Behavior of a GPI-anchored protein in phospholipid monolayers at the air–water interface

AbstractThe interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the π–A diagrams, allowed to deduct that the AP-GPI–phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein–lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane

Interaction entre une protéine à ancre glycosylphosphatidyl inositol et des membranes biomimétiques

Les protéines à ancre glycosylphosphatidyl inositol (GPI) sont ancrées dans le feuillet externe des membranes plasmiques. L'objectif du travail a été de caractériser le comportement de la phosphatase alcaline (AP) à ancre GPI dans des monocouches, en présence ou en absence de lipides, à l'interface air/eau et dans des liposomes. Nous avons étudié le comportement de la phosphatase alcaline seule à l'interface air/eau en utilisant la technique de Langmuir et la spectroscopie IR de modulation (PM-IRRAS). L'AP avec l'ancre GPI présente une plus forte affinité pour l'interface air/eau que la forme dépourvue de ce motif. Dans les deux cas, l'activité enzymatique est conservée au niveau de la surface. Les résultats obtenus à partir de l'analyse des isothermes de compression de Langmuir ou des spectres IR suggèrent une orientation particulière de l'AP/GPI à l'interface air/eau telle que le site actif est dirigé vers le milieu aqueux. On a également mis en évidence des mouvements réversibles de domaines intramoléculaires qui pourraient rendre compte des propriétés allostériques de ces enzymes. La microscopie à angle de Brewster a permis de mettre en évidence la formation réversible d'agrégats résultant d'interactions protéine-protéine très fortes.L'insertion de la protéine dans des monocouches de phospholipides se fait en plusieurs étapes et dépend de la pression de surface initiale de la monocouche. De plus, l'interfaction de la protéine avec les phospholipides diffère selon la nature de la tête polaire. Par PM-IRRAS, des mouvements de domaines sont également observés montrant que le comportement de la protéine est sans doute identique en présence ou en l'absence de phospholipides. La phosphatase alcaline insérée dans des liposomes montre une hétérogénéité de distribution à la surface des liposomes et la protéine peut être mesurée, ce qui correspond à la longueur de l'ancre.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems

Three dimensional morphology of rabies virus studied by cryo-electron tomography

The rabies virus (RABV) continues to be a worldwide health problem. RABV contains a single-stranded RNA genome that associates with the nucleoprotein N. The resulting ribonucleoprotein complex is surrounded by matrix protein M, lipid bilayer and glycoprotein G. RABV was reported to organize in bullet-like virions, but the role of each viral component in adopting this morphology is unclear. We present here a cryo-electron tomography study of RABV showing additional morphologies consisting in bullet-like virions containing a tubular, lipidic appendage having G-protein at its apex. In addition, there was evidence for an important fraction of pleomorphic particles. These pleomorphic forms differed in the amount of membrane-associated M-, M/N-protein providing interesting insight into its role in viral morphogenesis. In the absence of membrane-associated M-, M/N-protein viral morphology was almost spherical. Other images, showing straight membrane portions, correlate with the M-protein recruitment at the membrane independently of the presence of the G-protein. The viral membrane was found to contain a negative net charge indicating that M-, M/N-protein-membrane charge attraction drives this interaction

PrPd accumulation in organs of ARQ/ARQ sheep experimentally infected with BSE by peripheral routes

To study the pathogenesis of bovine spongiform encephalopathy infection in small ruminants, two Lacaune sheep with the AA136RR154QQ171 and one with the AA136RR154RR171 genotype for the prion protein, were inoculated with a brain homogenate from a French cattle BSE case by peripheral routes. Sheep with the ARQ/ARQ genotype are considered as susceptible to prion diseases contrary to those with the ARR/ARR genotype. The accumulation of disease-associated prion protein (PrPd) was analysed by biochemical and immunohistochemical methods. No PrPd accumulation was detected in samples from the ARR/ARR sheep 2 years post inoculation. In the two ARQ/ARQ sheep that had scrapie-like clinical symptoms, PrPd was found in the central, sympathetic and enteric nervous systems and in lymphoid organs. Remarkably, PrPd was also detected in some muscle types as well as in all peripheral nerves that had not been reported previously thus revealing a widespread distribution of BSE-associated PrPd in sheep tissues