Antibody Cross-Linking of Myelin Oligodendrocyte Glycoprotein Leads to Its Rapid Repartitioning into Detergent-Insoluble Fractions, and Altered Protein Phosphorylation and Cell Morphology

Abstract

Myelin oligodendrocyte glycoprotein (MOG) is, quantitatively, a relatively minor component of the myelin membrane. Nevertheless, peritoneal administration of MOG evokes potent cellular and humoral immunoreactivity, resulting in an experimental allergic encephalitis with immunopathology similar to multiple sclerosis. Moreover, antibodies against MOG cause myelin destruction in situ. Therefore, it appears that MOG-related demyelination is dependent on anti-MOG antibody, but the mechanism(s) by which it occurs is unclear. Of potential significance are observations that some proteins are selectively partitioned into specialized plasma membrane microdomains rich in glycosphingolipids and cholesterol ("lipid rafts"). In particular, during ligand or antibody cross-linking, various plasma membrane receptors undergo enhanced partitioning into rafts as an obligatory first step toward participation in early signal transduction events. In contrast to mature myelin, in oligodendrocytes (OLs) in culture MOG is not raft associated [Triton X-100 (TX-100) soluble, 4 degrees C]. However, in this study we show that antibody cross-linking (anti-MOG plus secondary antibody) of MOG on the surface of OLs results in the repartitioning of approximately 95% of MOG into the TX-100-insoluble fraction. This repartitioning of MOG is rapid (<or=1 min), antibody dose dependent, requires an intact cytoskeleton, leads to phosphorylation or dephosphorylation of tyrosine, serine, and threonine residues in specific proteins (e.g., beta-tubulin, Gbeta1-2), and invokes a rapid retraction of OL processes. After removal of the cross-linking antibodies, these events are reversed. We hypothesize that antibody-mediated repartitioning of MOG into TX-100-insoluble glycosphingolipid-cholesterol-rich microdomains initiates specific cellular signaling that could be related to initial steps of MOG-mediated demyelination.

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

“…6). This is consistent with previous obsevations that an intact cytoskeleton is necessary for the integrity of rafts in OLs (Holowka et al, 2000;Fassett et al, 2001;Nebl et al, 2002;Marta et al, 2003).…”

“…1A). Additional control studies for cross-linking experiments are shown in (Marta et al 2003). We conclude that cross-linking MAG on the surface of differentiated OLs in culture results in a major change in its detergent solubility properties.…”

“…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.…”

“…In general, raft-associated proteins that are insoluble in TX-100 at 4°C become solubilized when the extraction is carried out at either 37°C (Brown and Rose, 1992) or 4°C following treatment with the cholesterol-binding agent saponin (Rothberg et al, 1990;Cerneus et al, 1993;Hanada et al, 1995;Stulnig et al, 1997;Ledesma et al, 1998). It has been reported (Marta et al, 2003) that markers of lipid rafts, such as GM1 ganglioside and caveolin (a marker for caveolae, a subgroup of lipid rafts) (Abrami et al, 2001), are enriched in low-density, detergent-insoluble fractions derived from OLs on TX-100 extraction at 4°C, and that they are completely solubilized following either extraction at 37°C or 4°C after pretreatment with saponin. Applying these three additional criteria, we found that detergent-insoluble MAG (TX-100, 4°C…”

“…For example, we have shown that whereas ∼40% of MOG in myelin is associated with detergent-insoluble complexes that are characteristic of rafts, MOG in OLs in culture is nearly entirely excluded from rafts (Marta et al, 2003). However, antibody cross-linking of MOG in OLs in culture results in its increased association with lipid rafts, and leads to rapid, novel signal-transduction events and pronounced morphological changes in OLs (Marta et al, 2003).…”