Lipid raft formation and peptide-lipid interactions in myelin model membranes

Multiple sclerosis (MS), a demyelinating disease affecting 75,000 Canadians and almost 400,000 Americans, is one of the most prevalent diseases in young adults. Unfortunately, there exist no known cures to date and the pathways involved in the progression of the disease remain relatively obscure. The demyelination process triggered by the onset of MS, affects the lipid composition of the myelin membrane and causes a loss in viable myelin which can in turn greatly impact the proper functioning of the central nervous system (CNS). The cholesterol content of myelin fluctuates during MS and consequently this could affect the fluidity as well as the lipid microdomain profile in the membranes. Using model membranes such as a fluid palmitoyl-oleoyl-glycerophosphocholine (POPC) system and the “canonical’ raft system (POPC/sphingomyelin/cholesterol 1:1:1) to represent the two extremes of the fluidity scale, as well as myelin-mimicking membranes (healthy and diseased) which have intermediate lipid fluidities, the lipid domains in each of the systems were probed using fluorescence resonance energy transfer (FRET). FRET analysis indicated that the myelin membrane models contained lipid domains than were overall smaller that those found in the raft system. The addition of melittin, a positively-charged (+6) peptide, to these lipid systems triggered lipid rearrangement to yield larger lipid domains in the myelin systems, whereas in the raft system a change from large domains to smaller ones in the presence of the peptide was observed. Melittin interacted differently with each of the lipid systems where the lipid composition determined the overall conformation of the peptide which, according to circular dichroism (CD) spectroscopy, was shown to be mostly α-helical in membrane environments. It is believed that melittin can exist in a number of different states; a monomer at low concentrations, and as the concentration increases self-association results in either dimers consisting of two parallel α-helices or even a tetramer which would be a dimer of dimers (four α-helices). Tryptophan fluorescence indicated that melittin had a strong affinity for the myelin mimicking models which were negatively-charged and the peptide appeared to be buried relatively deep into the hydrocarbon core of the bilayer, whereas melittin was located nearer the interfacial region in the raft system which was not charged. Furthermore, melittin appeared to interact more strongly with the healthy myelin model compared to the diseased model which contained a higher level of cholesterol. This would indicate that melittin’s interaction is strongly dictated by electrostatic interactions as well as the presence of cholesterol which would affect the fluidity and physical properties of the lipid bilayer, thus making it harder for the peptide to penetrate deeply. Since the lipid composition is affected during demyelination and the cholesterol content has been shown to increase, this could suggest that similarly to melittin, other basic proteins found in the CNS could also suffer reduced lipid interactions which would affect their overall structure and consequently their function. The proteolipid protein (PLP) is amongst one of the most abundant proteins found in the myelin membrane. This extremely hydrophobic integral membrane protein consisting of four transmembrane α-helices is known to induced encephalomyelitis in experimental animals and is consequently of great interest in MS research. Among the different loops that connect the helices, the C-terminus located on the cytoplasmic side of the membrane, is of particular interest since it has been hypothesized that it could be involved in guiding and properly positioning PLP in myelin, as well as potentially interacting with other myelin proteins. A PLP C-terminus peptide (residues 258-277 in the human sequence) was synthesized and its interaction and secondary structure were studied using CD spectroscopy. CD measurements indicated that this loop region adopted different conformations in the different lipid systems, but all in all, it appeared to be mostly randomly coiled and it had a limited amount of interaction with the lipid vesicles. The structure of the peptide also seemed unaffected by the change in lipid composition of the healthy and diseased myelin model systems. This would suggest that the lack of structure of the peptide could allow it to interact with other cytosolic myelin proteins. These studies on the lipid domains in myelin membrane models as well as their interactions with melittin and the PLP C-terminus peptide suggest that within the myelin sheath protein structure and protein-lipid interactions are affected during MS.

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