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Glycosylphosphatidylinositol (GPI) is a glycolipid that tethers more than 150 different proteins to the cell surface. Aberrations in biosynthesis of GPI anchors cause congenital disorders of glycosylation with clinical features including intellectual disability (ID), seizures, and facial dysmorphism. Here, we present two siblings with ID, cerebellar hypoplasia, cerebellar ataxia, early-onset seizures, and minor facial dysmorphology. Using exome sequencing, we identified a homozygous nonsense variant (NM_001127178.1:c.1640G>A, p.Trp547*) in the gene Phosphatidylinositol Glycan Anchor Biosynthesis, Class G (PIGG) in both the patients. Variants in several other GPI anchor synthesis genes lead to a reduced expression of GPI-anchored proteins (GPI-APs) that can be measured by flow cytometry. No significant differences in GPI-APs could be detected in patient granulocytes, consistent with recent findings. However, fibroblasts showed a reduced global level of GPI anchors and of specific GPI-linked markers. These findings suggest that fibroblasts might be more sensitive to pathogenic variants in GPI synthesis pathway and are well suited to screen for GPI-anchor deficiencies. Based on genetic and functional evidence, we confirm that pathogenic variants in PIGG cause an ID syndrome, and we find that loss of function of PIGG is associated with GPI deficiency.
GPI-linked proteins coassociate with intracellular tyrosine kinases in "lipid rafts" proposed to function as platforms for signal transduction and cytoskeletal reorganization. TCR activation requires both tyrosine kinase signals and cytoskeletal reorganization. How receptor engagement initiates cytoskeletal changes remains poorly understood. We investigated the consequences of recruiting GPI-linked CD48 and associated rafts to the site of T cell:APC contact by stimulating T cells with APCs that express the CD48 ligand CD2. We demonstrate that CD2:CD48 interactions enhance TCR-mediated functions. CD48/TCR coengagement qualitatively and quantitatively enhances lipid raft-dependent zeta association with the actin cytoskeleton and zeta tyrosine phosphorylation. This implicates lipid rafts as sites where receptor-induced signals and cytoskeletal reorganization are integrated and reveals a novel component of accessory molecule function.
Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly.
Reverse signaling via glycosylphosphatidylinositol (GPI)-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), whereas adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways.
Little is known about glycosylphosphatidylinositol (GPI)-linked surface proteins in the coccidian parasite Eimeria tenella. Examination of 28,550 EST sequences from the sporozoite and second merozoite developmental stages of the parasite led to the identification of 37 potential GPI-linked variant surface proteins, termed EtSAGs. Analysis of the complete nucleotide sequences of 23 EtSAG genes separated them into two multi-gene families. All the predicted EtSAG proteins (which vary in length from 228 to 271 residues) have an N-terminal hydrophobic signal peptide, a C-terminal hydrophobic GPI signal-anchor peptide and an extracellular domain organised around six cysteine residues, the positions of which are conserved within each family. Using specific antibodies against a small number of recombinant-expressed EtSAGs, the surface localisation and GPI-anchorage of members of both families was confirmed experimentally. Expression of EtSAGs is differentially regulated between the oocyst/sporozoite and second generation merozoite stages, with only one expressed specifically in the sporozoite, a small number expressed in both stages and the majority expressed specifically in the second generation merozoite. Preliminary data support a model in which multiple variant surface antigens are co-expressed on individual parasites, rather than a model of antigenic switching. The biological role(s) of EtSAGs and the effect(s) that expression of a complex repertoire of variant surface antigens by the second generation merozoite has on host adapted immunity are unknown.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome-wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. The exostosin-1 (EXT1) and sulfatase-1 (SULF1) are key enzymes contributing to the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in the initiation and polymerisation of the growing HS chain. SULF1 removes 6-O-sulfate groups from HS chains, affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study, we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case-control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.
Carbonic anhydrase (CA) isozymes CA IV and CA XV are anchored on the extracellular cell surface via glycosylphosphatidylinositol (GPI) linkage. Analysis of evolution of these isozymes in vertebrates reveals an additional group of GPI-linked CAs, CA XVII, which has been lost in mammals. Our work resolves nomenclature issues in GPI-linked fish CAs. Review of expression data brings forth previously unreported tissue and cancer types in which human CA IV is expressed. Analysis of collective glycosylation patterns of GPI-linked CAs suggests functionally important regions on the protein surface.
The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae.
While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.
The ULBPs are a family of MHC class I-related molecules. We have previously shown that ULBPs 1, 2, and 3 are functional ligands of the NKG2D/DAP10 receptor complex on human natural killer (NK) cells. Here, we describe a new member of the ULBP family, ULBP4, which contains predicted transmembrane and cytoplasmic domains, unlike the other ULBPs, which are GPI-linked proteins. Transduction of ULBP4 into EL4 cells confers the ability to bind recombinant NKG2D and mediates increased cytotoxic activity by human NK cells, consistent with the role of ULBPs as ligands for the NKG2D/DAP10 activating receptors. Tissue expression of ULBP4 differs from other members of the family, in that it is expressed predominantly in the skin.
The motility and invasion of Plasmodium parasites is believed to require a cytoplasmic actin-myosin motor associated with a cell surface ligand belonging to the TRAP (thrombospondin-related anonymous protein) family. Current models of invasion usually invoke the existence of specific receptors for the TRAP-family ligands on the surface of the host cell; however, the identities of these receptors remain largely unknown. Here, we identify the GPI-linked protein Semaphorin-7A (CD108) as an erythrocyte receptor for the P. falciparum merozoite-specific TRAP homolog (MTRAP) by using a systematic screening approach designed to detect extracellular protein interactions. The specificity of the interaction was demonstrated by showing that binding was saturable and by quantifying the equilibrium and kinetic biophysical binding parameters using surface plasmon resonance. We found that two MTRAP monomers interact via their tandem TSR domains with the Sema domains of a Semaphorin-7A homodimer. Known naturally-occurring polymorphisms in Semaphorin-7A did not quantitatively affect MTRAP binding nor did the presence of glycans on the receptor. Attempts to block the interaction during in vitro erythrocyte invasion assays using recombinant proteins and antibodies showed no significant inhibitory effect, suggesting the inaccessibility of the complex to proteinaceous blocking agents. These findings now provide important experimental evidence to support the model that parasite TRAP-family ligands interact with specific host receptors during cellular invasion.
In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.
A major theme of host against invading pathogens lies in multiple regulatory nodes that ensure sufficient signals for protection while avoiding excessive signals toward over-inflammation. The TLR4/MD-2/CD14 complex receptor-mediated response to bacterial lipopolysaccharide (LPS) represents a paradigm for understanding the proper control of anti-pathogen innate immunity. In this study, we studied the mechanism by which the glycosylphosphatidylinositol (GPI)-linked LY6E protein constrains LPS response via downregulating CD14. We first showed that LY6E downregulated CD14 via ubiquitin-dependent proteasomal degradation. The subsequent profiling of LY6E protein interactome led to the revelation that the degradation of CD14 by LY6E requires PHB1, which interacts with CD14 in a LY6E-dependent manner. Finally, we identified the PHB1-interacting TRIM21 as the major ubiquitin E3 ligase for the LY6E-mediated ubiquitination of CD14. Together, our study elucidated the molecular basis of LY6E-mediated governance of LPS response, alongside providing new insights to regulatory mechanisms controlling the homeostasis of membrane proteins.
Beta-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the beta-secretase responsible for generating the amyloid-beta protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few beta-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative beta-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.
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.
Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 μm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 μm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.
Experiences during early development can influence neuronal functions and modulate adult behaviors [1, 2]. However, the molecular mechanisms underlying the long-term behavioral effects of these early experiences are not fully understood. The C. elegans ascr#3 (asc-ΔC9; C9) pheromone triggers avoidance behavior in adult hermaphrodites [3-7]. Here, we show that hermaphrodites that are briefly exposed to ascr#3 immediately after birth exhibit increased ascr#3-specific avoidance as adults, indicating that ascr#3-experienced animals form a long-lasting memory or imprint of this early ascr#3 exposure [8]. ascr#3 imprinting is mediated by increased synaptic activity between the ascr#3-sensing ADL neurons and their post-synaptic SMB motor neuron partners via increased expression of the odr-2 glycosylated phosphatidylinositol (GPI)-linked signaling gene in the SMB neurons. Our study suggests that the memory for early ascr#3 experience is imprinted via alteration of activity of a single synaptic connection, which in turn shapes experience-dependent plasticity in adult ascr#3 responses.
The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.
Even though antigenic variation is employed among parasitic protozoa for host immune evasion, Tetrahymena thermophila, a free-living ciliate, can also change its surface protein antigens. These cysteine-rich glycosylphosphatidylinositol (GPI)-linked surface proteins are encoded by a family of polymorphic Ser genes. Despite the availability of T. thermophila genome, a comprehensive analysis of the Ser family is limited by its high degree of polymorphism. In order to overcome this problem, a new approach was adopted by searching for Ser candidates with common motif sequences, namely length-specific repetitive cysteine pattern and GPI anchor site. The candidate genes were phylogenetically compared with the previously identified Ser genes and classified into subtypes. Ser candidates were often found to be located as tandem arrays of the same subtypes on several chromosomal scaffolds. Certain Ser candidates located in the same chromosomal arrays were transcriptionally expressed at specific T. thermophila developmental stages. These Ser candidates selected by the motif analysis approach can form the foundation for a systematic identification of the entire Ser gene family, which will contribute to the understanding of their function and the basis of T. thermophila antigenic variation.
Biochemical similarities have been noted between the natively unstructured region of the cellular prion protein, PrP(C), and a GPI-linked glycoprotein called Shadoo (Sho); these proteins are encoded by the Prnp and Sprn genes, respectively. Both proteins are expressed in the adult central nervous system and they share overlapping partners, including each other, in interactome studies. As prior studies have ascribed neuroprotective properties to the N-terminal region of PrP(C), specifically the octarepeat region, we investigated Sho's neuroprotective properties. To this end we assessed Sho-null (Sprn(0/0)) and hemizygous (Sprn(0/+)) mice in the middle cerebral artery occlusion (MCAO) model versus wild type mice and also vs. transgene-rescued Sprn(0/0)-TgSprn mice. Sprn(0/0) mice had a tendency to greater fragility in reaching endpoint and deficits in parameters including infarct volume and neurogenesis, with a reciprocal trend noted in transgene-rescued mice; however these effects did not reach significance. Loss of both PrP(C) and Sho immunostaining occurred in parallel to neuronal loss on the ipsilateral side of MCAO-lesioned animals; while focal elevations in immunostaining in the penumbra region were sometimes evident for PrP(C), they were not noted for Sho. Our studies argue against discernible neuroprotective action of Sho in the genetic backgrounds used for this MCAO paradigm. Whether or not the positively charged N-terminal regions in Sho and PrP(C) fulfil different roles in vivo remains to be determined.
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