Glycosyl phosphatidylinositol-anchored ceruloplasmin is expressed by rat Sertoli cells and is concentrated in detergent-insoluble membrane fractions.

The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.     

Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.     

Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, including human Cripto-1 (hCFC2/hCR-1) and human Cryptic (hCFC1), are membrane-associated Nodal co-receptors, which have critical roles in vertebrate development. Most of the EGF-CFC proteins have been experimentally proven or predicted to be glycosylphosphatidylinositol (GPI)-anchored proteins. However, unlike other EGF-CFC proteins, hCFC1 does not exhibit a typical GPI-signal sequence, containing a 32-amino acid hydrophilic extension in its COOH-terminal end. Here we experimentally demonstrate that the COOH-terminal sequence of hCFC1 functions as a GPI-anchoring signal. Moreover, addition of a hydrophilic epitope tag of 55-amino acids (V5-His) after the GPI signal of hCR-1 interfered with generation of a GPI-anchored form of hCR-1. In contrast, addition of the same epitope tag to the end of GPI signal of hCFC1 did not affect the GPI-attachment of hCFC1. The COOH-terminal signal of hCFC1 could produce two different forms of the protein; a GPI-anchored form and an unprocessed form which was more prone to be secreted into the conditioned medium. The hydrophilic extension of hCFC1 negatively regulates the activity of hCFC1 as a Nodal co-receptor. These results demonstrate the presence of endogenous GPI-signal sequence with a hydrophilic extension, which can generate both GPI-anchored and soluble forms of the protein.     

N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.     

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.     

Biochemical analysis of a missense mutation in aceruloplasminemia.

Aceruloplasminemia is an inherited neurodegenerative disease characterized by parenchymal iron accumulation secondary to loss-of-function mutations in the ceruloplasmin gene. To elucidate the molecular pathogenesis of aceruloplasminemia, the biosynthesis of a missense mutant ceruloplasmin (P177R) occurring in an affected patient was examined. Chinese hamster ovary cells transfected with cDNAs encoding secreted and glycosylphosphatidylinositol (GPI)-linked wild-type or P177R human ceruloplasmin were examined by pulse-chase metabolic labeling. These experiments, as well as immunofluorescent analysis and N-linked glycosylation studies, indicate that both the secreted and GPI-linked forms of the P177R mutant are retained in the endoplasmic reticulum (ER). The P177R mutation resides within a novel motif, which is repeated six times in human ceruloplasmin and is conserved in the homologous proteins hephaestin and factor VIII. Analysis of additional mutations in these motifs suggests a critical role for this region in ceruloplasmin trafficking and indicates that substitution of the arginine residue is critical to the ER retention of the P177R mutant. Metabolic labeling of transfected Chinese hamster ovary cells with (64)Cu indicates that the P177R mutant is retained in the ER as an apoprotein and that copper is incorporated into both secreted and GPI-linked ceruloplasmin as a late event in the secretory pathway. Taken together, these studies reveal new insights into the determinants of holoceruloplasmin biosynthesis and indicate that aceruloplasminemia can result from retention of mutant ceruloplasmin within the early secretory pathway.     

Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor

Secreted forms of the sialoglycoprotein designated cellularprion protein (PrP^C) have been identified that cannot be explainedby alternative splicing. We report that secreted forms of PrP^Cderive from precursors that are bound to the plasma membraneby glycoinositol phospholipid (GPI) anchors. Secreted PrP^C slowlyappeared in the culture medium of metabolically radiolabelledcells after incubations of 8—24 h. Digestion of nascentPrP^C with phosphatidylinositol-specific phospholipase C (PIPLC)prevented the appearance of secreted PrP^C. Secreted PrP^c partitionedinto the aqueous phase of Triton X–114 like PIPLC-releasedPrP^C. While the M_r of PIPLC-released PrP^C was reduced 2–4kDa after treatment with aqueous hydroflouric acid, which removesthe entire GPI anchor modification, the M_r of secreted PrP^Cwas unchanged. Both PIPLC-released and secreted PrP^c were recognizedby antiserum raised against a synthetic C-terminal peptide correspondingto residues 220–233 (amino acid 231 is the site of GPIattachment). We conclude that GPI-anchored PrP^C is post-translationallyprocessed to remove most, if not all, of the GPI modificationand then shed into culture medium. Whether PrP^C is shed afterproteolysis near the C-terminus remains to be established. Aminority of PrP^C in normal Syrian hamster brain partitionedinto the aqueous phase of Triton X–114 like shed PrP^Csuggesting physiological significance. post-translational prion protein secretion sialoglycoprotein     

Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.     

Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion of GPI8 (to give Deltagpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non-protein-linked GPI molecules, some of which are surface located. Procyclic Deltagpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.     

Processing and trafficking of Leishmania mexicana GP63. Analysis using GP18 mutants deficient in glycosylphosphatidylinositol protein anchoring

GPI8 is a clan CD, family C13 cysteine protease and the catalytic core of the GPI-protein transamidase complex. In Leishmania mexicana, GPI8 is nonessential, and Deltagpi8 mutants lack the GPI-anchored metalloprotease GP63, which is the major surface protein of promastigotes. We have identified the active site histidine and cysteine residues of leishmanial GPI8 and generated Deltagpi8 lines expressing modified GPI8 proteins. This has allowed us to study the processing and trafficking of GP63 in wild type and Deltagpi8 mutants. We show using pulse-chase labeling that in Deltagpi8 non-GPI-anchored GP63 was glycosylated and secreted without further processing from the cell with a t(12) of 120 min. This secretion was prevented by growth of cells in the presence of tunicamycin, indicating that glycosylation is necessary for secretion of non-GPI-anchored proteins. In contrast, in wild type cells the majority of GP63 was rapidly glycosylated, GPI-anchored, and trafficked to the surface with defined processing intermediate forms. Tunicamycin inhibited glycosylation but did not prevent GPI anchor addition or trafficking. These results show that GPI-anchored and unanchored GP63 are trafficked via different pathways. In addition, the balance between GPI anchor addition and secretion of GP63 in Leishmania can vary depending on the activity of the GPI-protein transamidase, which has implications for the host-parasite interaction.     

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in?vivo assay

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.     

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.     

Metabolism of glycosylphosphatidylinositol-anchored proteins in Arabidopsis

Although glycosylphosphatidylinositol (GPI)-anchored proteins have now been found in several plants, very little is known regarding their metabolism there. This report describes studies of the biosynthesis and turnover of arabinogalactan proteins, a class of abundant GPI-anchored proteins secreted by cultured Arabidopsis cells.     

Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the beta-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins.     

GPI-anchored proteins: now you see 'em, now you don't.

Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.     

PER1 is required for GPI-phospholipase A2 activity and involved in lipid remodeling of GPI-anchored proteins

Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway. We found that GPI-anchored proteins could not associate with the detergent-resistant membranes in per1Delta cells. In addition, the mutant cells had a defect in the lipid remodeling from normal phosphatidylinositol (PI) to a C26 fatty acid-containing PI in the GPI anchor. In vitro analysis showed that PER1 is required for the production of lyso-GPI, suggesting that Per1p possesses or regulates the GPI-phospholipase A2 activity. We also found that human PERLD1 is a functional homologue of PER1. Our results demonstrate for the first time that PER1 encodes an evolutionary conserved component of the GPI anchor remodeling pathway, highlighting the close connection between the lipid remodeling of GPI and raft association of GPI-anchored proteins.     

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.     

Glycosylphosphatidylinositol-anchored ceruloplasmin is required for iron efflux from cells in the central nervous system

Ceruloplasmin (Cp) is a ferroxidase that converts highly toxic ferrous iron to its non-toxic ferric form. A glycosylphosphatidylinositol (GPI)-anchored form of this enzyme is expressed by astrocytes in the mammalian central nervous system, whereas the secreted form is expressed by the liver and found in serum. Lack of this enzyme results in iron accumulation in the brain and neurodegeneration. Herein, we show using astrocytes purified from the central nervous system of Cp-null mice that GPI-Cp is essential for iron efflux and not involved in regulating iron influx. We also show that GPI-Cp colocalizes on the astrocyte cell surface with the divalent metal transporter IREG1 and is physically associated with IREG1. In addition, IREG1 alone is unable to efflux iron from astrocytes in the absence of GPI-Cp or secreted Cp. We also provide evidence that the divalent metal influx transporter DMT1 is expressed by astrocytes and is likely to mediate iron influx into these glial cells. The coordinated actions of GPI-Cp and IREG1 may be required for iron efflux from neural cells, and disruption of this balance could lead to iron accumulation in the central nervous system and neurodegeneration.     

A mutation in the second intracellular loop of the pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation

A recently identified membrane-type 6 matrix metalloproteinase (MT6-MMP) has a hydrophobic stretch of 24 amino acids at the C-terminus. This hydrophobicity pattern is similar to glycosyl-phosphatidyl inositol (GPI)-anchored MMP, MT4-MMP, and other GPI-anchored proteins. Thus, we tested the possibility that MT6-MMP was also a GPI-anchored proteinase. Our results showed that MT6-MMP as well as MT4-MMP were labeled with [³H]ethanolamine indicating the presence of a GPI unit with incorporated label. In addition, phosphatidyl inositol-specific phospholipase C treatment released MT6-MMP from the surface of transfected cells. These results strongly indicate that MT6-MMP is a GPI-anchored protein. Since two members of MT-MMPs are now assigned as GPI-anchored proteinase, MT-MMPs can be subgrouped into GPI type and transmembrane type.     

New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins

Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I (GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG-N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG-N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.