Membrane Properties of D-erythro-N-acyl Sphingomyelins and Their Corresponding Dihydro Species

AbstractWe have prepared acyl chain-defined D-erythro-sphingomyelins and D-erythro-dihydrosphingomyelins and compared their properties in monolayer and bilayer membranes. Surface pressure/molecular area isotherms of D-erythro-N-16:0-sphingomyelin (16:0-SM) and D-erythro-N-16:0-dihydrosphingomyelin (16:0-DHSM) show very similar packing properties, except that the expanded-to-condensed phase transition (crystallization) occurs at a lower surface pressure for 16:0-DHSM. The measured surface potential was generally about 100mV less for 16:0-DHSM monolayers compared to 16:0-SM monolayers. The condensed domains (crystals) that formed in 16:0-SM monolayers as a function of compression displayed star-shaped morphology when viewed under an epifluorescence microscope. 16:0-DHSM monolayers did not form similar crystals upon compression. 16:0-DHSM was degraded much faster by sphingomyelinase from Staphylococcus aureus than 16:0-SM (10-fold difference in enzyme activity needed for comparable hydrolytic rate). Cholesterol desorption from 16:0-DHSM to cyclodextrin was slightly slower (∼20%) than the rate measured from 16:0-SM monolayers (at 60mol % cholesterol). The bilayer melting temperature of 16:0-DHSM was 47.7°C (ΔH 8.3kcal/mol) whereas it was 41.2°C for 16:0-SM (ΔH 8.1kcal/mol). Cholesterol/16:0-DHSM bilayers (15mol % sterol) had more condensed domains than comparable 16:0-SM bilayers, as evidenced from the quenching resistance of DPH in DHSM membranes. We conclude that cholesterol interacts more favorably with 16:0-DHSM and that the membranes are more condensed than comparable 16:0-SM-containing membranes

Human Glycolipid Transfer Protein (GLTP) Expression Modulates Cell Shape

Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ∼70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.

Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors