Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse Model

Abstract

Multiple sclerosis has complex pathogenesis encompassing a variety of components (immunologic, genetic, and environmental). The autoimmunogenicity against the host's myelin basic protein is a major contributor. An increase in myelin basic protein deimination (a post-translational modification) and a change in phospholipid composition have been associated with multiple sclerosis. The interaction of myelin basic protein with phospholipids in the myelin membrane is an important contributor to the stability and maintenance of proper myelin sheath function. The study of this aspect of multiple sclerosis is an area that has yet to be fully explored and that the present study seeks to understand. Several biochemical methods, a capillary electrophoresis coupled system and mass spectrometry, were used in this study. These methods identified four specific phospholipids complexing with myelin basic protein. We show that lysophosphatidylcholine 18:1 provides a robust competitive effect against hyper-deimination. Our data suggest that lysophosphatidylcholine 18:1 has a different biochemical behavior when compared to other phospholipids and lysophosphatidylcholines 14:0, 16:0, and 18:0.

Figure 1

Myelin basic protein in EAE is deiminated. (A) Representative sucrose density ultracentrifugation fractions of mouse brains probing for myelin basic protein (Abcam-ab7349). *Sucrose fraction 32 (SF 32) from each group was used for further analysis (coomassie, Western blot, mass spectrometry, and capillary electrophoresis (CE)). EAE, n = 15; sham-injection control, n = 10; and no-injection control, n = 10. Each immunoblot was repeated three times. (B) Coomassie blue stain and Western blot for sucrose fraction 32. Bands were excised from coomassie gel and analyzed by mass spectrometry. Western blot probed for levels of deimination (citrulline, Millipore-MABS54887). (C) Mass spectrometry analysis of excised bands from coomassie blue stain gel. PSM = peptide-spectrum match.

Figure 2

Myelin basic protein isolated from EAE has different phospholipid compositions than controls. (A) Representative capillary electrophoresis (CE) chromatogram of sucrose fraction 32 (SF 32). Arrow points toward the peak collected for protein and phospholipid analysis. (B) Dot immunoblot for MBP in CE fractions, 10 min before the peak, peak (as noted by the arrow), and 10 min after the peak. MBP was probed with an anti-MBP antibody (Abcam, ab7349). (C) Phospholipid analysis of CE peak fractions, three replicates, and bars correspond to standard error mean (SEM) (*p < 0.05).

Figure 3

Confirmation of MBP–phospholipid complexation. (A) Structure of candidate lipids: PC 16:0/22:6, 1-((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine, LMGP01011116; PC 16:1/16:1, 1,2-di-((9Z)-hexadecanoyl)-sn-glycero-3-phosphocholine, LMGP01010684; PI 18:0/20:4, 1-octadecanoyl-2-((5Z,8Z,11Z,14Z)-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol), LMGP06010010; PS 18:0/18:1, 1-octadecanoyl-2-((9Z)-octadecanoyl)-sn-glycero-3-phosphoserine, LMGP03010025; LPC 18:1, 2-((9Z)-octadecanoyl)-sn-glycero-3-phosphocholine, LMGP01050082. (B) Liposome flotation assay (LFA) of candidate phospholipids demonstrating MBP complexation with phospholipids. B, bottom fraction; M, middle fraction; and T, top fraction (MBP, Abcam, ab7349). (C) Protein–lipid overlay assay (PLOA) of candidate phospholipids demonstrating MBP complexation with phospholipids (MBP, Abcam, ab7349).

Figure 4

Complexation of LPC 18:1 with MBP is competitive for MBP hyper-deimination. (A) Dot blot analysis of deimination reaction for MBP (MBP, Abcam-ab7349) and deiminated MBP (citrulline, Millipore-MABS54887) in the presence of candidate phospholipids. PAD, peptidyl arginine deiminase. (B) Deimination fold change analysis of Western blot, three replicates, and densitometry using ImageJ was normalized to MBP (***p < 0.0005, comparison to MBP–PAD). (C) Representative circular dichroism (CD) spectra of deimination reaction for MBP in the presence of candidate phospholipids, three replicates per spectra. (D) CD spectra analysis of secondary structure probability, utilizing CD analysis and plotting tool (CAPITO) software (mean with SEM).

Figure 5

LPC 18:1 competes with hyper-deimination of MBP arginine residues and has differential complexation with MBP. (A) Mass spectrometry analysis of MBP arginine residues that are being deiminated in in vitro deimination assay (Millipore, 13-104, Burlington, MA). The deiminated Myelin basic protein (UniProt accession: P02687) peptides were identified by mass spectrometry (see Table 3). Gray highlight, deiminated arginine; red bold square, arginine not deiminated in LPC 18:1 reaction (residues 63, 112, and 129). (B) Protein–lipid overlay assay of LPC 18:1, LPC 14:0, LPC 16:0, and LPC 18:0 MBP was probed with an anti-MBP antibody (Abcam, ab7349).

Figure 6

Change in MBP conformations and solvent accessibility of arginine residues. Representative structures are shown from simulations only in water, water with LPC, and water with PC. Helices are shown in red, β-strands in yellow, three N-terminal residues are colored green, and three C-terminal residues are colored cyan. Arginine residues are shown as green/blue sticks, while the lipid is shown as magenta/blue/orange sticks. Water was included in the simulations but omitted here for visualization purposes. In the case of water-only, the arginine residues are exposed on the surface; the presence of LPC protects a number of these residues, and in the presence of PC, MBP adopts an extended conformation, exposing several arginine residues to the solvent.

Figure 7

Hypothesis model of MBP–phospholipid complexation. (A) Working hypothesis for membrane disruption, incorporating key events associated with unstable myelin. The order of events has yet to be determined. (B) Model of phospholipid (PC 16:1/16:1, PI 18:0/20:4, PS 18:0/18:1, LPC 18:0, LPC 16:0, and LPC 14:0) competitive effects against PAD-mediated hyper-deimination and potential cascade of events. (C) Model of LPC 18:1 competitive effects against PAD-mediated hyper-deimination. Δ, “change in”; MBP, myelin basic protein; and PAD, peptidyl arginine deiminase. Open and C-shaped conformations are consistent with the previously published reports.