Radhia Mahfoud

Identification of a common sphingolipid-binding domain in Alzheimer, prion, and HIV-1 proteins.

The V3 loop of the human immunodeficiency virus (HIV)-1 surface envelope glycoprotein gp120 is a sphingolipid-binding domain mediating the attachment of HIV-1 to plasma membrane microdomains (rafts). Sphingolipid-induced conformational changes in gp120 are required for HIV-1 fusion. Galactosylceramide and sphingomyelin have been detected in highly purified preparations of prion rods, suggesting that the prion protein (PrP) may interact with selected sphingolipids. Moreover, a major conformational transition of the Alzheimer β-amyloid peptide has been observed upon interaction with sphingolipid-containing membranes. Structure similarity searches with the combinatorial extension method revealed the presence of a V3-like domain in the human prion protein PrP and in the Alzheimer β-amyloid peptide. In each case, synthetic peptides derived from the predicted V3-like domain were found to interact with monomolecular films of galactosylceramide and sphingomyelin at the air-water interface. The V3-like domain of PrP is a disulfide-linked loop (Cys179–Cys214) that includes the E200K mutation site associated with familial Creutzfeldt-Jakob disease. This mutation abrogated sphingomyelin recognition. The identification of a common sphingolipid-binding motif in gp120, PrP, and β-amyloid peptide underscores the role of lipid rafts in the pathogenesis of HIV-1, Alzheimer, and prion diseases and may provide new therapeutic strategies.

A major fraction of glycosphingolipids in model and cellular cholesterol-containing membranes is undetectable by their binding proteins.

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based “aglycone” interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb3), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb3, HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb3, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb3/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb3, suggesting GSL-GSL interaction can counter cholesterol masking of Gb3. The similar separation of Vero cell membrane-derived vesicles into minor “binding,” and major “non-binding” fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of “available” and “cholesterol-masked” plasma membrane Gb3 pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.

Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies

Although verotoxin‐1 (VT1) and verotoxin‐2 (VT2) share a common receptor, globotriaosyl ceramide (Gb3), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb3 assemblies. At 4°C, fluorescent‐VT1 and VT2 bound both coincident and distinct punctate surface Gb3 microdomains. After 10 min at 37°C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3–6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport‐dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential ‘raft’ association. >90% 125I‐VT1 cell surface bound, or added to detergent‐resistant cell membrane extracts (DRM), was in the Gb3‐containing sucrose gradient ‘insoluble’ fraction, whereas only 30% 125I‐VT2 was similarly DRM‐associated. VT1 bound more efficiently to Gb3/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb3 ratios. VT2 competed less effectively for 125I‐VT1/Gb3 DRM‐binding but only VT2‐Gb3/cholesterol DRM‐binding was augmented by sphingomyelin. Differential VT1/VT2 Gb3 raft‐binding may mediate differential cell binding/intracellular trafficking and cytopathology. J. Cell. Physiol. 216: 750–763, 2008,

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes.

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb3/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb3 water-air interface monolayers in a similar manner. VT2 bound Gb3/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb3vesicles. C18/C20Gb3 were dominant negative in mixed Gb3 fatty acid isoform vesicles, but including C24:1Gb3 gave maximal binding. VT1 bound C18Gb3 vesicles after cholesterol removal, but C20Gb3vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb3 vesicles. C18 and C20Gb3 were, in mixtures without C24:1Gb3, dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb3 mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb3 at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb3 binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.