Membrane-anchoring and Charge Effects in the Interaction of Myelin Basic Protein with Lipid Bilayers Studied by Site-directed Spin Labeling

Abstract

Myelin basic protein (MBP) maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers and may also play a role in signal transduction. Site-directed spin labeling was done at eight matching sites in each of two recombinant murine MBPs, qC1 (charge ؉19) and qC8 charge (؉13), which, respectively, emulate the native form of the protein (C1) and a posttranslationally modified form (C8) that is increased in multiple sclerosis. When interacting with large unilamellar vesicles, most spin-labeled sites in qC8 were more mobile than those in qC1. Depth measurement via continuous wave power saturation indicated that the Nterminal and C-terminal sites in qC1 were located below the plane of the phospholipid headgroups. In qC8, the C-terminal domain dissociated from the membrane, suggesting a means by which the exposure of natural C8 to cytosolic enzymes and ligands might increase in vivo in multiple sclerosis. The importance of two Phe-Phe pairs in MBP to its interactions with lipids was investigated by separately mutating each pair to Ala-Ala. The mobility at F42A/F43A and especially F86A/F87A increased significantly. Depth measurements and helical wheel analysis indicated that the Phe-86/Phe-87 region could form a surface-seeking amphipathic ␣-helix.The 18.5-kDa isoform of myelin basic protein (MBP) 1 is a stabilizing factor in the myelin sheath. A major function of MBP is to bind to the apposing cytoplasmic faces of the myelin membrane and maintain compaction for efficient nerve transmission (1, 2), but it may also be involved in signal transduction (3). Because of a diversity of post-translational modifications, MBP exists as a number of charge isomers denoted C1-C8 with a net positive charge decreasing from ϩ19 to ϩ13 at pH 7.0 (4 -6). The C8 component is characterized by the enzymatic deimination of arginine to citrulline. Each conversion results in the loss of one positive charge, and C8 is thus the least basic form of the protein and has a diminished ability to cause adhesion of lipid bilayers (7-9). Component C8 occurs in greater amounts in patients with the demyelinating disease, multiple sclerosis (9, 10).We have previously produced and characterized a recombinant murine 18.5-kDa MBP (11). Here, we will denote this protein quasi-C1 (qC1), because it is unmodified post-translationally (with the exception of an LEH 6 tag) and emulates the least-modified, most basic charge isomer C1. We have also generated by site-directed mutagenesis a quasi-deiminated form of recombinant murine 18.5-kDa MBP that we call qC8, since it was designed to mimic the less cationic natural form C8. The recombinant qC8 consists of Arg/Lys 3 Gln substitutions at the same deimination sites in human MBP that predominate in chronic multiple sclerosis and has properties similar to those of natural C8 (7). The net charge of qC1 is ϩ19 at neutral pH, whereas that of qC8 is ϩ13 as for their natural counterparts.In this study, we investigated the electrostatic and hydro...

An Immunodominant Epitope of Myelin Basic Protein Is an Amphipathic α-Helix

“…The calibration of the ⌽ value with the depth of penetration was especially important for the Cyt-LUVs, because they consisted of six different lipid components, including 40% cholesterol. The effect of the cholesterol was to increase the penetration of NiEDDA up to the fifth carbon of the acyl group of the phospholipid, as reported previously (22). The hyperbolic tangent function (Eq.…”

“…Preparation of Large Unilamellar Vesicles (LUVs)-Aliquots of the chloroform solutions of various lipids were combined in the following molar ratios to form large unilamellar vesicles (LUVs) with a lipid composition similar to that estimated for the cytoplasmic face of the myelin membrane (Cyt-LUVs): 44 mol % cholesterol, 27 mol % PE, 13 mol % phosphatidylserine, 11 mol % PC, 3 mol % sphingomyelin, and 2 mol % PI (22). The solvent was evaporated under a stream of nitrogen, dried in a vacuum desiccator overnight, and then hydrated in 20 mM HEPES-NaOH, pH 7.4, and 10 mM NaCl.…”

“…The preparation of the Cys-containing mutants was done as described previously (22) using the QuikChange® protocol (Stratagene, La Jolla, CA). Mutant murine MBP expression and purification were carried out as described (11).…”

“…We have previously used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy (20,21) to compare the membrane interactions of normal murine MBP (mMBP) with that of a less cationic form that predominates in MS (22). In that study, H85R1 was found to be motionally restricted, yet situated more than 3 Å above the lipid phosphate groups.…”

The Multiple Sclerosis Degradome: Enzymatic Cascades in Development and Progression of Central Nervous System Inflammatory Disease

“…Far less is known regarding the role of aspartic proteases, cathepsins D and E, in MS. Cathepsin D degrades myelin basic protein (MBP) [15,291], and at least one study has shown that pepstain, an inhibitor of cathepsin D, suppresses clinical and histological signs of EAE [29]. Of interest, cathepsin D was among the genes upregulated by more than 2.5-fold in a large-scale cDNA sequencing study of MS plaques [39].…”