PGAP3 regulates human bronchial epithelial cell mRNAs present in asthma and respiratory virus reference data sets.

PGAP3 is a glycosylphosphatidylinositol (GPI) phospholipase gene localized within chromosome 17q12-21, a region highly linked to asthma. Although much is known about the function of other chromosome 17q12-21 genes expressed at increased levels in bronchial epithelium such as ORMDL3 and GSDMB, little is known about the function of increased PGAP3 expression in bronchial epithelium in the context of asthma. The aim of this study was therefore to determine whether increased PGAP3 expression in human bronchial epithelial cells regulated expression of mRNA pathways important to the pathogenesis of asthma by utilizing RNA-sequencing and bioinformatic analysis. We performed RNA-sequencing on normal human bronchial epithelial cells transfected with PGAP3 for 24 and 48 hours. PGAP3 regulated genes were compared to asthma and respiratory virus (influenza A, rhinovirus, respiratory syncytial virus) reference data sets to identify PGAP3 target genes and pathways. Approximately 9% of the upregulated PGAP3-induced genes were found in an asthma reference data set, 41% in a rhinovirus reference data set, 33% in an influenza A reference data set, and 3% in a respiratory syncytial virus reference data set. PGAP3 significantly upregulated the expression of several genes associated with the innate immune response and viral signatures of respiratory viruses associated with asthma exacerbations. Two of the highest expressed genes induced by PGAP3 are RSAD2, OASL, and IFN-λ, which are anti-viral genes associated with asthma. PGAP3 also upregulated the antiviral gene BST2, which like PGAP3 is a GPI-anchored protein. We conclude that PGAP3 expression in human bronchial epithelial cells regulates expression of genes known to be linked to asthma, and also regulates the bronchial epithelial expression of genes pertinent to the pathogenesis of respiratory viral triggered asthma exacerbations.

AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models.

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins, and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis, and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate CNS-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno-associated virus (AAV). It expressed efficacy within 6 days, and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for approximately 1 year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter.

Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

Functional partnerships between GPI-anchored proteins and adhesion GPCRs.

Specific extracellular interaction between glycophosphatidylinositol (GPI)-anchored proteins and adhesion G protein-coupled receptors (aGPCRs) plays an important role in unique biological functions. GPI-anchored proteins are derived from a novel post-translational modification of single-span membrane molecules, while aGPCRs are bona fide seven-span transmembrane proteins with a long extracellular domain. Although various members of the two structurally-distinct protein families are known to be involved in a wide range of biological processes, many remain as orphans. Interestingly, accumulating evidence has pointed to a complex interaction and functional synergy between these two protein families. I discuss herein current understanding of specific functional partnerships between GPI-anchored proteins and aGPCRs.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Multi-omic characterization of the maize GPI synthesis mutant gwt1 with defects in kernel development.

BACKGROUND: Glycosylphosphatidylinositol (GPI) and GPI-anchored proteins (GAPs) are important for cell wall formation and reproductive development in Arabidopsis. However, monocot counterparts that function in kernel endosperm development have yet to be discovered. Here, we performed a multi-omic analysis to explore the function of GPI related genes on kernel development in maize. RESULTS: In maize, 48 counterparts of human GPI synthesis and lipid remodeling genes were identified, in which null mutation of the glucosaminyl-phosphatidylinositol O-acyltransferase1 gene, ZmGWT1, caused a kernel mutant (named gwt1) with defects in the basal endosperm transport layer (BETL). We performed plasma membrane (PM) proteomics to characterize the potential GAPs involved in kernel development. In total, 4,981 proteins were successfully identified in 10-DAP gwt1 kernels of mutant and wild-type (WT), including 1,638 membrane-anchored proteins with different posttranslational modifications. Forty-seven of the 256 predicted GAPs were differentially accumulated between gwt1 and WT. Two predicted BETL-specific GAPs (Zm00001d018837 and Zm00001d049834), which kept similar abundance at general proteome but with significantly decreased abundance at membrane proteome in gwt1 were highlighted. CONCLUSIONS: Our results show the importance of GPI and GAPs for endosperm development and provide candidate genes for further investigation of the regulatory network in which ZmGWT1 participates.

A systematic survey of LU domain-containing proteins reveals a novel human gene, LY6A, which encodes the candidate ortholog of mouse Ly-6A/Sca-1 and is aberrantly expressed in pituitary tumors.

The Ly-6 and uPAR (LU) domain-containing proteins represent a large family of cell-surface markers. In particular, mouse Ly-6A/Sca-1 is a widely used marker for various stem cells; however, its human ortholog is missing. In this study, based on a systematic survey and comparative genomic study of mouse and human LU domain-containing proteins, we identified a previously unannotated human gene encoding the candidate ortholog of mouse Ly-6A/Sca-1. This gene, hereby named LY6A, reversely overlaps with a lncRNA gene in the majority of exonic sequences. We found that LY6A is aberrantly expressed in pituitary tumors, but not in normal pituitary tissues, and may contribute to tumorigenesis. Similar to mouse Ly-6A/Sca-1, human LY6A is also upregulated by interferon, suggesting a conserved transcriptional regulatory mechanism between humans and mice. We cloned the full-length LY6A cDNA, whose encoded protein sequence, domain architecture, and exon-intron structures are all well conserved with mouse Ly-6A/Sca-1. Ectopic expression of the LY6A protein in cells demonstrates that it acts the same as mouse Ly-6A/Sca-1 in their processing and glycosylphosphatidylinositol anchoring to the cell membrane. Collectively, these studies unveil a novel human gene encoding a candidate biomarker and provide an interesting model gene for studying gene regulatory and evolutionary mechanisms.

The New GPI-Anchored Protein, SwgA, Is Involved in Nitrogen Metabolism in the Pathogenic Filamentous Fungus Aspergillus fumigatus.

GPI-anchored proteins display very diverse biological (biochemical and immunological) functions. An in silico analysis has revealed that the genome of Aspergillus fumigatus contains 86 genes coding for putative GPI-anchored proteins (GPI-APs). Past research has demonstrated the involvement of GPI-APs in cell wall remodeling, virulence, and adhesion. We analyzed a new GPI-anchored protein called SwgA. We showed that this protein is mainly present in the Clavati of Aspergillus and is absent from yeasts and other molds. The protein, localized in the membrane of A. fumigatus, is involved in germination, growth, and morphogenesis, and is associated with nitrogen metabolism and thermosensitivity. swgA is controlled by the nitrogen regulator AreA. This current study indicates that GPI-APs have more general functions in fungal metabolism than cell wall biosynthesis.

FT-GPI, a highly sensitive and accurate predictor of GPI-anchored proteins, reveals the composition and evolution of the GPI proteome in Plasmodium species.

BACKGROUND: Protozoan parasites are known to attach specific and diverse group of proteins to their plasma membrane via a GPI anchor. In malaria parasites, GPI-anchored proteins (GPI-APs) have been shown to play an important role in host-pathogen interactions and a key function in host cell invasion and immune evasion. Because of their immunogenic properties, some of these proteins have been considered as malaria vaccine candidates. However, identification of all possible GPI-APs encoded by these parasites remains challenging due to their sequence diversity and limitations of the tools used for their characterization. METHODS: The FT-GPI software was developed to detect GPI-APs based on the presence of a hydrophobic helix at both ends of the premature peptide. FT-GPI was implemented in C ++and applied to study the GPI-proteome of 46 isolates of the order Haemosporida. Using the GPI proteome of Plasmodium falciparum strain 3D7 and Plasmodium vivax strain Sal-1, a heuristic method was defined to select the most sensitive and specific FT-GPI software parameters. RESULTS: FT-GPI enabled revision of the GPI-proteome of P. falciparum and P. vivax, including the identification of novel GPI-APs. Orthology- and synteny-based analyses showed that 19 of the 37 GPI-APs found in the order Haemosporida are conserved among Plasmodium species. Our analyses suggest that gene duplication and deletion events may have contributed significantly to the evolution of the GPI proteome, and its composition correlates with speciation. CONCLUSION: FT-GPI-based prediction is a useful tool for mining GPI-APs and gaining further insights into their evolution and sequence diversity. This resource may also help identify new protein candidates for the development of vaccines for malaria and other parasitic diseases.

Single-Molecule Imaging of Ganglioside Probes in Living Cell Plasma Membranes.

Gangliosides play a variety of physiological roles and are one of the most important lipid raft constituents. However, their dynamic behaviors have scarcely been investigated in living cells because of the lack of fluorescent probes that behave like their parental molecules. Recently, fluorescent ganglioside probes that mimic native ganglioside behaviors have been developed. In this chapter, I discuss the recent advances in research related to the lateral localization and dynamic behaviors of gangliosides in the plasma membranes of living cells.

The glycoprotein gp63- a potential pan drug target for developing new antileishmanial agents.

Leishmaniasis is a tropical parasitic disease caused by Leishmania spp. They cause several presentations of illness ranging from cutaneous leishmaniasis to visceral leishmaniasis. The current arsenal of drugs to treat leishmaniasis is limited, and drug resistance further impedes the problem. Therefore, it is necessary to revisit the available information to identify an alternative or new target for treatment. The glycoprotein 63 (gp63), is a potential anti-leishmanial target that plays a significant role in host-pathogen interaction and virulence. Many studies are ongoing to develop gp63 inhibitors or use it as a vaccine target. In this review, we will discuss the potential of gp63 as a drug target. This review summarises the studies focusing on gp63 as a drug target and its inhibitors identified using in silico approaches.

A fungal GPI-anchored protein gene functions as a virulence and antiviral factor.

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.

The regulation of the cell wall by glycosylphosphatidylinositol-anchored proteins in Arabidopsis.

A polysaccharides-based cell wall covers the plant cell, shaping it and protecting it from the harsh environment. Cellulose microfibrils constitute the cell wall backbone and are embedded in a matrix of pectic and hemicellulosic polysaccharides and glycoproteins. Various environmental and developmental cues can regulate the plant cell wall, and diverse glycosylphosphatidylinositol (GPI)-anchored proteins participate in these regulations. GPI is a common lipid modification on eukaryotic proteins, which covalently tethers the proteins to the membrane lipid bilayer. Catalyzed by a series of enzymic complexes, protein precursors are post-translationally modified at their hydrophobic carboxyl-terminus in the endomembrane system and anchored to the lipid bilayer through an oligosaccharidic GPI modification. Ultimately, mature proteins reach the plasma membrane via the secretory pathway facing toward the apoplast and cell wall in plants. In Arabidopsis, more than three hundred GPI-anchored proteins (GPI-APs) have been predicted, and many are reported to be involved in diverse regulations of the cell wall. In this review, we summarize GPI-APs involved in cell wall regulation. GPI-APs are proposed to act as structural components of the cell wall, organize cellulose microfibrils at the cell surface, and during cell wall integrity signaling transduction. Besides regulating protein trafficking, the GPI modification is potentially governed by a GPI shedding system that cleaves and releases the GPI-anchored proteins from the plasma membrane into the cell wall.

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

FocECM33, a GPI-anchored protein, regulates vegetative growth and virulence in Fusarium oxysporum f. sp. cubense tropical race 4.

ECM33, a glycosylphosphatidylinositol (GPI)-anchored protein, is important for fungal development and infection through regulating fungal cell wall integrity, however, the functions of its orthologs in pathogenesis have not been characterized in Fusarium oxysporum. Here, we discovered a GPI-anchored protein, FocECM33, which is required for vegetative growth and virulence of Fusasium oxysporum f. sp. cubense tropical race 4 (Foc TR4). FocECM33 was highly upregulated during the early infection process of Foc TR4 in banana roots. The targeted disruption of FocECM33 led to decreased hyphal growth, increased sensitivity to cell wall stresses and reduced virulence on banana plantlets. Furthermore, ΔFocECM33 mutant demonstrated a cell morphology defect, elevated ROS production and increased chitin content. Transcriptome analysis showed that FocECM33 has a significant influence on the production of various secondary metabolites and regulation of many biosynthetic processes in Foc TR4. Taken together, it seems FocECM33 contributes to the virulence of Foc TR4 through regulating the process of hyphal growth, ROS production and chitin synthesis.

Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive ß-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support ß-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis.

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis.

Unveiling the structure of GPI-anchored protein of Malassezia globosa and its pathogenic role in pityriasis versicolor.

Glycosylphosphatidylinositols (GPI)-anchored proteins (GpiPs) are related to the cell wall biogenesis, adhesion, interactions, protease activity, mating, etc. These proteins have been identified in many organisms, including fungi such as Neurospora crassa, Candida albicans, Saccharomyces cerevisiae, and Fusarium graminearum. MGL-3153 gene of Malassezia globosa (M. globosa) encodes a protein which is homologous of the M. restricta, M. sympodialis, M. Pachydermatis, and U. maydis GpiPs. Real-time PCR assay showed that the expression of MGL_3153 gene was significantly up-regulated among M. globosa isolated from patients with pityriasis versicolor (PV) compared to a healthy individual, suggesting the contribution of this gene in the virulence of M. globosa. Accordingly, the sequence of this protein was analyzed by bioinformatics tools to evaluate the structure of that. The conservation analysis of MGL-3153 protein showed that the C-terminal region of this protein, which is responsible for GPI-anchor ligation, was highly conserved during evolution while the N-terminal region just conserved in Malassezia species. Moreover, the predicted tertiary structure of this protein by homology modeling showed that this protein almost has alpha helix structure and represented a stable structure during 150 ns of molecular dynamic simulation. Our results revealed that this protein potentially belongs to GPI-anchored proteins and may contribute to the virulence of M. globosa which warrants further investigations in this area.

The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

The membrane of platelets contains at least one uncharacterized glycosylphosphatidylinositol (GPI)-anchored protein according to the literature. Moreover, there is not enough knowledge on the receptor of low-density lipoproteins (LDL) mediating rapid Ca2+ signaling in platelets. Coincidentally, expression of a GPI-anchored protein T-cadherin increases LDL-induced Ca2+ signaling in nucleated cells. Here we showed evidence that supports the hypothesis about the presence of T-cadherin on platelets. The presence of T-cadherin on the surface of platelets and megakaryocytes was proven using antibodies whose specificity was tested on several negative and positive control cells by flow cytometry and confocal microscopy. Using phosphatidylinositol-specific phospholipase C, the presence of glycosylphosphatidylinositol anchor in the platelet T-cadherin form as well as in other known forms was confirmed. We showed by immunoblotting that the significant part of T-cadherin was detected in specific membrane domains (detergent Triton X-114 resistant) and the molecular weight of this newly identified protein was greater than that of T-cadherin from nucleated cells. Nevertheless, polymerase chain reaction data confirmed only the presence of isoform-1 of T-cadherin in platelets and megakaryocytes, which was also present in nucleated cells. We observed the redistribution of this newly identified protein after the activation of platelets, but only further work may explain its functional importance. Thus, our data described T-cadherin with some post-translational modifications as a new GPI-anchored protein on human platelets.

Potential Physiological Relevance of ERAD to the Biosynthesis of GPI-Anchored Proteins in Yeast.

Misfolded and/or unassembled secretory and membrane proteins in the endoplasmic reticulum (ER) may be retro-translocated into the cytoplasm, where they undergo ER-associated degradation, or ERAD. The mechanisms by which misfolded proteins are recognized and degraded through this pathway have been studied extensively; however, our understanding of the physiological role of ERAD remains limited. This review describes the biosynthesis and quality control of glycosylphosphatidylinositol (GPI)-anchored proteins and briefly summarizes the relevance of ERAD to these processes. While recent studies suggest that ERAD functions as a fail-safe mechanism for the degradation of misfolded GPI-anchored proteins, several pieces of evidence suggest an intimate interaction between ERAD and the biosynthesis of GPI-anchored proteins.