Prion Rods Contain Small Amounts of Two Host Sphingolipids as Revealed by Thin-Layer Chromatography and Mass Spectrometry

Abstract

Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.

Raft lipids as common components of human extracellular amyloid fibrils

“…In addition, amyloidogenic monocytes interact with SAA aggregates through raft-like microdomains of their plasma membrane, and amyloid formation can be attenuated with CHdepleting agents. These data reconcile the single previous observations of lipids in tissue-extracted amyloids (15)(16)(17) with the presence of a common pattern of lipids. Altogether, raft lipids have been demonstrated in the following types of amyloidosis: AA, ATTR, AL , AL , and previously in the prion protein-dependent amyloidosis APr (16).…”

“…These data reconcile the single previous observations of lipids in tissue-extracted amyloids (15)(16)(17) with the presence of a common pattern of lipids. Altogether, raft lipids have been demonstrated in the following types of amyloidosis: AA, ATTR, AL , AL , and previously in the prion protein-dependent amyloidosis APr (16). In addition, amyloid deposits are enriched in lyso-PC, free fatty acids, and CEs, i.e., lipids that occur rarely in biological membranes and accumulate characteristically in the course of tissue degradation or necrosis (31,32).…”

“…The myoglobin fragment MYO(101-118) and serum amyloid A (SAA)1.1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) were obtained from Jerini (Berlin). Transthyretin was a gift from M. Krebs and C. Robinson, (University of Cambridge, Cambridge, U.K.) The coding region of full-length murine SAA1.1 was cloned in fusion to the gene of the maltose-binding protein in the pMAL-c2X vector (NEB, Beverly, MA) and separated by a cleavage site for tobacco etch virus protease.…”

“…Indeed, amyloid structures can have an annular organization (10) or the ability to interact with lipids (12)(13) or to disrupt lipid bilayers in vitro (14). Analysis of a few tissue-extracted amyloid fibril samples provided evidence that these polypeptide aggregates can be associated with lipids (15)(16)(17). Based on these single observations, we analyzed whether this evidence is of general nature and determined the chemical composition of the lipid components from disease-associated amyloid deposits.…”

Dynamics of membrane lipid domains in neuronal cells differentiated in culture

“…The quantity of cholesterol was determined by densitometry and comparison with 0.1-2 g of standard compound using the Molecular Analyst program (Bio-Rad Laboratories). In the cholesterol, the SD of the procedure was Ϯ 20% in the range of 0.1-0.5 g, and Ϯ 10% in the range of 1-2 g. These values were similar to those already reported for analytical methods based on HPTLC separation and charring of cholesterol (44).…”