Precursors of select GPI-anchored proteins positively regulate GPI biosynthesis under the control of ERAD

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is regulated by endoplasmic reticulum associated degradation (ERAD); however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR–Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 together upregulate GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, remaining the uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 32 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55 and CD48 enhance GPI biosynthesis. ARV1 is essential for the GPI upregulation by CD55 precursor. Our data demonstrate an ARV1-dependent regulatory connection between GPI biosynthesis and precursors of select GPI-anchored proteins that are under the control of ERAD.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Inherited glycosylphosphatidylinositol defects cause the rare Emm-negative blood phenotype and developmental disorders

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors more than 150 proteins to the cell surface. Pathogenic variants in several genes that participate in GPI biosynthesis cause inherited GPI deficiency (IGD) disorders. Here, we reported that homozygous null alleles of PIGG, a gene involved in GPI modification, are responsible for the rare Emm-negative blood phenotype. Using a panel of K562 cells defective in both the GPI-transamidase and GPI remodeling pathways, we demonstrate that the Emm antigen, whose molecular basis has remained unknown for decades, is carried only by free GPI and that its epitope is composed of the second and third ethanolamine of the GPI backbone. Importantly, we show that the decrease in Emm expression in several IGD patients is indicative of GPI defects. Overall, our findings establish Emm as a novel blood group system and have important implications for understanding the biological function of human free GPI.

Function of AtPGAP1 in GPI anchor lipid remodeling and transport to the cell surface of GPI-anchored proteins

ABSTRACTGPI-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signalling and cell wall biosynthesis. The GPI-anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodelled. In mammals and yeast, this remodelling is required for GPI-APs to be included in Coat Protein II (COPII) coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodelling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and PGAP1 (mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis PGAP1 localizes to the ER and probably functions as the GPI inositol-deacylase which cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.One sentence summaryGPI anchor lipid remodeling in GPI-anchored proteins is required for their transport to the cell surface in Arabidopsis.

The consensus Nglyco-X-S/T motif and a previously unknown Nglyco-N -linked glycosylation are necessary for growth and pathogenicity of Phytophthora

Asparagine (Asn, N) -linked glycosylation within the glycosylation motif (Nglyco-X-S/T; X≠P) is a ubiquitously distributed post-translational modification that participates in diverse eukaryotic cellular processes. However, little is known about the characteristic features and roles of N-glycosylation in oomycetes. In this work, it found that 2.5 μg/ml tunicamycin (N-glycosylation inhibitor) completely inhibited Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map all N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cysts, and sexual oospores. A total of 355 N-glycosylated proteins were found, containing 496 glycosites that likely participate in glycan degradation, carbon metabolism, glycolysis, or other central metabolic pathways. To verify the glycoproteomic results and further examine the function of N-glycosylation in P. sojae, two proteins were selected for PNGase F deglycosylation assays and CRISPR/Cas9-mediated site-directed mutagenesis, including a GPI transamidase protein (GPI16) up-regulated in cysts, with the consensus Nglyco-X-S/T motif at Asn 94, and a heat shock protein 70 (HSP70) up-regulated in cysts and oospores with a previously unknown Nglyco-N motif at Asn 270. We demonstrated that the GPI16 and HSP70 are both N-glycosylated proteins, confirming that the Nglyco-N motif is a target site for asparagine - oligosaccharide N-glycosidic linkage. Glycosite mutations of Asn 94 in the GPI16 led to impaired cyst germination and pathogenicity, while HSP70 mutants exhibited decreased cyst germination and oospore production. This work describes an integrated map of oomycete N-glycoproteomes and advances our understanding of N-glycosylation in oomycetes. Moreover, we confirm that the consensus Nglyco-X-S/T and the Nglyco-N -linked glycosites are both essential for the growth of Phytophthora sojae, indicating that there are multiple N-glycosylation motifs in oomycetes.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase Subunit, is critical for pollen tube growth in Arabidopsis

BackgroundGlycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG, yet is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:PIGS-GFP transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings, but seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:PIGS-GFP seedlings died soon after germination.ConclusionsCharacterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step. Our study also provided further evidence that GPI transamidation is essential in seedling development.