Detergent‐insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

Abstract

Glycosphingolipids and cholesterol form lateral assemblies, or lipid ÔraftsÕ, within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergentinsoluble complexes (in non-ionic detergents at 4°C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/ cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4°C or (ii) 37°C, or at 4°C after pre-extraction with (iii) saponin or (iv) methyl-b-cyclodextrin (MbCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbCD. Furthermore, pre-extraction with MbCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.

Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes

“…In agreement with previous results using purified myelin (Kim and Pfeiffer, 1999;Taylor et al, 2002), when cultures enriched for OLs were extracted with 1% TX-100 at 4°C, nearly all MAG was detected in the soluble fraction (Fig. 1A).…”

“…Triton X-100 (1% final concentration) was added to cell lysates and incubated for 30 minutes at either 4°C or 37°C (Kim and Pfeiffer, 1999;Taylor et al, 2002). The TX-100 extracts were centrifuged (13 000×g, 4°C for 10 minutes) to separate detergent-insoluble pellet and detergent-soluble supernatant fractions.…”

“…Insolubility of a protein in TX-100 at 4°C can be caused by association with lipid rafts and/or through protein-protein interactions that often involve cytoskeletal elements (Pfeiffer et al, 1993;De Angelis and Braun, 1996;Kim and Pfeiffer, 1999;Taylor et al, 2002;Marta et al, 2003). Therefore, we applied three, additional, well-established biochemical criteria to analyze the properties of detergent-resistant membrane complexes.…”

“…Glycosphingolipids and cholesterol form microdomains in the plasma membrane of cells (termed rafts) into which some proteins can partition and others are excluded (Simons and Ikonen, 1997;Brown and London, 1998;Friedrichson and Kurzchalia, 1998;Varma and Mayor, 1998;Taylor et al, 2002;Taylor et al, 2004). Lipid rafts have an important role as platforms for the initiation of signal transduction by favoring specific protein-protein interactions (Simons and Toomre, 2000).…”

“…Lipid rafts have an important role as platforms for the initiation of signal transduction by favoring specific protein-protein interactions (Simons and Toomre, 2000). Using biochemical criteria to identify proteins in rafts, it has been shown that in myelin the GPI-linked proteins NCAM-120 and contactin, the doubly acylated proteins Fyn and Lyn kinases, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) and myelin oligodendrocyte glycoprotein (MOG) partition into rafts (Kim et al, 1995;Kramer et al, 1997;Kim and Pfeiffer, 1999;Kramer et al, 1999;Taylor et al, 2002), whereas MAG does not. Cross-linking of some proteins to either ligand or antibody can result in their enhanced partitioning into rafts and participation in early signaltransduction events (Simons and Toomre, 2000).…”