GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice.

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease.

The release of glycosylphosphatidylinositol-anchored proteins from the cell surface.

Starting with the first description of the anchorage of a subset of cell surface proteins in eukaryotic cells from yeast to mammals with the aid of a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein, experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released into the extracellular environment has been accumulating. GPI-AP are released as soluble monomers or multimers having lost their anchor or within hetero-/multimeric assemblies with their complete anchor remaining attached. The configurations reported so far for those assemblies encompass carrier protein-bound monomers, phospholipid- and cholesterol-harboring micelle-like complexes as well as membrane vesicles and particles. Each of these configurations prevents direct contact of the GPI anchor with the aqueous environment. Their structural diversity is reflected in the different molecular mechanisms underlying their release, which involve (i) proteolytic or lipolytic cleavage of the protein or GPI moiety, respectively, (ii) masking of the GPI anchor in the binding pocket of carrier proteins or in the phospholipid mono- or bilayers of particles or vesicles, respectively, and (iii) direct transfer of anchor-harboring GPI-AP from donor to acceptor cells through intimate contact of their plasma membranes. Release of GPI-AP may occur spontaneously or in response to certain endogenous or environmental stress signals and exert specific roles in the (patho)physiology of eukaryotic organisms which, however, are only incompletely understood so far.

Communicative functions of GPI-anchored surface proteins in unicellular eukaryotes.

Research on several unicellular eukaryotes has identified communicative surface proteins, which are anchored to the outer membrane by glycosylphosphatidylinositol (GPI). Surprisingly, these surface proteins are also released into the environment, raising questions regarding the underlying adaptive advantages and the physical mechanisms that allow for this shedding. This article reviews the current knowledge on several GPI-proteins of different protist species, assembles the puzzling data on the different functions of surface bound and released forms of these proteins, and summarizes their contribution to intra- and interspecific signaling. Recent advances in biochemistry and glycobiology indicate that the GPI-anchor is one of the prerequisites of protein function of membrane bound as well as of released proteins, and hence is a crucial invention for microbial molecular communication. The sensitivity of GPI-anchors (e.g. to phospholipase C) requires consideration of environmental lipase activity of different sources in microbial communities, as these may represent exogenous factors involved in surface protein release. We hypothesize a complex surface protein based communication network and discuss the known facts on protist GPIs in an evolutionary context.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2.

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Galphai2 and Lyn via protein-protein and raft interactions. Lyn is activated probably by the Galphai2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLCgamma2 and CD59 clusters revealed that PLCgamma2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP(3)-Ca(2+) signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP(3)-Ca(2+) signal, via PLCgamma2 recruitment. The prolonged, analogue, bulk IP(3)-Ca(2+) signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP(3) bursts, each created by the transient recruitment of PLCgamma2 molecules to STALLed CD59.

GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1.

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Reverse signaling by glycosylphosphatidylinositol-linked Manduca ephrin requires a SRC family kinase to restrict neuronal migration in vivo.

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.

Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2.

During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T cells is induced upon activation and that anti-TSA-1 mAb inhibits the T cell receptor (TCR) signaling pathway in activated T cells. In the present study, we have analyzed a role of TSA-1 in thymic selection events, especially in TCR-mediated apoptosis. In in vitro experiments, anti-TSA-1 blocked anti-CD3-induced cell death of T cell hybridomas. When anti-TSA-1 was injected into newborn mice in vivo together with anti-CD3 epsilon or anti-TCR-beta, TCR/CD3-mediated apoptosis of thymocytes was almost completely blocked. The blockade of apoptosis was defined by the inhibition of, first, the decrease in total number of thymocytes; second, the decrease in percentages of CD4+ CD8+ thymocytes; and third, the induction of DNA fragmentation. However, anti-TSA-1 did not block either steroid- or radiation-induced apoptosis, indicating that a signal via TSA-1 does not inhibit a common pathway of thymocyte apoptosis. Since TCR-mediated apoptosis is pivotal in thymic ontogeny, these results suggest that TSA-1/Sca-2 is an important cell surface molecule regulating the fate of a developing T cell.

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

Inherited GPI deficiency: a disorder of histone hypoacetylation.

Co-operative interaction of transcription factors (TF) with epigenetic processes, such as chromatin remodeling and modification (acetylation or methylation), as well as DNA methylation, determine transcriptional activity, activation or repression of a given gene. Mutations disrupting binding of TF to their cognate DNA motifs would be expected to alter the epigenetic landscape of the promoter and selectively affect transcription of the given gene. We review here the transcriptional, epigenetic, biochemical, and clinical consequences of a constitutional mutation in the promoter of PIGM, a housekeeping gene that disrupts binding of the general TF, SP1, thus causing the autosomal recessive disease, inherited glycosylphosphatidylinositol (GPI) deficiency. We suggest that detailed dissection of the function of the mutated PIGM promoter provides important lessons pertinent to the transcriptional and epigenetic control of housekeeping genes as a whole and might have wider therapeutic implications.

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro, such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens. Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlalpha1/beta2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I(max) of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlalpha1/beta2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlalpha1(Y151S) mutation was included (Nlalpha1(Y151S)/beta2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlalpha1/beta2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlalpha1(Y151S)/beta2 (3.25-fold and 2.86-fold) than in Nlalpha1/beta2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens. These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.

The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration.

The proximodistal identity of a newt limb regeneration blastema is respecified by exposure to retinoic acid, but its molecular basis is unclear. We identified from a differential screen the cDNA for Prod 1, a gene whose expression in normal and regenerating limbs is regulated by proximodistal location and retinoic acid: Prod 1 is the newt ortholog of CD59. Prod 1/CD59 was found to be located at the cell surface with a GPI anchor which is cleaved by PIPLC. A proximal newt limb blastema engulfs a distal blastema after juxtaposition in culture, and engulfment is specifically blocked by PIPLC, and by affinity-purified antibodies to two distinct Prod 1/CD59 peptides. Prod 1 is therefore a cell surface protein implicated in the local cell-cell interactions mediating positional identity.

Integrins take partners: cross-talk between integrins and other membrane receptors.

This review discusses an emerging theme in the understanding of how an integrin contributes to the life of a cell. Previously, integrins have been thought to 'go it alone', but it is now appreciated that their duties extend beyond that of being 'sticky' receptors. By interacting in cis with other receptors on the cell membrane, integrins and their partner receptors inteact to form distinct membrane complexes that recruit signalling molecules to each receptor's mutual benefit. Here, Joanna Porter and Nancy Hogg discuss a few of the best characterized of these specialist integrin partnerships.

Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent.

CD59, an 18-20-kD complement inhibitor anchored to the membrane via glycosyl phosphatidylinositol (GPI), can induce activation of T cells and neutrophils upon cross-linking with antibody. GPI-anchored molecules cocluster in high mol wt detergent-resistant complexes containing tyrosine kinases that are implicated in the signaling pathway. Exogenous, incorporated GPI-anchored molecules are initially unable to induce activation, presumably because they are not associated with kinases. Here we demonstrate that erythrocyte-derived CD59 incorporated in a CD59-negative cell line acquires signaling capacity in a time-dependent manner. Confocal microscopy revealed an initial diffuse distribution of CD59 that became clustered within 2 h to give a pattern similar to endogenous GPI-anchored molecules. Gel filtration of detergent-solubilized cells immediately after incorporation revealed that CD59 was mainly monomeric, but after 3 h incubation all was in high mol wt complexes and had become associated with protein kinases. Newly incorporated CD59 did not deliver a Ca2+ signal upon cross-linking, but at a time when it had become clustered and associated with kinase activity, cross-linking induced a large calcium transient, indicating that CD59 had incorporated in a specialized microenvironment that allowed it to function fully as a signal-transducing molecule.

GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.

The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells.

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST-Rho39Tr) to the apical membrane. The targeting of GST-Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST-Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST-Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells.

GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP-binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells.

A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy of ultracryosections of mucosal tissue, the protein was localized to the apical surface of the enterocytes, whereas it was absent from the basolateral plasma membrane. Interestingly, it was mainly found in patches of flat or invaginated apical membrane domains rather than at the surface of microvilli. Caveolae were not found in association with these labeled microdomains. In addition, the 80-kD protein was seen in apical endocytic vacuoles and in tubulo-vesicular structures, suggesting that the apical microdomains are involved in endocytosis of the 80-kD protein. By its NH2-terminal amino acid sequence, iron-binding capacity and partial immunological cross-reactivity with serum transferrin, the 80-kD protein was shown to belong to the transferrin family, and it is probably homologous to melanotransferrin, a human melanoma-associated antigen. The 80-kD iron-binding protein was fully detergent-soluble immediately after synthesis and only became insoluble after gaining resistance to endo H, supporting a mechanism for exocytic delivery to the apical cell surface by way of detergent-insoluble glycolipid "rafts" that fuse with the plasmalemma at restricted sites devoid of microvilli.

Pathogenic mutations in the glycosylphosphatidylinositol signal peptide of PrP modulate its topology in neuroblastoma cells.

Point mutations M232R (PrP(232R)), M232T (PrP(232T)), and P238S (PrP(238S)) in the glycosylphosphatidylinositol signal peptide (GPI-SP) of the prion protein (PrP(C)) segregate with familial Creutzfeldt-Jakob disease (CJD). However, the mechanism by which these mutations induce cytotoxicity is unclear since the GPI-SP is replaced by a GPI anchor within 5 min of PrP synthesis and translocation into the endoplasmic reticulum (ER). To examine if mutations in this region interfere with translocation of nascent PrP into the ER or anchor addition, the metabolism of PrP(232R) and PrP(232T) was investigated in transfected human neuroblastoma cells. In this report, we demonstrate that PrP mutations M232R and M232T do not interfere with GPI anchor addition. Instead, these mutations increase the stability and transport of GPI-SP mediated post-translationally translocated PrP to the plasma membrane, where it is linked to the lipid bilayer in a potentially neurotoxic C-transmembrane ((Ctm)PrP) orientation. Furthermore, we demonstrate that the GPI-SP of PrP functions as an efficient co-translational and inefficient post-translational ER translocation signal when tagged to an unrelated protein, underscoring the functional versatility of this peptide. These data uncover an alternate pathway of ER translocation for nascent PrP, and provide information on the possible mechanism(s) of neurotoxicity by mutations in the GPI-SP.