Membrane lipid frustration : Contribution of ceramide and its effects on lateral segregation

Ceramides are both sphingolipid precursor molecules and breakdown products with effects on membranes that are of interest both from biophysical and physiological points of view. When present in a membrane, ceramide induces the formation of highly ordered domains. Ceramide accumulation in biological membranes has in turn been suggested to be involved in committing the cell to death, possibly through effects on membrane fluidity or as a signalling molecule involved in the apoptotic cascade. Though ceramide aggregates at low concentrations in the membrane, this effect can be promoted by other major membrane lipids. Notably some of these are lipids typical of mitochondria, which also is the proposed site of ceramide mediated apoptotic induction. In the interaction of ceramide with its co-lipids the proportion of the headgroup size to the volume occupied by acyl chains appeared as an important factor to consider along with alterations of the hydrogen bonding competency of ceramide itself to other surrounding lipids. The promoted lateral segregation of ceramide by co-lipid molecular shape seems to be driven by different mechanisms. Lipids with proportionally small headgroups interact unfavourably with ceramide which as a result is pushed away, while lipids with large headgroups and small acyl chain volume provides shielding from the aqueous environment. ---------- Ceramider är både byggstenar och nedbrytningsprodukter av sfingolipider vars inverkan på membraner är intressanta ur både biofysiskt och fysiologiskt perspektiv. Vid närvaro av ceramid i ett membran bildas domäner med ökad ordningsgrad i förhållande till omgivande membran. Ansamling av ceramider i cellens membraner har påvisats ha en koppling till att förpassa cellen till att genomgå programmerad celldöd, möjligen genom att påverka membranets fluiditet eller som en signalmolekyl som utlöser en apoptotisk kaskad. Ceramider aggregeras vid låga halter i membraner, men denna effekt kan förstärkas av andra betydande lipidklasser. Anmärkningsvärt är att vissa av dessa lipider är typiska för mitokondrier, som också anses vara startpunkten för utlösandet av apoptos. I ceramiders samverkan med andra lipider i membranet visade vi att förhållandet mellan huvudgruppens storlek och acylkedjornas volym samt ceramidens vätebindningspotential är viktiga faktorer att ta i beaktande. Den förstärkta laterala segregeringen av ceramider som förmedlas av omgivande lipiders form verkar ha olika bakomliggande mekanismer. Lipider med förhållandevis små huvudgrupper samverkar ofördelaktigt med ceramid och har en frånstötande effekt, medan lipider med stora huvudgrupper och liten acylkedjevolym ger ceramiden skydd från omgivande vatten

Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP

Natural Ceramides and Lysophospholipids Cosegregate in Fluid Phosphatidylcholine Bilayers

The mode of interactions between palmitoyl lysophosphatidylcholine (palmitoyl lyso-PC) or other lysophospholipids (lyso-PLs) and palmitoyl ceramide (PCer) or other ceramide analogs in dioleoylphosphatidylcholine (DOPC) bilayers has been examined. PCer is known to segregate laterally into a ceramide-rich phase at concentrations that depend on the nature of the ceramides and the co-phospholipids. In DOPC bilayers, PCer forms a ceramide-rich phase at concentrations above 10 mol%. In the presence of 20 mol% palmitoyl lyso-PC in the DOPC bilayer, the lateral segregation of PCer was markedly facilitated (segregation at lower PCer concentrations). The thermostability of the PCer-rich phase in the presence of palmitoyl lyso-PC was also increased compared to that in the absence of palmitoyl lyso-PC. Other saturated lyso-PLs (e.g., palmitoyl lyso-phosphatidylethanolamine and lyso-sphingomyelin) also facilitated the lateral segregation of PCer in a similar manner as palmitoyl lyso-PC. When examined in the DOPC bilayer, it appeared that the association between palmitoyl lyso-PC and PCer was equimolar in nature. It is proposed that the interaction of PCer with lyso-PLs was driven by the need of ceramide to obtain a large-headgroup co-lipid, and saturated lyso-PLs were preferred co-lipids over DOPC because of the nature of their acyl chain. Structural analogs of PCer (1- or 3-deoxy-PCer) were also associated with palmitoyl lyso-PC, similarly to PCer, suggesting that the ceramide/lyso-PL interaction was not sensitive to structural alterations in the ceramide molecule. Binary complexes containing palmitoyl lyso-PC and ceramide were prepared, and these had a bilayer structure as ascertained by transmission electron microscopy. It is concluded that ceramides and lyso-PLs associated with each other both in binary bilayers and in ternary systems based on the DOPC bilayers. This association may have biological relevance under conditions in which both sphingomyelinases and phospholipase A2 enzymes are activated, such as during inflammatory processes

List of putative FFAT-like domains found in proteins related to the sphingolipid synthesis.

List of putative FFAT-like domains found in proteins related to the sphingolipid synthesis.</p

Changes in GLTP Trp emission maxima versus GlcCer concentration.

(A) Trp emission maxima versus increasing amounts of GlcCer. (B) C8:0-GlcCer, C12:0-GlcCer, and C16:0-GlcCer created blueshifts between 12 and 13 nm, whereas the longer GlcCers, as well as POPC and DOPC, showed no significant blueshifts. The data are from at least three different experiments.</p

GLTP structure and CPM thermal shift assays.

The crystal structure of GLTP with the three cysteine (Cys) residues in yellow, bound ganglioside GM3 (2BV7) in green, and the tryptophan Trp-96 in red. The left structure shows the opening of the hydrophobic tunnel, with the N-linked acyl chain and saccharide headgroup of GM3 visible. On the right, GLTP is turned horizontally 180 degrees, exposing the opening of the tunnel end. The third structure show the exposed Cys-36 on the surface of GLTP and the two more embedded Cys-112 and Cys-176. The lower graphs show the increase in the CPM fluorescence intensity (λexc. 400 nm) at 500 nm as a function of a temperature increase. The black trace corresponds to wildtype apo-GLTP (0.42 μM) in PBS buffer. The different colored traces are GLTP incubated with GlcCer of different lengths (ethanol injected) 30 min prior to heating. The curves are averages of at least 4 independent measurements. The curves were normalized to the intensity maximum for the apo-GLTP melting curve. The curve maximas are shown for each representative GlcCer-GLTP mixture.</p