Neural cell recognition molecule F11: membrane interaction by covalently attached phosphatidylinositol

The mode of membrane insertion of F11 130 kDa protein, a neural chick cell surface glycoprotein involved in neurite fasciculation, has been investigated. Up to 41% of total F11 130 kDa is released from adult chick brain plasma membranes by phosphatidylinositol specific phospholipase C (PI-PLC), whereas no release is mediated by lecithin/cephalin specific phospholipase C (PLC). PI-PLC dependent release of F11 is also observed from embryonal chick brain plasma membranes and from the surface of intact retinal cells. Biosynthetic labelling experiments demonstrate that F11 contains ethanolamine. Taken together, these results suggest that F11 interacts with the plasma membrane at least partially through covalently linked glycosyl-phosphatidylinositol (GPI) or a structurally similar lipid.     

Molecular Cloning and in Situ Localization of the Human Contactin Gene (CNTN1) on Chromosome 12q11-q12

Chick contactin/F11 (also known as F3 in mouse) is a neuronal cell adhesion molecule of the immunoglobulin (Ig) gene family that is implicated in playing a role in the formation of axon connections in the developing nervous system. In human brain, contactin was first identified by amino terminal and peptide sequencing of the lentil-lectin-binding glycoprotein Gp135. We now report the isolation and characterization of cDNA clones encoding human contactin. Human contactin is composed of six C2 Ig-domains and four fibronectin type III (FNIII) repeats and is anchored to the membrane via a glycosyl phosphatidylinositol moiety, as shown by PI-PLC treatment of cells transfected with contactin cDNA and metabolic labeling with [3H]-ethanolamine. At the amino acid level, h-contactin is 78% identical to chick contactin/F11 and 94% to mouse F3. Independent cDNAs encoding two putative contactin isoforms were isolated and sequenced: h-contactin 1 cDNA encodes a protein with the amino-terminal sequence of purified Gp135, while the putative h-contactin 2 gene has a deletion of 33 nucleotides that predicts a protein with a shortened amino terminus. Northern analysis with a probe common for both isoforms detects one mRNA species of approximately 6.6 kb in adult human brain. Fluorescence in situ hybridization maps the gene for human contactin to human chromosome 12q11-q12. The h-contactin gene locus is thus in close proximity to homeobox 3, integrin subunit α5, several proto-oncogene genes, a chromosomal breakpoint associated with various tumors, and the gene locus for Stickler syndrome. The cloning of human contactin now permits the study of its role in disorders of the human nervous system.     

Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains

Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains.     

Identification of a GPI-anchored type HDL-binding protein on human macrophages

To identify the HDL3-binding proteins on human macrophages, we examined the involvement of GPI-anchored protein in the binding of HDL3, and tried to purify HDL3-binding protein. From membrane fractions of macrophages, we obtained 80- and 130-kDa HDL3-binding proteins by ligand blotting. Treatment of macrophages with phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreased the specific HDL3-binding in a dose-dependent manner. Furthermore, treatment with mannosamine, which blocks GPI-anchor formation, decreased specific HDL3-binding in a dose-dependent manner. PI-PLC treatment released from the cells the proteins with an M(r) of 80 kDa, which could also bind HDL3. PI-PLC as well as mannosamine treatment markedly reduced cholesterol efflux from macrophages in association with the decreased HDL-binding. Using HDL3-affinity chromatography, we purified 80-kDa GPI-anchored type HDL3-binding protein. In summary, we demonstrate the implication of 80-kDa GPI-anchored protein in the binding of HDL3 to human macrophages, which might have some role in reverse cholesterol transport.     

Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.     

Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.     

A neuronal surface glycoprotein associated with the cytoskeleton

A cytoskeleton-associated glycoprotein of 130-kilodalton molecular mass (GP 130) was purified from a nonionic detergent-insoluble fraction of 10-16-d-old chicken embryo brains. GP 130 is tightly associated with other proteins in actin-containing complexes (Moss, D.J., 1983, Eur. J. Biochem., 135:291-297); thus, pure protein preparations were obtained only after the partial dissociation of the complexes with the zwitterionic detergent, dimethyl dodecyl glycine (EMPIGEN BB), followed by ion-exchange chromatography and electrophoresis on preparative SDS polyacrylamide gels. Specific monoclonal and polyclonal antibodies were raised to GP 130 and used to examine its distribution in the developing nervous system. Experiments with these antibodies revealed that GP 130 is confined to nervous tissue and is restricted to the surface of neurons in cultures derived from both the central and peripheral nervous systems. This novel glycoprotein is immunologically unrelated to the neuronal cell adhesion molecule (N-CAM), or to vinculin, a protein of similar molecular mass which has been suggested to link actin filaments to the plasma membrane. In the developing chicken embryo brain, GP 130 is first detectable around day 8 after fertilization and increases to approximately 50% of its adult level by embryonal day 13. In contrast, no increase is observed over a similar developmental period in sciatic nerve. In the adult chicken, GP 130 is most abundant in brain and has a particularly high content in areas rich in dendrites and synapses.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.     

F3/contactin,a neuronal cell adhesion molecule implicated in axogenesis and myelination

A general feature of the cell adhesion molecules belonging to the immunoglobulin family (Ig-CAMs) is to display a modular structure that provides a framework for multiple binding sites for other recognition molecules. Among this family, F3/contactin is a glycan phosphatidyl-inositol (GPI)-anchored molecule expressed by neurons that displays the distinctiveness to exert heterophilic but no homophilic binding activities. The Ig domains of F3/contactin were shown to interact with the L1 family of Ig-CAMs, including L1, NrCAM, and neurofascin. Binding between F3/contactin and NrCAM is known to modulate axonal elongation of the cerebellar granule cells and to control sensory axon guidance. F3/contactin mediates neuron-glial contacts through its association with extracellular matrix components (tenascin-R, tenascin-C) and RPTPbeta/phosphacan, influencing axonal growth and fasciculation. Another major role of F3/contactin is to organize axonal subdomains at the node of Ranvier of myelinated fibers in interplay with other Ig-CAMs, through its binding with caspr/paranodin at paranodes and the voltage-gated sodium channels in the nodal region. The F3/contactin deficient mice display a severe ataxia correlated with defects in axonal and dendritic projections in the cerebellum. These mice also display defects in nerve influx conduction due to the disruption of the axo-glial contacts at paranodes. Finally, the recent identification of a Drosophila homologue of F3/contactin indicated that this family of GPI-anchored CAMs plays a conserved function in axonal insulation.     

On the same cell type GPI-anchored normal cellular prion and DAF protein exhibit different biological properties

Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.     

A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor.

Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment.     

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.     

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.     

Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule.

The rodent neural cell adhesion molecule (NCAM) consists of three glycoproteins with Mr of 180,000, 140,000 and 120,000. The Mr 120,000 protein (NCAM-120) has been shown to exist in membrane-bound and soluble forms but the nature of its membrane association and release has remained obscure. We show here that phosphatidylinositol-specific phospholipase C (PI-PLC), but not a phospholipase C of different specificity, releases a substantial proportion of NCAM-120 from brain membranes and solubilizes almost quantitatively NCAM-120 present at the surface of C6 astroglial cells. The PI-PLC effect was highly selective since only one other protein species was detectably released from C6 cells. These results suggest that NCAM-120 is held in the membrane by covalently bound phosphatidylinositol or a closely related lipid in a way similar to several other surface proteins from eukaryotic cells. The presence of NCAM in a form which can be released from the cell surface by a highly selective mechanism raises additional possibilities for modulation and control of cell--cell adhesion.     

Phospholipase-C sensitive GPI-anchored proteins of goat sperm: possible role in sperm protection

The role of glycosylphosphatidylinositol (GPI)-anchored sperm proteins in reproduction has been investigated. SDS-polyacrylamide gels (PAGE) analysis of goat sperm (Capra indica) indicated that several GPI-anchored proteins were released by phosphatidylinositol-specific phospholipase-C (PI-PLC) treatment. The distribution of this category of PI-PLC-sensitive GPI-anchored proteins on the surface of sperm was examined by indirect immunofluorescence. The fluorescence microscopic study clearly demonstrated that the PI-PLC-sensitive GPI-anchored proteins are confined predominantly to the head region of goat sperm. Further experiments were conducted on intact and PI-PLC treated sperm in order to decipher the function of GPI proteins. Co-incubation of sperm with peritoneal macrophages led to the enhanced phagocytosis of PI-PLC treated sperm by macrophages compared with the untreated intact sperm. Transmission electron micrographs of the macrophages acquired from the phagocytosis assay are provided to corroborate the same. From the results obtained it is inferred that one or more of the PI-PLC-sensitive GPI-anchored proteins on the sperm surface could act as protection factor(s) that shield the sperm from macrophages.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.     

Role of GPI-anchored Enzyme in Liposome Detergent-Resistance

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.