Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase

The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles.     

Naphthalene uptake by a Pseudomonas fluorescens isolate

The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.     

Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane

Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.     

Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events.

The gag precursor polyprotein of Moloney murine leukemia virus (MuLV) is normally modified by myristylation of the N-terminal glycine. Previous work showed that the Pr65gag lacking the myristylation site does not associate with cellular membranes or assemble into virus particles. We now report that it also is not cleaved to the mature gag cleavage products within the cell and that it sediments as a free 65-kilodalton monomer in detergent-free cell extracts containing 0.3 M NaCl. Even when the cells containing the mutant are productively infected with wild-type MuLV, the mutant Pr65gag is not processed into cleavage products and is not incorporated into the virions produced by these cells. Thus, the mutant gag molecules seem unable to participate in the normal processes of self-assembly and maturation. We propose that myristate-mediated membrane association is an essential first step in MuLV assembly. This association may also play a role in budding of MuLV.     

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

The effects of Al on red spruce ( Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereological and microscopic methods. Exposure to Al for 24–48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al. Using energy-dispersive X-ray microanalysis on sections of freeze-substituted cells that had no obvious disruption in cytoplasmic or cell wall structure, Al (always in the presence of P) was detected in dense regions in cell walls, cytoplasm, plastids and vacuoles after 48 h exposure to Al. Stereological quantification of spruce cell structure showed that, after 24 h of Al treatment, intact cells had increased vacuolar and total cell volume, but the nuclear volume did not change. In addition, Al treatment resulted in increased surface area of Golgi membranes and endoplasmic reticulum. The biochemical and ultrastructural alterations in red spruce cells, in combination with the presence of Al in cellular organelles of visually intact cells, suggest that Al movement occurred across the plasma membrane without major cellular disruption. Detailed short-term time course studies are needed to determine if intracellular Al in these cells results from its passage into cells through submicroscopic lesions in the plasma membrane or it is taken up into the symplast through the intact membrane by an active, but slow, process.     

Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. Evidence for a protein-mediated and ATP-dependent process(es)

The internalization of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine following their insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes containing the fluorescent lipid 1,2-(palmitoyl-N-4-nitrobenzo-2-oxa-1,3-diazole-amino-caproyl) phosphatidylserine [palmitoyl-C6-NBD)-PS), were incubated with monolayer cell cultures at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells occurred, resulting in prominent labeling of the plasma membrane. However, if the cells were washed and warmed to 7 degrees C for 30 min, the (palmitoyl-C6-NBD)-PS also labeled numerous intracellular membranes. Evidence is presented suggesting that this internalization was not due to endocytosis, but was the result of transmembrane movement of the (palmitoyl-C6-NBD)-PS at the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. This transmembrane movement was reversibly inhibited by depletion of cellular ATP levels and was blocked by treatment with structural analogs of the lipid or by pretreatment of cells with glutaraldehyde or N-ethyl-maleimide. A fluorescent analog of phosphatidylethanolamine [palmitoyl-C6-NBD)-PE), which also exhibits transmembrane movement at the plasma membrane at 7 degrees C (Sleight, R. G., and Pagano, R. E. (1985) J. Biol. Chem. 260, 1146-1154), was further studied. Its transmembrane movement was also inhibited by depletion of cellular ATP levels, or by pretreatment of cells with N-ethylmaleimide. The transmembrane movement of the fluorescent phosphatidylserine and phosphatidylethanolamine analogs was inhibited when the unnatural D-isomers of these lipids were used, further suggesting that this process was stereospecific and therefore likely to have been protein-mediated.     

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.     

Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.     

Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis.

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.     

Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

Increased binding of a hydrophobic, photolabile probe to Escherichia coli inversely correlates to membrane potential but not adenosine 5'-triphosphate levels.

We describe conditions for a quantitative determination of azidopyrene binding to Escherichia coli cells. In addition, we define conditions whereby irradiation of azidopyrene in the presence of cells leads to irreversible association of probe with cells. This is presumably due to the light-dependent generation of reactive nitrenes and subsequent incorporation of nitrenopyrene moieties into cellular components. These methods allowed us to determine that the amount of azidopyrene bound to cells was inversely correlated with the magnitude of the cellular membrane potential, but was not correlated with high or low adenosine 5-triphosphate levels per se. Cells bound more azidopyrene if the delta psi was low. Cell-bound azidopyrene was found to be entirely associated with the inner and outer membrane. We suggest that the decreased association of hydrophobic probes upon energization of whole cells reflects a rapid transition in structural properties of the cell envelope.     

Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus

The release of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A (BFA) action, preceding the movement of Golgi membrane into the ER. ATP depletion also causes the reversible redistribution of the 110-kD protein from Golgi membrane into the cytosol, although no Golgi disassembly occurs. To further define the effects of BFA on the association of the 110-kD protein with the Golgi apparatus we have used filter perforation techniques to produce semipermeable cells. All previously observed effects of BFA, including the rapid redistribution of the 110-kD protein and the movement of Golgi membrane into the ER, could be reproduced in the semipermeable cells. The role of guanine nucleotides in this process was investigated using the nonhydrolyzable analogue of GTP, GTP gamma S. Pretreatment of semipermeable cells with GTP gamma S prevented the BFA-induced redistribution of the 110-kD protein from the Golgi apparatus and movement of Golgi membrane into the ER. GTP gamma S could also abrogate the observed release of the 110-kD protein from Golgi membranes which occurred in response to ATP depletion. Additionally, when the 110-kD protein had first been dissociated from Golgi membranes by ATP depletion, GTP gamma S could restore Golgi membrane association of the 110-kD protein, but not if BFA was present. All of these effects observed with GTP gamma S in semipermeable cells could be reproduced in intact cells treated with AlF4-. These results suggest that guanine nucleotides regulate the dynamic association/dissociation of the 110-kD protein with the Golgi apparatus and that BFA perturbs this process by interfering with the association of the 110-kD protein with the Golgi apparatus.     

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.     

Uptake and Active Efflux of Polycyclic Aromatic Hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).     

Prediction of naphthalene bioaccumulation using an adipocyte cell line model

A long-term goal of this research is to develop an in vitro model to study the metabolism, distribution, and fate of chemicals or pharmaceuticals in animals and humans. An important component of such a system is an in vitro model to study bioaccumulation of specific chemicals in adipose tissue. Due to the difficulties in maintaining primary adipocytes in culture and conducting reproducible experiments, transformed adipocyte cell lines have been used as an alternative. In this paper, several rodent preadipocyte cell lines (3T3-L1, 3T3-F442A, and TA1 cells) that differentiate into adipocytes when exposed to the appropriate stimuli are tested as an investigative tool to study naphthalene accumulation. The in vitro model is tested by comparison of its performance to that of primary adipocytes. All the experimental evidence supports the hypothesis that naphthalene accumulation is primarily dependent on the level of intracellular lipid. Furthermore, the level of naphthalene bioaccumulation is linearly correlated with the amount of triglyceride content with the slope of 37.7 +/- 0.5 microg of naphthalene/(mg of triglyceride). Indomethacin/dexamethasone/insulin are shown to be more effective in promoting preadipocyte differentiation than methylisobutylxanthine/dexamethasone/insulin. Additionally, external factors, such as the presence of albumin and serum in the medium, affect the cellular naphthalene uptake by decreasing the amount of naphthalene transported into fat cells. Among the three cell lines tested, 3T3-L1 adipocytes accumulated the highest intracellular lipid and, hence, yielded the highest level of naphthalene accumulation. Its ability to accumulate naphthalene is comparable to that of primary adipocytes. The 3T3-L1 adipocyte model is appropriate for studying the bioaccumulation of xenobiotics that are aromatic hydrocarbons.     

Myristoyl-based transport of peptides into living cells

Translocation of membrane-impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to uptake of a cell-penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid, reaching a maximum within 30 min. Cellular uptake of the peptide lagged the membrane association but occurred within a similar time frame. Experiments performed at 37 versus 4 degrees C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 muM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cell-based biochemical studies.     

Transfer of cholesterol from its site of synthesis to the plasma membrane

We have followed the transfer of newly synthesized cholesterol to the plasma membrane in cultured fibroblasts using cholesterol oxidase as a probe. Since the enzyme has access only to the plasma membrane in intact cells, it permits the discrimination of cell surface and endogenous cholesterol. Cholesterol synthesized from radiolabeled acetate was transferred to the plasma membrane in a strictly first order fashion with a half-time of 1-2 h at 37 degrees C. The rate of transfer was similar in rapidly growing and confluent cells and was not affected by preincubating the cells in lipoprotein-deficient serum which greatly stimulated cholesterol synthesis. We used equilibrium density gradient centrifugation of homogenates from cholesterol oxidase-treated cells to examine further the distribution of newly synthesized cholesterol between cellular pools. We identified membrane fractions enriched in newly synthesized cholesterol yet inaccessible to cholesterol oxidase. The cholesterol in these membranes eventually moved to the plasma membrane. The movement of exogenous radiocholesterol from the plasma membrane to the cell interior also was examined by this method. No detectable transfer was observed over several hours, during which time endogenous cholesterol moved to the plasma membrane. We conclude that the transfer of newly synthesized cholesterol to the plasma membrane is a vectorial process and is not mediated by a simple diffusional equilibrium.     

Physico-chemical requirements for cellular uptake of pAntp peptide. Role of lipid-binding affinity.

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor-independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide-lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.     

Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.