Glycosylphosphatidylinositol Anchor Research Articles

Novel insights into the function of CD24: A driving force in cancer.

CD24 is a highly glycosylated protein with a small protein core that is linked to the plasma membrane via a glycosyl-phosphatidylinositol anchor. CD24 is primarily expressed by immune cells but is often overexpressed in human tumors. In cancer, CD24 is a regulator of cell migration, invasion and proliferation. Its expression is associated with poor prognosis and it is used as cancer stemness marker. Recently, CD24 on tumor cells was identified as a phagocytic inhibitor ("do not eat me" signal) having a suppressive role in tumor immunity via binding to Siglec-10 on macrophages. This finding is reminiscent of the demonstration that soluble CD24-Fc can dampen the immune system in autoimmune disease. In the present review, we summarize recent progress on the role of the CD24-Siglec-10 binding axis at the interface between tumor cells and the immune system, and the role of CD24 genetic polymorphisms in cancer. We describe the specific function of cytoplasmic CD24 and discuss the presence of CD24 on tumor-released extracellular vesicles. Finally, we evaluate the potential of CD24-based immunotherapy.

Evolutionary analysis and protein family classification of chitin deacetylases in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen causing cryptococcal meningoencephalitis. Interestingly, the cell wall of C. neoformans contains chitosan, which is critical for its virulence and persistence in the mammalian host. C. neoformans (H99) has three chitin deacetylases (CDAs), which convert chitin to chitosan. Herein, the classification of the chitin-related protein (CRP) family focused on cryptococcal CDAs was analyzed by phylogenetics, evolutionary pressure (dN/dS), and 3D modeling. A phylogenetic tree of 110 CRPs revealed that they can be divided into two clades, CRP I and II with bootstrap values (> 99%). CRP I clade comprises five groups (Groups 1-5) with a total of 20 genes, while CRP II clade comprises sixteen groups (Groups 6-21) with a total of 90 genes. CRP I comprises only fungal CDAs, including all three C. neoformans CDAs, whereas CRP II comprises diverse CDAs from fungi, bacteria, and amoeba, along with other carbohydrate esterase 4 family proteins. All CDAs have the signal peptide, except those from group 11. Notably, CDAs with the putative O-gycosylation site possess either the glycosylphosphatidylinositol (GPI)-anchor motif for CRP I or the chitin-binding domain (CBD) for CRP II, respectively. This evolutionary conservation strongly indicates that the O-glycosylation modification and the presence of either the GPI-anchor motif or the chitin-binding domain is important for fungal CDAs to function efficiently at the cell surface. This study reveals that C. neoformans CDAs carrying GPI anchors have evolved divergently from fungal and bacterial CDAs, providing new insights into evolution and classification of CRP family.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls

The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.

Abstract 1820: Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth

Abstract Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian ARTs are extracellular with glycosylphosphatidylinositol (GPI) anchors. ARTs can target cell surface proteins on bystander cells or soluble proteins in the tumor microenvironment, particularly P2RX7 on T cells1. We hypothesized that an evolutionarily conserved parallel protective role may be provided by ART1 expression in lung cancer cells, particularly during times of cellular stress2. A mouse KP1 lung tumor cell line with ART1 expression was established from KRASG12D/P53-/- mice and transduced with shRNA targeting ART1 (KP1-sh) or an empty vector (KP1-ev). For in vitro studies, KP1 WT cells were treated with an ER stress inducer thapsigargin, with radiation, or with chemotherapy. Using human materials, we evaluated ART1 expression in NSCLC tumors by whole tumor RT-PCR/RNAseq, immunofluorescence (IF), and immunohistochemistry (IHC). For evaluation of tumor growth in immune competent models, KP1 cells were injected into syngeneic C57BL/6 mice into the flank or intravenously to generate orthotopic lung tumors. Following in vitro treatment with thapsigargin, expression of ART1 increased 11.8-fold in KP1 WT cells (24 hrs). Similarly, following radiation (8Gy x 3,) ART1 expression increased 1.93-fold by RT-PCR and surface protein expression increased by 1.87 fold (p<0.0005) by IF in KP1 cells. Similar increases were seen following cisplatin (10uM, 24 hrs) treatment (1.87-fold). Radiation (2.26-fold) and cisplatin (25uM, 3.30-fold) also induced ART1 expression in human A549 cells (24 hrs). In a human tissue microarray of patients who received neoadjuvant chemotherapy or radiation therapy (n=200), all tumors expressed ART1 and 38% of strongly expressed ART1. We also compared pre-treatment biopsy and post-treatment surgical resection tumor samples (n=21) in an ongoing clinical trial (NCT02904954) of neoadjuvant durvalumab +/- stereotactic radiation (8Gy x 3). ART1 was expressed at low levels by RNAseq, but trended upward (4-fold) after treatment, particularly in those patients who received radiation therapy (6-fold). Expression trended higher in nonresponders. Of these tumors without major pathologic response (n=13), 44% had moderate-strong ART1 expression by IHC. In murine flank tumor models, KP1-ev cells grew significantly faster than KP1-sh cells (688 mm3 vs. 331mm3 at 4 weeks, p=0.004). Similarly, following tail vein injection, mice injected with KP1-ev cells had heavier tumor burdens by micro-CT scan and significantly greater total lung weights (p=0.036) at sacrifice than did mice injected with KP1-sh cells. In conclusion, ART1 is broadly expressed in human lung tumors and is upregulated by cellular stress invoked by common treatments such as radiation and chemotherapy. Knockdown of ART1 abrogates tumor growth and may be a potential therapeutic target for combination therapy in lung cancer patients. 1. PMID: 7930612 2. PMID: 25292337 Citation Format: Sumit Mukherjee, Erik Wennerberg, Clarey Hung, Najla Saadallah, Shashi Kariyawasam, Christopher Agrusa, Amanda Valeta, Nasser Altorki, Timothy McGraw, Sandra Demaria, Brendon Stiles. Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1820.

Subtyping Cryptosporidium ryanae: A Common Pathogen in Bovine Animals.

Cryptosporidium ryanae is one of the most common species for cryptosporidiosis in cattle. However, little is known of the genetic characteristics of C. ryanae due to the lack of subtyping tools. In the present study, the 60-kDa glycoprotein (gp60) gene of C. ryanae was identified in whole genome sequence data and analyzed for sequence characteristics using bioinformatics tools. The protein it encodes had some of the typical characteristics of GP60 proteins, with a signal peptide, a furin cleavage site, and a glycosylphosphatidylinositol anchor at the C terminus of the protein, and numerous O-glycosylation sites. The gene sequence was used in the development of a subtyping tool, which was used in characterizing C. ryanae from 110 specimens from dairy cattle, 2 from beef cattle, 6 from yaks, and 4 from water buffaloes in China. Altogether, 17 subtypes from 8 subtype families were recognized, namely XXIa to XXIh. Possible host adaption was identified within this species, reflected by the unique occurrence of XXIa, XXIc, and XXIh in dairy cattle, yaks, and water buffaloes, respectively. Some geographical differences were detected in the distribution of subtype families in dairy cattle; specimens from southern China showed higher genetic diversity than from northern China, and the XXIa subtype family was only seen in dairy cattle in southern and eastern China. The gp60-based subtyping tool should be useful in molecular epidemiological studies of the transmission of C. ryanae.

Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells’ plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes’ catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.

Analysis of GPI-anchored proteins involved in germline stem cell proliferation in theCaenorhabditis elegansgermline stem cell niche

Stem cells divide and undergo self-renewal depending on the signals received from the stem cell niche. This phenomenon is indispensable to maintain tissues and organs in individuals. However, not all the molecular factors and mechanisms of self-renewal are known. In our previous study, we reported that glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) synthesized in the distal tip cells (DTCs; the stem cell niche) are essential for germline stem cell proliferation in Caenorhabditis elegans. Here, we characterized the GPI-APs required for proliferation. We selected and verified the candidate GPI-APs synthesized in DTCs by RNA interference screening and found that F57F4.3 (GFI-1), F57F4.4 and F54E2.1 are necessary for germline proliferation. These proteins are likely involved in the same pathway for proliferation and activated by the transcription factor PQM-1. We further provided evidence suggesting that these GPI-APs act through fatty acid remodelling of the GPI anchor, which is essential for association with lipid rafts. These findings demonstrated that GPI-APs, particularly F57F4.3/4 and F54E2.1, synthesized in the germline stem cell niche are located in lipid rafts and involved in promoting germline stem cell proliferation in C. elegans. The findings may thus shed light on the mechanisms by which GPI-APs regulate stem cell self-renewal.

The Role of Glypicans in Cancer Progression and Therapy.

Glypicans are a family of heparan sulfate proteoglycans that are attached to the cell membrane via a glycosylphosphatidylinositol anchor. Glypicans interact with multiple ligands, including morphogens, growth factors, chemokines, ligands, receptors, and components of the extracellular matrix through their heparan sulfate chains and core protein. Therefore, glypicans can function as coreceptors to regulate cell proliferation, cell motility, and morphogenesis. In addition, some glypicans are abnormally expressed in cancers, possibly involved in tumorigenesis, and have the potential to be cancer-specific biomarkers. Here, we provide a brief review focusing on the expression of glypicans in various cancers and their potential to be targets for cancer therapy.

Age-dependent membrane release and degradation of full-length glycosylphosphatidylinositol-anchored proteins in rats

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are associated with the surface of eucaryotic cells only through a covalently coupled carboxy-terminal GPI glycolipid structure which is anchored at the outer leaflet of plasma membranes. This mode of membrane association may be responsible for the recent observations that full-length GPI-APs harbouring the complete GPI anchor are (i) released from isolated rat adipocytes in vitro and (ii) expressed in rat and human serum. The upregulation of the adipocyte release in response to increased cell size and blood glucose/insulin levels of the donor rats and downregulation of the expression in serum of insulin resistant and diabetic rats have been reconciled with enhanced degradation of the full-length GPI-APs released into micelle-like complexes together with (lyso) phospholipids and cholesterol by serum GPI-specific phospholipase D (GPI-PLD).Here by using a sensitive and reliable sensing method for full-length GPI-APs, which relies on surface acoustic waves propagating over microfluidic chips, the upregulation of (i) the release of the full-length GPI-APs CD73, alkaline phosphatase and CD55 from isolated adipocyte plasma membranes monitored in a “lab-on-the-chip” configuration, (ii) their release from isolated rat adipocytes into the incubation medium and (iii) the lipolytic cleavage of their GPI anchors in serum was demonstrated to increase with age (3–16 weeks) and body weight (87−477 g) of (healthy) donor rats. In contrast, the amount of full-length GPI-APs in rat serum, as determined by chip-based sensing, turned out to decline with age/body weight. These correlations suggest that age-/weight-induced alterations (in certain biophysical/biochemical characteristics) of plasma membranes are responsible for the release of full-length GPI-APs which becomes counteracted by elevated GPI-PLD activity in serum. Thus, sensitive and specific measurement of these GPI-AP-relevant parameters may be useful for monitoring of age-related cell surface changes, in general, and diseases, in particular.

A novel variant in GPAA1, encoding a GPI transamidase complex protein, causes inherited vascular anomalies with various phenotypes.

Vascular anomalies (VAs), comprising wide subtypes of tumors and malformations, are often caused by variants in multiple tyrosine kinase (TK) receptor signaling pathways including TIE2, PIK3CA and GNAQ/11. Yet, a portion of individuals with clinical features of VA do not have variants in these genes, suggesting that there are undiscovered pathogenic factors underlying these patients and possibly with overlapping phenotypes. Here, we identified one rare non-synonymous variant (c.968A > G) in the seventh exon of GPAA1 (Glycosylphosphatidylinositol Anchor Attachment Protein 1), shared by the four affected members of a large pedigree with multiple types of VA using whole-exome sequencing. GPAA1 encodes a glycosylphosphatidylinositol (GPI) transamidase complex protein. This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum (ER). We showed such variant led to scarce expression of GPAA1 protein in vascular endothelium and induced a localization change from ER membrane to cytoplasm and nucleus. In addition, expressing wild-type GPAA1 in endothelial cells had an effect to inhibit cell proliferation and migration, while expressing variant GPAA1 led to overgrowth and overmigration, indicating a loss of the quiescent status. Finally, a gpaa1-deficient zebrafish model displayed several types of developmental defects as well as vascular dysplasia, demonstrating that GPAA1 is involved in angiogenesis and vascular remodeling. Altogether, our results indicate that the rare coding variant in GPAA1 (c.968A > G) is causally related to familial forms of VAs.

Cold-sensitive phenotypes of a yeast null mutant of ARV1 support its role as a GPI flippase.

Glycosylphosphatidylinositol (GPI) is synthesized in the endoplasmic reticulum (ER) and added onto proteins to form GPI-anchored proteins. Among the many proteins involved in this process, ACAT-related enzyme-2 required for viability 1 (Arv1) is a candidate, functioning as a flippase that translocates GPI intermediates from the cytoplasmic side into the luminal side of the ER membranes. Here, we show that the deletion of the ARV1 gene in yeast leads to cold-sensitive defects in cell growth and GPI anchor synthesis. Furthermore, complementation assays show that the overexpression of a missense human ARV1-G189R mutant does not completely restore the cold-sensitive phenotypes of the yeast arv1 mutant. Our results support the proposed role of Arv1 in GPI anchor synthesis and suggest that ARV1-linked human diseases result from defective GPI anchor synthesis.

Role of Folic Acid in the Therapeutic Action of Nanostructured Porous Silica Functionalized with Organotin(IV) Compounds Against Different Cancer Cell Lines.

The synthesis, characterization and cytotoxic activity against different cancer cell lines of various mesoporous silica-based materials containing folate targeting moieties and a cytotoxic fragment based on a triphenyltin(IV) derivative have been studied. Two different mesoporous nanostructured silica systems have been used: firstly, micronic silica particles of the MSU-2 type and, secondly, mesoporous silica nanoparticles (MSNs) of about 80 nm. Both series of materials have been characterized by different methods, such as powder X-ray diffraction, X-ray fluorescence, absorption spectroscopy and microscopy. In addition, these systems have been tested against four different cancer cell lines, namely, OVCAR-3, DLD-1, A2780 and A431, in order to observe if the size of the silica-based systems and the quantity of incorporated folic acid influence their cytotoxic action. The results show that the materials are more active when the quantity of folic acid is higher, especially in those cells that overexpress folate receptors such as OVCAR-3 and DLD-1. In addition, the study of the potential modulation of the soluble folate receptor alpha (FOLR1) by treatment with the synthesized materials has been carried out using OVCAR-3, DLD-1, A2780 and A431 tumour cell lines. The results show that a relatively high concentration of folic acid functionalization of the nanostructured silica together with the incorporation of the cytotoxic tin fragment leads to an increase in the quantity of the soluble FOLR1 secreted by the tumour cells. In addition, the studies reported here show that this increase of the soluble FOLR1 occurs presumably by cutting the glycosyl-phosphatidylinositol anchor of membrane FR-α and by the release of intracellular FR-α. This study validates the potential use of a combination of mesoporous silica materials co-functionalized with folate targeting molecules and an organotin(IV) drug as a strategy for the therapeutic treatment of several cancer cells overexpressing folate receptors.

Diastereoselective desymmetric 1,2-cis-glycosylation of meso-diols via chirality transfer from a glycosyl donor

Chemical desymmetrization reactions of meso-diols are highly effective for the precise and efficient synthesis of chiral molecules. However, even though enzyme-catalyzed desymmetric glycosylations are frequently found in nature, there is no method for highly diastereoselective desymmetric chemical glycosylation of meso-diols. Herein, we report a highly diastereoselective desymmetric 1,2-cis-glycosylation of meso-diols found in myo-inositol 1,3,5-orthoesters using a boronic acid catalyst based on predictions of regioselectivity by density functional theory (DFT) calculations. The enantiotopic hydroxyl groups of the meso-diols are clearly differentiated by the stereochemistry at the C2 position of the glycosyl donor with excellent regioselectivities. In addition, the present method is successfully applied to the synthesis of core structures of phosphatidylinositolmannosides (PIMs) and glycosylphosphatidylinositol (GPI) anchors, and common β-mannoside structures of the LLBM-782 series of antibiotics.

Coarse-Grained Molecular Model for the Glycosylphosphatidylinositol Anchor with and without Protein.

Glycosylphosphatidylinositol (GPI) anchors are a unique class of complex glycolipids that anchor a great variety of proteins to the extracellular leaflet of plasma membranes of eukaryotic cells. These anchors can exist either with or without an attached protein called GPI-anchored protein (GPI-AP) both in vitro and in vivo. Although GPIs are known to participate in a broad range of cellular functions, it is to a large extent unknown how these are related to GPI structure and composition. Their conformational flexibility and microheterogeneity make it difficult to study them experimentally. Simplified atomistic models are amenable to all-atom computer simulations in small lipid bilayer patches but not suitable for studying their partitioning and trafficking in complex and heterogeneous membranes. Here, we present a coarse-grained model of the GPI anchor constructed with a modified version of the MARTINI force field that is suited for modeling carbohydrates, proteins, and lipids in an aqueous environment using MARTINI’s polarizable water. The nonbonded interactions for sugars were reparametrized by calculating their partitioning free energies between polar and apolar phases. In addition, sugar–sugar interactions were optimized by adjusting the second virial coefficients of osmotic pressures for solutions of glucose, sucrose, and trehalose to match with experimental data. With respect to the conformational dynamics of GPI-anchored green fluorescent protein, the accessible time scales are now at least an order of magnitude larger than for the all-atom system. This is particularly important for fine-tuning the mutual interactions of lipids, carbohydrates, and amino acids when comparing to experimental results. We discuss the prospective use of the coarse-grained GPI model for studying protein-sorting and trafficking in membrane models.

OR24-02 Identification of Membrane Proteins That Enhance the Responsiveness of the Ghrelin Receptor

Ghrelin, a 28-amino acid peptide gut hormone, occurs in acylated (AG) and unacylated (UAG) variants. AG is a GH secretagogue as well as being orexigenic and diabetogenic, acting via the growth hormone secretagogue receptor (GHSR1a) in the hypothalamus and pituitary. UAG counteracts these metabolic effects through unknown mechanisms. While screening for potential UAG receptor(s) we discovered previously uncharacterised interactions of AG and UAG with five cell membrane proteins (MPs), three of which are known to modulate metabolism. Here, we studied if two of these MPs (MP1 & 2) could modulate GHSR1a signalling by expressing their transgenes in HEK293 cells. As GHSR1a is coupled with calcium signalling via Gq proteins, aequorin luminescence was used to evaluate Ca2+ influx into the cells. Transfected cells were treated with either AG, UAG, or soluble parts of the MPs, or combinations thereof.MP2 markedly enhanced the efficacy (~5.5-fold), but not the potency, of AG-induced Ca2+ influx, whereas MP1 had no effect on Ca2+ influx. Neither MP1 nor MP2 overexpression altered cellular GHSR1a levels. In the absence of GHSR1a, MP2 was unable to stimulate an AG-induced Ca2+ influx. UAG treatment (100nM) had no effect on GHSR1a-mediated Ca2+ influx in the presence or absence of MP2. MP2 is post-translationally modified and we suspected this to be important for its activity. However, removal or blockade of these modifications had no effect on the ability of MP2 to enhance GHSR1a signalling. Moreover, incubating the cells with soluble ectodomain of MP2 did not alter its effect on GHSR1a signalling. Nevertheless, induction of ectodomain shedding with PMA (0.4-1µM) dose-dependently reduced the AG-induced Ca2+ response to 0.5-0.2 of control levels (DMSO) in MP2-GHSR1a co-transfected cells. Unlike MP2, which has a transmembrane and intracellular domain, MP1 is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor and lacks an intracellular domain. Since MP1 is otherwise structurally similar to MP2, we suspected that the intracellular domain of MP2 may be important for its function. Therefore, we expressed chimeras of MP2 and MP1 in which the GPI linkage site and the transmembrane/intracellular domains were exchanged. The MP2 ectodomain with a GPI-anchor had similar stimulatory effects on GHSR1a signalling as full-length MP2, whereas the MP1 ectodomain with MP2 transmembrane and intracellular domain only enhanced GHSR1a signalling by approximately 3-fold.In conclusion, we have identified a membrane protein as a novel component of the ghrelin signalling pathway that markedly enhances the response of the ghrelin receptor to AG. Our current data suggest its ectodomain is important in mediating this effect. Studies are ongoing to fully delineate the mode of interaction and to determine the role of MP2 in ghrelin signalling in vivo.

The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin

Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.

α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.

SELENOI is important for maintenance of phosphatidylethanolamine and glycosylphosphatidylinositol anchor protein during T cell activation and immune response

Abstract Selenoproteins are important for both innate and adaptive immune responses. As one of the selenoprotein members, SELENOI is characterized by its ethanolamine phosphotransferase activity in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis and PE is also the donor of phosphoethanolamine moiety in glycosylphosphatidylinositol anchor protein (GPI-AP). To explore how SELENOI is involved in the immune response, we generated three mouse models with different SELENOI expression levels, including defined selenium diets, doxycycline inducible SELENOI knockdown (KD) and T cell specific SELENOI knockout (KO) mice models. We analyzed both the quantity of PE species on activated CD3 T cell membrane by LCMS and GPI-AP expression on activated T cell surface by FACS in all these three models. The SELENOI KO T cells exhibit a lower level of PE species on T cell membrane compared to WT T cells, and reduced expression of GPI-anchored proteins including CD52, CD73, CD48, CD109 and CD44 on cell surface upon activation as well. We also detected the same pattern of reduced PE species and GPI-anchor proteins on defined selenium diets and doxycycline-inducible SELENOI KD mouse models. Furthermore, the SELENOI KO T cells exhibited a reduced proliferation rate upon CD3/CD28 co-stimulation. Vaccination with LCMV peptides in SELENOI deficient mice results in less CD8+ and CD4+ LCMV specific T cells and reduced IFN-γ production, indicating an impaired immune response when SELENOI is absent. These results indicate that SELENOI is crucial for both PE and GPI-AP synthesis during T cell activation and proliferation due to the importance of PE during the activation process.