Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species

In the past decade, the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (two to six C atoms) also exist in Nature, apart from being extensively used in experimentation, because they can be dispersed easily in water. Long-chain ceramides are among the most hydrophobic molecules in Nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent.

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Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2.

The Journal of Cell Biology ◽

10.1083/jcb.101.4.1455 ◽

1985 ◽

Vol 101 (4) ◽

pp. 1455-1462 ◽

Cited By ~ 32

Author(s):

A Christiansson ◽

F A Kuypers ◽

B Roelofsen ◽

J A Op den Kamp ◽

L L van Deenen

Keyword(s):

Phospholipase A2 ◽

Morphological Changes ◽

Acyl Chain ◽

Molecular Shape ◽

Lipid Monolayer ◽

Intact Cells ◽

Erythrocyte Morphology ◽

Transfer Protein ◽

Hydrolysis Of ◽

Normal Erythrocyte

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.

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Microstructural morphology of sphingolipid dispersions as investigated by freeze-fracture EM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141093 ◽

1995 ◽

Vol 53 ◽

pp. 944-945

Author(s):

Vitthal S. Kulkarni ◽

Wayne H. Anderson ◽

Rhoderick E. Brown

Keyword(s):

Acyl Chain ◽

Biological Significance ◽

Freeze Fracture ◽

Fatty Acyl ◽

Fatty Acyl Moiety ◽

Aqueous Dispersions ◽

Head Groups ◽

Lipid Species ◽

Microstructural Morphology ◽

Layer Chromatography

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.

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Structure of rattlesnake venom lectin determined by single particle high-resolution electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162776 ◽

1996 ◽

Vol 54 ◽

pp. 62-63

Author(s):

Peter D. Moisiuk ◽

Daniel R. Beniac ◽

Ross A. Ridsdale ◽

Martin Young ◽

Bhushan Nagar ◽

...

Keyword(s):

High Resolution Electron Microscopy ◽

Lipid Monolayer ◽

Outer Diameter ◽

X Ray Diffraction ◽

Single Particles ◽

Crotalus Atrox ◽

3D Reconstructions ◽

X Ray ◽

Rattlesnake Venom ◽

Covalently Linked

Venom from the rattlesnake Crotalus atrox contains a mixture of enzymes that induce a localized effect leading to hemorrhaging, necrosis and edema. As a member of the crotalid family of snake venoms, Crotalus atrox venom contains a C-type lectin that will agglutinate blood cells in a Ca2+-dependent fashion. The lectin is a hydrophilic protein, consisting of two covalently linked, 135 amino acid residues, identical subunits that are rich in aspartic acid, glutamic acid and lysine. Sequence homology with known carbohydrate recognition domains (CRDs) indicates that rattlesnake venom lectin (RSLV) contains a CRD motif that is not linked to accessory domains. Preliminary X-ray diffraction and sedimentation analysis has indicated that lectin from Crotalus atrox forms decamers composed of two five-fold symmetric pentamers. Single particles of RSVL imaged at – 171°C displayed two distinct orientations on the specimen support (Figure a) following incubation in a crystallization Teflon well, coated with a lipid monolayer consisting of phosphatidylcholine and monosialoganglioside. When lying in an end-on orientation, the lectin exhibited a “pentagonal ring” with an outer diameter of 6.7 nm and an inner hollow core of 1.7 nm. A side orientation was also seen, whereby a thickness of 5.8 nm was measured for the lectin. Image processing of 2280 single particles placed in 100 classes (Figure b) led to 3D reconstructions of RSVL (Figure c). Density limited 3D reconstructions showed the lectin to be made of two five-fold symmetrical rings covalently linked between the five subunits that constitute each ring of this homodimer. These results are consistent with sedimentation and preliminary X-ray diffraction analysis on the shape of RSVL and provide the framework for structural verification by 2D electron crystallography.

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145.v1 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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