) but not expressed in normal B lymphocytes. Its expression in CLL enhances tumor cell proliferation and resistance to apoptosis. CD160 is also a potential prognostic marker for the detection of minimal residual disease (MRD) in CLL, which is important for the clinical management of CLL, the prevention of disease relapse, and the achievement of complete remission. In this review, we present an overview of CD160 and its involvement in the pathophysiology of CLL. We also discuss its use as a prognostic marker for the assessment of MRD in CLL.

During pearl culture, the excess immune responses may induce nucleus rejection and death of pearl oysters after transplantation. To better understand the immune response and pearl formation, lipidomic analysis was applied to investigate changes in the serum lipid profile of pearl oyster

Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic β3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed.

Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by

African trypanosomes evade the immune system of the mammalian host by the antigenic variation of the predominant glycosylphosphatidylinositol (GPI)-anchored surface protein, variant surface glycoprotein (VSG). VSG is a very stable protein that is turned over from the cell surface with a long half-life (~26 h), allowing newly synthesized VSG to populate the surface. We have recently demonstrated that VSG turnover under normal growth is mediated by a combination of GPI hydrolysis and direct shedding with intact GPI anchors. VSG synthesis is tightly regulated in dividing trypanosomes, and when subjected to RNA interference (RNAi) silencing, cells display rapid cell cycle arrest in order to conserve VSG density on the cell surface (K. Sheader, S. Vaughan, J. Minchin, K. Hughes, et al., Proc Natl Acad Sci U S A 102:8716-8721, 2005, https://doi.org/10.1073/pnas.0501886102). Arrested cells also display an altered morphology of secretory organelles-engorgement of the

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by a mutation of the X-linked PIGA gene, resulting in a deficient expression of glycosylphosphatidylinositol (GPI)-anchored proteins. While large clonal expansions of GPI(-) cells cause hemolytic symptoms, tiny GPI(-) cell populations can be found in healthy individuals and remain miniscule throughout life. The slight expansion of PNH clones often occurs in patients with acquired aplastic anemia (AA), an autoimmune bone marrow (BM) failure caused by autoreactive cytotoxic T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs). The presence of PNH clones is thought to represent the immune pathophysiology of BM failure and be derived from GPI(-) HSPCs that evaded immune attack against HSPCs. However, which mechanisms underlie the selection of GPI(-) HSPCs as well as their overwhelming clonal expansion remains unclear. Ancestral or secondary somatic mutations in GPI(-) HSPCs contribute to the clonal expansion of the aberrant HSPCs in certain patients with PNH; however, it remains unclear whether such driver mutations are responsible for clonal expansion of all patients. Increased sensitivity to TGF-β in GPI(-) HSPCs partly explains the predominance of GPI(-) erythrocytes in immune-mediated BM failure. CD4

Aim: To investigate the short-term efficacy and safety of high-dose pyridoxine and pyridoxal 5-phosphate (P5P) in the treatment of inherited glycosylphosphatidylinositol (GPI) deficiency-associated epilepsy. Method: Participants with genetically confirmed GPI deficiency were treated with oral pyridoxine or P5P as compassionate use in an agreed-upon clinical regimen. Pyridoxine (20–30 mg/kg/day) was used for 3 months. Baseline evaluation included 4 weeks of prospective seizure data and one video electroencephalogram (EEG). Seizure frequency was captured daily. The EEG was repeated after reaching maximum dosage of pyridoxine. Pyridoxine was switched to P5P (20–30 mg/kg/day) if seizure burden was unchanged after 3 months' treatment. Another EEG was done after 3 months of P5P treatment. Primary outcome measures were reduction of seizure frequency and EEG improvements. Results: Seven participants (one female, six males; age range 5–23 year; mean age 11 years 10 months, SD 5 year 2 months) were included. The genetic causes of inherited GPI deficiency were phosphatidylinositol N-acetylglucosaminyltransferase subunit A/T/V deficiency. All had drug-resistant epilepsy and neurodevelopmental impairment. We observed more than 50% seizure frequency reduction in 2 out of 7 and less than 50% reduction in another 3 out of 7 participants. No participants reached seizure freedom. No remarkable changes in electrophysiological findings were observed in 6 out of 7 participants treated with pyridoxine or P5P when comparing the baseline and follow-up EEGs. Interpretation: We observed no long-lasting electrophysiological improvements during treatment but pyridoxine may reduce seizure frequency or burden in inherited GPI deficiency. What this paper adds: Inherited glycosylphosphatidylinositol (GPI) deficiency often causes early-onset and drug-resistant epilepsy. Vitamin B 6 is a potential disease-specific treatment; however, efficacy and safety are ill-defined. Pyridoxine may reduce seizure frequency or burden in inherited GPI deficiency. Pyridoxine and P5P could prove to be a useful treatment in some individuals with inherited GPI deficiency and epilepsy.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI and the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show that EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the Ect-5'-nucleotidase and Netrin G2. We find that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3, and with a small fraction in wild-type cells. Our findings modify the current view of GPI anchoring and provide a mechanistic basis for IGDs caused by PIGG mutations.

Glypican 1 (Gpc1) is a heparan sulfate proteoglycan attached to the cell membrane via a glycosylphosphatidylinositol anchor, where it holds glycosaminoglycans nearby. We have recently shown that Gpc1 knockout (Gpc1We used echocardiography and in vivo (electrocardiographic recordings) and in vitro (patch clamping) electrophysiology to study mechanical and electric properties of mice hearts. We used RT-PCR to probe KGpc1Our data reveals an unprecedented connection between Gpc1 and voltage-gated K

Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. Covalent addition of structurally diverse GPI anchorages at the carboxyl termini of target proteins is catalyzed by an endoplasmic reticulum integral membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Despite its important role in developmental and cancer biology, a detailed picture of this intricate multi-component machinery remains elusive. Here, we report the cryo-electron microscopy (EM) structure of the human GPI-T complex at a global 2.53 Å resolution, revealing an assembly mechanism whereby the catalytic subunit PIGK is optimally positioned to accommodate its characteristic amphipathic substrates. Further, structural and functional characterizations suggest a previously unrecognized composite GPI-binding site formed by PIGU/PIGK/PIGT subunits and rationalize genetic diseases associated with GPI-T point mutations. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.

Wnt signalling pathways play pivotal roles in development, homeostasis and human diseases, and are tightly regulated. We previously identified Tiki as a novel family of Wnt inhibitory proteases. Tiki proteins were predicted as type I transmembrane proteins and can act in both Wnt-producing and Wnt-responsive cells. Here, we characterize Tiki proteins as glycosylphosphatidylinositol (GPI)-anchored proteases. TIKI1/2 proteins are enriched on the detergent-resistant membrane microdomains and can be released from the plasma membrane by GPI-specific glycerophosphodiesterases GDE3 and GDE6, but not by GDE2. The GPI anchor determines the cellular localization of Tiki proteins and their regulation by GDEs, but not their inhibitory activity on Wnt signalling. Our study uncovered novel characteristics and potential regulations of the Tiki family proteases.

Glycosylphosphatidylinositol (GPI) anchoring is a common post-translational modification in eukaryotic cells and has been demonstrated to have a wide range of biological functions, such as signal transduction, cellular adhesion, protein transport, immune response, and maintaining cell wall integrity. More than 25 proteins have been proven to participate in the GPI anchor synthesis pathway which occurs in the cytoplasmic and the luminal face of the ER membrane. However, the essential proteins of the GPI anchor synthesis pathway are still less characterized in maize pathogen

Glycosylphosphatidylinositol (GPI)-anchored proteins are found in all eukaryotes and are especially abundant on the surface of protozoan parasites such as Trypanosoma brucei. GPI-mannosyltransferase I (GPI-MT-I) catalyses the addition of the first of three mannoses that make up the glycan core of GPI. Mammalian and yeast GPI-MT-I consist of two essential subunits, the catalytic subunit Gpi14/Pig-M and the accessory subunit Pbn1/Pig-X (mammals/yeast). T. brucei GPI-MT-I has been highlighted as a potential anti-trypanosome drug target but has not been fully characterised. Here, we show that T. brucei GPI-MT-I also has two subunits, TbGPI14 and TbPBN1. Using TbGPI14 deletion, and TbPBN1 RNAi-mediated depletion, we show that both proteins are essential for the mannosyltransferase activity needed for GPI synthesis and surface expression of GPI-anchored proteins. In addition, using native PAGE and co-immunoprecipitation analyses, we demonstrate that TbGPI14 and TbPBN1 interact to form a higher-order complex. Finally, we show that yeast Gpi14 does not restore GPI-MT-I function in TbGPI14 knockout trypanosomes, consistent with previously demonstrated species specificity within GPI-MT-I subunit associations. The identification of an essential trypanosome GPI-MT-I subcomponent indicates wide conservation of the heterodimeric architecture unusual for a glycosyltransferase, leaving open the question of the role of the non-catalytic TbPBN1 subunit in GPI-MT-I function.

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.

Glycosylphosphatidylinositol (GPI)-linked molecules are surface-exposed membrane components that influence the infectivity, virulence and transmission of many eukaryotic pathogens. Procyclic (insect midgut) forms of Trypanosoma brucei do not require GPI-anchored proteins for growth in suspension culture. Deletion of TbGPI8, and inactivation of the GPI:protein transamidase complex, is tolerated by cultured procyclic forms. Using a conditional knockout, we show TbGPI8 is required for social motility (SoMo). This collective migration by cultured early procyclic forms has been linked to colonization of the tsetse fly digestive tract. The SoMo-negative phenotype was observed after a lag phase with respect to loss of TbGPI8 and correlated with an unexpectedly slow loss of procyclins, the major GPI-anchored proteins. Procyclins are not essential for SoMo, however, suggesting a requirement for at least one other GPI-anchored protein. Loss of TbGPI8 initiates the transition from early to late procyclic forms; this effect was observed in a subpopulation in suspension culture, and was more pronounced when cells were cultured on SoMo plates. Our results indicate two, potentially interlinked, scenarios that may explain the previously reported failure of TbGPI8 deletion mutants to establish a midgut infection in the tsetse fly: interference with stage-specific gene expression and absence of SoMo.

Glycosylphosphatidylinositols (GPIs) are complex glycolipids that act as membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) by generation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). The second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the luminal face for later biosynthetic steps and attachment to proteins. The mechanism of the luminal translocation of GlcN-PI is unclear. Here, we report a genome-wide CRISPR knockout screen of genes required for rescuing GPI-anchored protein expression after addition of chemically synthesized GlcNAc-PI to PIGA-knockout cells that cannot synthesize GlcNAc-PI. We identified CLPTM1L (cleft lip and palate transmembrane protein 1-like), an ER-resident multipass membrane protein, as a GlcN-PI scramblase required for efficient biosynthesis of GPIs. Knockout of CLPTM1L in PIGA-knockout cells impaired the efficient utilization of chemically synthesized GlcNAc-PI and GlcN-PI for GPI biosynthesis. Purified CLPTM1L scrambled GlcN-PI, GlcNAc-PI, PI, and several other phospholipids in vitro. CLPTM1L, a member of the PQ-loop family of proteins, represents a type of lipid scramblase having no structural similarity to known lipid scramblases. Knockout of CLPTM1L in various wild-type mammalian cultured cells partially decreased the level of GPI-anchored proteins. These results suggest that CLPTM1L is the major lipid scramblase involved in cytosol-to-lumen translocation of GlcN-PI across the ER membrane for efficient GPI biosynthesis.

We examined the frequencies and sizes of glycosylphosphatidylinositol (GPI)-deficient cells as per the International Clinical Cytometry Society/European Society for Clinical Cell Analysis (ICCS/ESCCA) consensus guidelines for the high-sensitivity detection of GPI-deficient cells. In 2018, the ICCS/ESCCA guidelines for the high-sensitivity detection of GPI-deficient cells were published. We evaluated frequencies and sizes of GPI-deficient red blood cells (RBCs), neutrophils, and monocytes as determined using the ICCS/ESCCA guidelines and Clinical and Laboratory Standards Institute (CLSI) guidelines in patients with a hematologic malignancy, aplastic anemia, or cytopenia. A total of 106 (38.7%) patients exhibited GPI deficiency in at least one blood cell lineage. GPI-deficient cells of one or more lineages were found in 62.7% of patients with a hematologic malignancy, 51.1% of patients with aplastic anemia, and 23.4% of patients with cytopenia. GPI-deficient monocytes were most frequently detected in all three groups. By population size, GPI-deficient clones (>1%) in monocytes were mostly detected in patients with a hematologic malignancy or aplastic anemia. Rare cells with GPI deficiency (

International audience; It is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature as well as decreased phagocytosis compared. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antifungals, treatment strategies or vaccines to manage C. neoformans disease.

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

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes., Gangliosides such as GM1 present in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. Here the authors show that the acyl chain structure of GM1 determines the establishment of nanodomains when actively clustered by actin, which depended on membrane cholesterol and phosphatidylserine or superimposed by the GM1-binding bacterial cholera toxin.

Carbohydrates have quite complicated micro heterogenic structure which may undergo different structural transitions. Due to their extreme flexibility it is very difficult to investigate such structural changes experimentally. In these studies we want to predict what structural and conformational changes are possible in the carbohydrate glycosylphosphatidylinositol anchor chain (GPI): the tetrasaccharide with the unique sequence Man-α(1 → 2)-Man-α(1 → 6)-Man-α(1 → 4)-GlcN-α. This is a very important biomolecule associated with the processes of transmitting various types of signals in the cells of living organisms. In order to investigate conformational and structural changes in GPI we use in these studies the theoretical Enforced Geometry Optimization (EGO) method. In this method a molecule is exposed to a mechanical stress caused by external forces applied to selected atoms. It turned out that under external stretching forces the mannopyranose unit can change its

The diverse applications of porphyrin-based nano-sized metal-organic frameworks (NMOFs) lead to great exposure risks to human and environment. Understanding the cellular biological effects (such as toxicity, distribution, and localization) of porphyrinic NMOFs is a prerequisite to the assessment of their health risk. However, the characteristics of distribution, localization, and immune response induced by porphyrinic NMOFs have not been studied yet. Here, we report the size-dependent biological effects of porphyrinic NMOFs under sublethal dose. Various sizes of PCN-224 (30, 90, and 180 nm) were taken as model porphyrinic NMOFs. We found that 30 nm PCN-224 gave the highest uptake content, followed by 90 and 180 nm PCN-224. The mechanism for uptake was clathrin-mediated for 30 and 90 nm PCN-224, but clathrin- and glycosylphosphatidylinositol-mediated for 180 nm PCN-224. All PCN-224 were localized in lysosome with size-dependent velocity of colocalization transport. 30 nm PCN-224 induced the highest released cytokines than 90 and 180 nm PCN-224 accompanied with the activation of NF-κB pathway. This work reveals the mechanisms for the endocytosis of PCN-224 and the release of cytokine induced by PCN-224, which is helpful for the health risk assessment of NMOFs.

Immunoglobulin-like cell adhesion molecule (IgLON4) is a glycosylphosphatidylinositol-anchored membrane protein that has been associated with neuronal growth and connectivity, and its deficiency has been linked to increased fat mass and low muscle mass. Adequate information on IgLON4 is lacking, especially in the context of skeletal muscle. In this study, we report that IgLON4 is profusely expressed in mouse muscles and is intensely localized on the cell membrane. IgLON4 expression was elevated in CTX-injected mouse muscles, which confirmed its role during muscle regeneration, and was abundantly expressed at high concentrations at cell-to-cell adhesion and interaction sites during muscle differentiation. IgLON4 inhibition profoundly affected myotube alignment, and directional analysis confirmed this effect. Additionally, results demonstrating a link between IgLON4 and lipid rafts during myogenic differentiation suggest that IgLON4 promotes differentiation by increasing lipid raft accumulation. These findings support the notion that a well-aligned environment promotes myoblast differentiation. Collectively, IgLON4 plays a novel role in myogenesis and regeneration, facilitates myotube orientation, and is involved in lipid raft accumulation.

We show that a gene (CpGap1) encoding a glycosylphosphatidylinositol-anchored protein (GPI-AP) of the chestnut blight fungus Cryphonectria parasitica is differentially expressed by Cryphonectria hypovirus 1 (CHV1) infection. Functional analysis using a CpGap1-null mutant results in no observed changes in cultural morphology other than hypersensitivity to ROS. Analysis of the protein product of the CpGap1 gene (CpGAP1) confirmed motifs with antioxidizing properties. The virulence of the CpGap1-null mutant is significantly decreased, and phytotoxic activity is seen in the peptides of CpGAP1. CHV1 transfer to the CpGap1-null mutant results in severely retarded colonial growth, and virus-titer is significantly increased in the mycelia of CHV1-infected CpGap1-null mutant. These results indicate that CpGAP1 functions as a protective barrier against plant defenses, but also acts as a virulence factor. Moreover, our study demonstrates that the CpGap1 gene is a host-tolerating antiviral factor that helps maintain fungal growth and suppress viral titer after CHV1 infection.

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.

Misfolded glycosylphosphatidylinositol-anchored prion protein (PrP) is primarily degraded in lysosomes but is often rapidly removed from the cell surface before endocytosis in a preemptive manner. However, this mechanism is poorly understood. In this study, we discovered a disease-causing prion mutation (Q212P) that exceptionally promoted the extracellular release of PrP. Spatiotemporal analyses combined with genome editing identified the role of sheddase ADAM10 in Q212P shedding from the cell surface. ADAM10 was observed to catalytically interacts with Q212P but non-catalytically with wild-type PrP (wtPrP). This intrinsic difference in the interaction of ADAM10 between Q212P and wtPrP allowed Q212P to selectively access the sheddase activity of ADAM10 in a redox-sensitive manner. In addition, redox perturbation instigated the latent misfolding propensity of Q212P and disrupted the catalytic interaction between PrP and ADAM10, resulting in the accumulation of misfolded PrP on the cell surface. Upon recovery, active ADAM10 was able to reversibly release the surface Q212P. However, it might prove detrimental if unregulated resulting in unexpected proteotoxicity. This study provides a molecular basis of the mutant-selective shedding of PrP by demonstrating the catalytic interaction of ADAM10 with Q212P.

GPI-APs are a family of proteins attached to the plasma membrane by a glycoplipid that undergoes remodeling of the glycan and lipid structure during transport to the cell surface. We describe a protocol to induce the synthesis of a GPI-anchored protein whereby EtNP is added to Man2 but not removed. By temporally manipulating the expression of Gpi7p, the enzyme that adds EtNP to Man2, in ted1Δ dcr2Δ cells prior to the expression of a canonical GPI-AP (mNeon-Gas1p), EtNP is attached to Man2 of de novo synthesized mNeon-Gas1p and cannot be removed. This strategy provides a means to temporally and spatially track the transport of remodeling-defective GPI-APs in yeast cells. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).