myelin-basic-protein and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14-21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.

Topics: Aging; Animals; Cell Fractionation; Cholic Acids; Detergents; Disease Models, Animal; Membrane Microdomains; Mice; Mice, Inbred Strains; Mice, Transgenic; Multiple Sclerosis; Myelin Basic Protein; Phosphorylation; Protein Isoforms; Protein Processing, Post-Translational; Severity of Illness Index; Silver Staining; Subcellular Fractions; Threonine

We have previously shown that CNS myelin basic protein (MBP) can be purified in the lipid-bound, native-like form by using a procedure based on myelin solubilization with detergents at pH above 7, and on the filter-like use of hydroxyapatite to separate non-adsorbed MBP from other myelin proteins. Here, we report on the isolation of MBP in the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate (CHAPS), which does not interfere at 280 nm and can be removed by dialysis. This detergent appears to improve MBP purification and to be suitable for fluorescence and reconstitution studies that can be useful to understand both structure and function of MBP in its natural environment.

Topics: Animals; Brain Chemistry; Cattle; Cholic Acids; Chromatography, Thin Layer; Detergents; Hydroxyapatites; Isomerism; Lipids; Myelin Basic Protein; Phospholipids; Spectrophotometry, Ultraviolet