Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER

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.

Deficiency of GPI Glycan Modification by Ethanolamine Phosphate Results in Increased Adhesion and Immune Resistance of Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in maintaining the function of the cell wall and participating in pathogenic processes. The addition and removal of phosphoethanolamine (EtN-P) on the second mannose residue in the GPI anchor are vital for maturation and sorting of GPI-anchored proteins. Previously, we have shown that deletion of the gpi7, the gene that encodes an EtN-P transferase responsible for the addition of EtN-P to the second mannose residue of the GPI anchor, leads to the mislocalization of GPI-anchored proteins, abnormal polarity, reduced conidiation, and fast germination in Aspergillus fumigatus. In this report, the adherence and virulence of the A. fumigatus gpi7 deletion mutant were further investigated. The germinating conidia of the mutant exhibited an increased adhesion and a higher exposure of cell wall polysaccharides. Although the virulence was not affected, an increased adherence and a stronger inflammation response of the mutant were documented in an immunocompromised mouse model. An in vitro assay confirmed that the Δgpi7 mutant induced a stronger immune response and was more resistant to killing. Our findings, for the first time, demonstrate that in A. fumigatus, GPI anchoring is required for proper organization of the conidial cell wall. The lack of Gpi7 leads to fast germination, stronger immune response, and resistance to macrophage killing.

PIGG defines the Emm blood group system

Emm is a high incidence red cell antigen with eight previously reported Emm− probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm− brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm− individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm− brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm− individuals had various PIGG mutations; deletion of Exons 2–3, deletion of Exons 7–9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm− phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.

The biosynthesis of phospholipids is linked to the cell cycle in a model eukaryote

The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. In this study, we tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell’s structure would form part the control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol over the G1/pre-replication phase boundary (+100%, phosphatidylglycerol synthase increased 22×). The relative abundance of phosphatidylcholine fell by ~35% during the G1. N-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. The relative abundance of both phosphatidylinositol and its synthase remained constant despite an eightfold increase in cell volume. We conclude that the biosynthesis of the three most abundant structural phospholipids is linked to the cell cycle in D. quadricauda.

Discrimination between Fresh and Frozen-Thawed Fish Involved in Food Safety and Fraud Protection

This study aims to discriminate fresh fish from frozen/thawed by identification of the key metabolites that are altered during the freezing/thawing processing. Atlantic salmon (Salmo salar) and bullet tuna (Auxis rochei) were selected as they are representative of broad consumption, and susceptible to pathogen contamination. Atlantic salmon samples were subjected to the following regimes: −20 °C (24h) and −35 °C (15 h) freezing, then thawed respectively in the blast chiller and in the cold room and analyzed immediately or after 10 days; (2) bullet tuna samples were frozen at −18 °C and thawed after 15, 30 and 90 days. High resolution mass spectrometry based on untargeted metabolomic analyses and statistical data treatment confirmed significant variations in the quantity of certain metabolites: the amount of l-phenylalanine in salmon increased immediately after thawing while that of anserine decreased. The concentration of l-arginine and its metabolites was altered at the 10th day after thawing rendering them promising markers of salmon freezing/thawing. As regards bullet tuna, compounds resulting from lipid degradation (l-α-Glyceryl-phosphoryl-choline and N-methyl-ethanolamine phosphate) increased notably during the storage period. This approach could be used to reveal common fraudulent incidents such as deliberate replacement of fresh fish with frozen/thawed, with food safety risks as the primary implication.

Cdc1p is a Golgi-localized glycosylphosphatidylinositol-anchored protein remodelase

Discrepancies in the prevailing role of the GPI-AP remodelase Cdc1p prompted us to reexamine the localization of this protein. Cdc1p is a Golgi-restricted membrane protein, rather than localized to the ER. Removal of ethanolamine phosphate appears to function in quality control surveillance of nascent GPI-APs in the ER and Golgi in yeast cells.

Ethanolamine phosphate on the second mannose as bridge in GPI anchored proteins: Towards understanding inherited PIGG deficiency

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. GPI precursor has three mannoses, which in mammalian cells, are all modified by the addition of ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI to the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient as postulated, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. We hypothesized that EthN-P-Man2 plays a role beyond what is now postulated. Indeed, EthN-P on Man2 is not only the alternate bridge in some GPI-APs but the actual bridge in others, among them, the ect-5’-nucleotidase and netrin G2. We found that CD59, a GPI-AP, is attached via EthN-P-Man2 in both 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 mechanistic bases of IGDs caused by PIGG mutations.

Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids

The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16–20:4 > DAG 18:0–20:4 > DAG 16:0–18:1 = AAG 16–18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0–18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.