C-terminal Glycosylphosphatidylinositol Research Articles

Eimeria tenella sporozoites and merozoites differentially express glycosylphosphatidylinositol-anchored variant surface proteins.

Little is known about glycosylphosphatidylinositol (GPI)-linked surface proteins in the coccidian parasite Eimeria tenella. Examination of 28,550 EST sequences from the sporozoite and second merozoite developmental stages of the parasite led to the identification of 37 potential GPI-linked variant surface proteins, termed EtSAGs. Analysis of the complete nucleotide sequences of 23 EtSAG genes separated them into two multi-gene families. All the predicted EtSAG proteins (which vary in length from 228 to 271 residues) have an N-terminal hydrophobic signal peptide, a C-terminal hydrophobic GPI signal-anchor peptide and an extracellular domain organised around six cysteine residues, the positions of which are conserved within each family. Using specific antibodies against a small number of recombinant-expressed EtSAGs, the surface localisation and GPI-anchorage of members of both families was confirmed experimentally. Expression of EtSAGs is differentially regulated between the oocyst/sporozoite and second generation merozoite stages, with only one expressed specifically in the sporozoite, a small number expressed in both stages and the majority expressed specifically in the second generation merozoite. Preliminary data support a model in which multiple variant surface antigens are co-expressed on individual parasites, rather than a model of antigenic switching. The biological role(s) of EtSAGs and the effect(s) that expression of a complex repertoire of variant surface antigens by the second generation merozoite has on host adapted immunity are unknown.

NYX (nyctalopin on chromosome X), the gene mutated in congenital stationary night blindness, encodes a cell surface protein.

The complete type of X-linked congenital stationary night blindness (CSNB1) in human and mouse is caused by mutations in the NYX gene. The human NYX protein has been predicted to contain an N-terminal endoplasmic reticulum (ER) signaling sequence and a C-terminal glycosylphosphatidylinositol (GPI) anchor. In the current study, these computer predictions were verified experimentally by expression of domain-specific cDNA constructs in COS-7 and HeLa cells. Moreover, computer-based analysis of the orthologue mouse amino acid sequence did not reveal a GPI anchor, which may result in a different protein localization compared with human NYX. Therefore, the cellular localization for the mouse Nyx protein was also examined. A new method was established that differentially visualizes both the protein at the surface of the living cell and subsequently in intracellular compartments. The localization of the human and mouse V5-tagged wild-type and mutant NYX protein were studied. Human and mouse V5-NYX proteins were dispersed in the form of speckles over the entire cell surface. Subsequent staining of the same cells after detergent extraction revealed that V5-NYX located to the ER and Golgi apparatus. Deletion of the GPI anchor domain of NYX resulted in a time-dependent loss of V5-NYX from the surface of living cells and accumulation of this truncated protein in the ER and Golgi apparatus. Deletion of the ER signal sequence in Nyx delocalized the intracellular V5-Nyx protein and caused its dispersion in the cytosol. Furthermore, mutations introduced in the leucine-rich repeat (LRR)-region, which has been described as a pathogenic variant of NYX, had no effect on subcellular localization of the protein. These data provide evidence that human and mouse nyctalopin are membrane-bound extracellular proteins and are functionally conserved.

Bst-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology.

An expression screen of a rat cDNA library for sequences encoding Golgi-localized integral membrane proteins identified a protein with an apparent novel topology, i.e. with both an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. Our data are consistent with this. Thus, the protein would have a topology that, in mammalian cells, is shared only by a minor, but pathologically important, topological isoform of the prion protein (PrP). The human orthologue of this protein has been described previously (BST-2 or HM1.24 antigen) as a cell surface molecule that appears to be involved in early pre-B-cell development and which is present at elevated levels at the surface of myeloma cells. We show that rat BST-2/HM1.24 has both a cell surface and an intracellular (juxtanuclear) location and is efficiently internalized from the cell surface. We also show that the cell surface pool of BST-2/HM1.24 is predominantly present in the apical plasma membrane of polarized cells. The fact that rat BST-2/HM1.24 apparently possesses a GPI anchor led us to speculate that it might exist in cholesterol-rich lipid microdomains (lipid rafts) at the plasma membrane. Data from several experiments are consistent with this localization. We present a model in which BST-2/HM1.24 serves to link adjacent lipid rafts within the plasma membrane.

Determinants of the in Vivo Folding of the Prion Protein: A BIPARTITE FUNCTION OF HELIX 1 IN FOLDING AND AGGREGATION

Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases. We analyzed wild type PrP in comparison with different PrP mutants and identified determinants of the in vivo folding pathway of PrP. The complete N terminus of PrP including the putative transmembrane domain and the first beta-strand could be deleted without interfering with PrP maturation. Helix 1, however, turned out to be a major determinant of PrP folding. Disruption of helix 1 prevented attachment of the glycosylphosphatidylinositol (GPI) anchor and the formation of complex N-linked glycans; instead, a high mannose PrP glycoform was secreted into the cell culture supernatant. In the absence of a C-terminal membrane anchor, however, helix 1 induced the formation of unglycosylated and partially protease-resistant PrP aggregates. Moreover, we could show that the C-terminal GPI anchor signal sequence, independent of its role in GPI anchor attachment, mediates core glycosylation of nascent PrP. Interestingly, conversion of high mannose glycans to complex type glycans only occurred when PrP was membrane-anchored. Our study indicates a bipartite function of helix 1 in the maturation and aggregation of PrP and emphasizes a critical role of a membrane anchor in the formation of complex glycosylated PrP.

Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein.

Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon-oligosaccharyltransferase complex.

The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery.

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins involves the action of a GPI trans-amidase, which replaces the C-terminal GPI signal sequence (GPI-SS) of the primary translation product with a preformed GPI lipid. The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p. Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI-SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue. This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon-specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins. Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI-anchoring machinery. However, in our hands, GPI attachment cannot be restored by the co-expression of human Gpi8p in yeast cells under any circumstances.

AFM Detection of GPI Protein Insertion into DOPC/DPPC Model Membranes

Membrane microdomains are involved in numerous cellular processes such as membrane transport and signal transduction. Among these functionally microdomains, rafts are enriched in sphingolipids and cholesterol and their existence is actually mainly explained by a fluid-ordered lipid lateral phase separation. They also concentrate proteins like these with a C-terminal glycosylphosphatidylinositol (GPI) modification that serves as an anchor to the plasma membrane. In order to better understand how GPI proteins partition into microdomains, the insertion of the GPI-anchored alkaline phosphatase (AP), added exogenously into supported phospholipid bilayers, was studied in real-time using atomic force microscopy (AFM). Fluid-gel (lipid ordered) phase-separated bilayers were made by fusion of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC, 1:1) vesicles. Time-dependent insertion of AP through its GPI anchor was observed at the periphery of the gel-phase domains. This demonstrates that GPI proteins can spontaneously localize in lipid ordered phases.

The circumsporozoite protein of Plasmodium falciparum is expressed and localized to the cell surface in the free-living ciliate Tetrahymena thermophila

Heterologous expression is an important tool for characterization of protein function, structural studies, and production of antigen. While many different host systems have been utilized for the expression of Plasmodium falciparum proteins, the extreme AT-richness of its genome represents an obstacle to efficient expression. In addition, primary sequence motifs such as glycosyl phosphatidyl-inositol (GPI) cleavage/attachment sites of P. falciparum are not recognized in currently used expression hosts. Recently, DNA-mediated transformation has been used for expression of heterologous genes in the ciliated protozoan Tetrahymena thermophila. We report the stable expression of full-length P. falciparum circumsporozoite (CS) protein in T. thermophila. The expressed gene utilized the native CS protein N-terminal secretory signal sequence and the C-terminal GPI anchoring signal. Immunofluorescence imaging demonstrated that the CS protein was localized to the cell surface of Tetrahymena. Metabolic labeling with tritiated myristate resulted in incorporation of label into the recombinant CS protein, indicating that the protein was bound to the cell surface via a GPI anchor. This is the first report of the recognition of targeting and GPI anchoring signals of the P. falciparum CS protein in a heterologous expression host.

Cell surface antigen CD109 is a novel member of the α2 macroglobulin/C3, C4, C5 family of thioester-containing proteins

Cell surface antigen CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein of approximately 170 kd found on a subset of hematopoietic stem and progenitor cells and on activated platelets and T cells. Although it has been suggested that T-cell CD109 may play a role in antibody-inducing T-helper function and it is known that platelet CD109 carries the Gov alloantigen system, the role of CD109 in hematopoietic cells remains largely unknown. As a first step toward elucidating the function of CD109, we have isolated and characterized a human CD109 cDNA from KG1a and endothelial cells. The isolated cDNA comprises a 4335 bp open-reading frame encoding a 1445 amino acid (aa) protein of approximately 162 kd that contains a 21 aa N-terminal leader peptide, 17 potential N-linked glycosylation sites, and a C-terminal GPI anchor cleavage-addition site. We report that CD109 is a novel member of the alpha 2 macroglobulin (alpha 2M)/C3, C4, C5 family of thioester-containing proteins, and we demonstrate that native CD109 does indeed contain an intact thioester. Analysis of the CD109 aa sequence suggests that CD109 is likely activated by proteolytic cleavage and thereby becomes capable of thioester-mediated covalent binding to adjacent molecules or cells. In addition, the predicted chemical reactivity of the activated CD109 thioester is complement-like rather than resembling that of alpha 2M proteins. Thus, not only is CD109 potentially capable of covalent binding to carbohydrate and protein targets, but the t(1/2) of its activated thioester is likely extremely short, indicating that CD109 action is highly restricted spatially to the site of its activation.

Metabolic labeling and structural analysis of glycosylphosphatidylinositols from parasitic protozoa.

Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-P-Manalpha1-2Manalpha1-6Manalpha1-4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phos-phatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence show that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially as this pathway, at least for Trypanosoma brucei, has been validated as a drug target.

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.

The Chaperone Protein BiP Binds to a Mutant Prion Protein and Mediates Its Degradation by the Proteasome

Familial prion diseases are thought to result from a change in structure of the mutant prion protein (PrP), which takes a pathogenic conformation. We have examined the role of molecular chaperones in the folding of normal and mutant PrP Q217R (PrP(217)) in transfected neuroblastoma cells. In a previous report we showed that, although most of the PrP(217) forms escape the endoplasmic reticulum quality control system and aggregate in post-Golgi compartments, a significant proportion of PrP(217) retains the C-terminal glycosylphosphatidyl inositol signal peptide (PrP32), and does not exit the endoplasmic reticulum (Singh, N., Zanusso, G., Chen, S. G., Fujioka, H., Richardson, S., Gambetti, P., and Petersen, R. B. (1997) J. Biol. Chem. 272, 28461-28470). We have now studied the folding and turnover of PrP32 to understand the mechanism by which abnormal PrP forms cause cellular toxicity in our cell culture model and in the human brain carrying the Gerstmann-Sträussler-Scheinker disease Q217R mutation. In this report, we show that PrP32 remains associated with the chaperone BiP for an abnormally prolonged period of time and is degraded by the proteasomal pathway. This study is the first demonstration that BiP is chaperoning the folding of PrP and plays a role in maintaining the quality control in the PrP maturation pathway. Our data provide new insight into the diverse pathways of mutant PrP metabolism and neurotoxicity.

Export of a misprocessed GPI-anchored protein from the endoplasmic reticulum in vitro in an ATP- and cytosol-dependent manner

Strict quality control mechanisms within the mammalian endoplasmic reticulum act to prevent misfolded and unprocessed proteins from entering post-endoplasmic reticulum (ER) compartments. Following translocation into the ER lumen via the Sec61p translocon, nascent polypeptide chains fold and are modified in an environment that contains numerous chaperones and other folding mediators. Recently it has emerged that polypeptides failing to acquire the native state are re-exported from the ER to the cytosol for ultimate degradation by the proteasome ubiquitin system, apparently mediated again via Sec61p. Substrates for this degradation pathway include proteins destined to become glycosyl phosphatidylinositol (GPI)-anchored, but which fail to be processed and retain the C-terminal GPI signal peptide. In order to characterise this process we have used a model GPI-anchored mutant protein, prepro mini human placental alkaline phosphatase (PLAP) W179, which cannot be processed efficiently on account of being a poor substrate for the transamidase which cleaves the GPI signal peptide and adds the GPI anchor in a coupled reaction. In vitro transcription, translation and translocation into canine pancreatic microsomes resulted in ER-targeting signal sequence cleavage and formation of prominiPLAP in the ER lumen. We were able to show that prominiPLAPW179 could be exported from the microsomes in a time-dependent manner and that release requires both ATP and cytosol. Export was not supported by GTP, indicating a biochemical distinction from glycopeptide export which we showed recently requires GTP hydrolysis. The process was not affected by redox, unlike several other GPI-anchored model proteins. These data demonstrate that misprocessed proteins can be exported in vitro from mammalian microsomes, facilitating identification of factors involved in this process.

Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins.

Glycosylphosphatidylinositol (GPI) anchors are attached to newly synthesized proteins in the ER by a transamidation reaction during which a C-terminal GPI attachment signal is replaced by a preformed GPI precursor lipid. This reaction depends on GAA1 and GPI8, the latter belonging to a novel cysteine protease family. Homologies between this family and other Cys proteinases, such as caspases, pointed to Cys199 and His157 as potential active site residues. Indeed, gpi8 alleles mutated at Cys199 or His157 are nonfunctional, i.e., they are unable to suppress the lethality of Deltagpi8 mutants. The overexpression of these nonfunctional alleles in wild-type cells leads to the accumulation of the free GPI precursor lipid CP2, delays the maturation of the GPI protein Gas1p, and arrests cell growth. The dominant negative effect of the Cys199 mutant cannot be overcome by the simultaneous overexpression of Gaa1p. Most GPI8 alleles mutated in other conserved regions of the protein can complement the growth defect of Deltagpi8, but nevertheless accumulate CP2. CP2 accumulation, a delay in Gas1p maturation and a slowing of cell growth can also be observed when Gpi8p is depleted to 50% of its normal level in wild-type cells. The dominant negative effect of nonfunctional and partially functional mutant alleles can best be explained by assuming that Gpi8p works as part of a homo- or heteropolymeric complex.

Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells.

The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N -linked high mannose type structures and low levels of O -linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by approximately 14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove approximately 6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intra-cellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N -glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.

The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor.

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.

Solution structure of an EGF module pair from the Plasmodium falciparum merozoite surface protein 1

The solution structure of the 96-residue C-terminal fragment of the merozoite surface protein 1 (MSP-1) from Plasmodium falciparum has been determined using nuclear magnetic resonance (NMR) spectroscopic measurements on uniformly 13C/15N-labelled protein, efficiently expressed in the methylotrophic yeast Komagataella (Pichia) pastoris. The structure has two domains with epidermal growth factor (EGF)-like folds with a novel domain interface for the EGF domain pair interactions, formed from a cluster of hydrophobic residues. This gives the protein a U-shaped overall structure with the N-terminal proteolytic processing site close to the C-terminal glycosyl phosphatidyl inositol (GPI) membrane anchor site, which is consistent with the involvement of a membrane-bound proteinase in the processing of MSP-1 during erythrocyte invasion. This structure, which is the first protozoan EGF example to be determined, contrasts with the elongated structures seen for EGF-module pairs having shared Ca2+-ligation sites at their interface, as found, for example, in fibrillin-1. Recognition surfaces for antibodies that inhibit processing and invasion, and antibodies that block the binding of these inhibitory antibodies, have been mapped on the three-dimensional structure by considering specific MSP-1 mutants.

Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion.

Using transformed procyclic trypanosomes, the synthesis, intracellular transport and secretion of wild-type and mutant variant surface glycoprotein (VSG) is characterized. We find no impediment to the expression of this bloodstream stage protein in insect stage cells. VSG receives a procyclic-type phosphatidylinositol-specific phospholipase C-resistant glycosyl phosphatidylinositol (GPI) anchor, dimerizes and is N-glycosylated. It is transported to the plasma membrane with rapid kinetics (t(1/2) approximately 1 h) and then released by a cell surface zinc-dependent metalloendoprotease activity, a possible homolog of leishmanial gp63. Deletion of the C-terminal GPI addition signal generates a soluble form of VSG that is exported with greatly reduced kinetics (t(1/2) approximately 5 h). Fusion of the procyclic acidic repetitive protein (PARP) GPI anchor signal to the C-terminus of the truncated VSG reporter restores both GPI addition and transport competence, suggesting that GPI anchors play a critical role in the folding and/or forward transport of newly synthesized VSG. The VSG-PARP fusion is also processed near the C-terminus by events that do not involve N-linked oligosaccharides and which are consistent with GPI side chain modification. This unexpected result suggests that GPI processing may be influenced by adjacent peptide sequence or conformation.

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein.

The plasma membrane of rat erythrocytes contains a 47-kDa glycoprotein that binds the channel-forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N-terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N-termini of a group of membrane proteins that are thought to be involved in ADP-ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C-terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol-specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells' sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin-binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.

A novel splice variant of the cell adhesion molecule BIG-2 is expressed in the olfactory and vomeronasal neuroepithelia

We have cloned a mouse cDNA encoding a novel truncated form of the gene BIG-2 from the vomeronasal organ. The related proteins BIG-2 and BIG-1 possess a C-terminal glycosylphosphatidylinositol anchor, six immunoglobulin domains and four fibronectin type III repeats. They are related to certain axonal-associated cell adhesion molecules (AxCAMs) exhibiting most similarity to the TAG-1/F3 subgroup of neural cell adhesion molecules. The cDNA we have identified, termed BIG-2A, appears to represent a novel splice variant of BIG-2 possessing six Ig-like domains, a single fibronectin repeat and lacking the glycosylphosphatidylinositol-anchoring domain (GPI). To determine the expression of this gene, in situ hybridization analysis was performed in adult and developing mice using a riboprobe specific for BIG-2A. Maximum expression was observed in mature sensory cells of the vomeronasal neuroepithelium and a less intense signal was also evident in the olfactory neuroepithelium. These results suggest that alternative splicing of the BIG-2 gene transcript may play an important role in the organization of the vomeronasal and olfactory neuroepithelia.