Myelin proteolipid protein, basic protein, the small isoform of myelin‐associated glycoprotein, and p42MAPK are associated in the Triton X‐100 extract of central nervous system myelin

Abstract

To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (M(r) 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP-MBP-S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP-MBP-S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin-axonal interactions.

Co‐clustering of galactosylceramide and membrane proteins in oligodendrocyte membranes on interaction with polyvalent carbohydrate and prevention by an intact cytoskeleton

“…Because MAG does not co-cluster with GalC and is not in GalC-containing DIGs, it is less likely to transmit the signal received by GalC and CBS. It is probably involved in transmission of other signals, however, because it is associated with Fyn kinase in OLs (Umemori et al, 1994) and has been found in a complex with PLP, MBP, and MAPK from the Triton-soluble fraction of myelin (Arvanitis et al, 2002). There may be other transmembrane constituents in the co-clustering domains that also are possible candidates for the signal transmitter.…”

“…The detergent solubility of proteins and different membrane domains depends on the detergent (Cerny et al, 1996;Madore et al, 1999;Roper et al, 2000;Simons et al, 2000;Claas et al, 2001;Taylor et al, 2002) and conditions used, including the temperature and the salt concentration of the buffer (Pereyra et al, 1988;Arvanitis et al, 2002). Some MBP, MOG, PLP, and MAG are present in a low-density TX- 100-or CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid)-insoluble fraction of myelin (Kramer et al, 1997;Kim and Pfeiffer, 1999;Simons et al, 2000;Taylor et al, 2002), although most of each of these proteins is found in the TX-100-soluble phase.…”

Myelin basic protein: a multifunctional protein

“…Some MBP distributes to two low-density glycosphingolipid/cholesterol-enriched Triton X-100-insoluble fractions, one of which is associated with actin, tubulin, caveolin, and kinases [167,[194][195][196]. Some MBP is also found in the Triton X-100 supernatant associated with MAG, PLP, MAPK, and some phospholipid [197]. Although these isolated fractions do not necessarily come from different membrane domains in myelin in situ [198], the presence of MBP in several different fractions suggests that it can interact with different contituents or complexes in myelin, some of which interact with kinases.…”

“…4], myelin also contains mechanisms which control kinase activity, e.g. phospholipase C to generate DAG, adenyl cyclase to generate cAMP, and MEK to activate p42/p44 MAPK [4,197]. Myelin also contains relatively high amounts of the signal transduction lipids phosphatidylinositol (PI), phosphatidic acid, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) [200].…”

Membrane lipid domains and membrane lipid domain preparations: are they the same thing?

“…As mentioned above, the Brown and Rose method or its modiˆcations have been applied not only to cultured cells but also to various tissues, including chicken gizzard smooth muscle (64), mouse (67), rat and human (93,146) cerebral cortex, mouse (66,(147)(148)(149)(150)(151), rat (21,65,(152)(153)(154) and human (155) brain, rat cerebellum (152), bovine and mouse brain myelin (71,75,149,156), rat (157) and mouse (92) brain synaptosomes, rat (158), mouse (148) and rainbow trout (159) liver, rat (160), rabbit (161) and mouse (148) lung, rat lung endothelium (162), pig (67,70) and mouse kidney (148). However, additional care is necessary to evaluate the results obtained analyzing DRM prepared from tissues.…”