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Publications (9)30.28 Total impact

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    ABSTRACT: The two major glycosphingolipids of myelin, galactosylceramide (GalC) and sulfatide (SGC), interact with each other by trans carbohydrate-carbohydrate interactions. They face each other in the apposed extracellular surfaces of the multilayered myelin sheath produced by oligodendrocytes (OLs). Multivalent galactose and sulfated galactose, in the form of GalC/SGC-containing liposomes or silica nanoparticles conjugated to galactose and galactose-3-sulfate, interact with GalC and SGC in the membrane sheets of OLs in culture. This stimulus results in transmembrane signaling, loss of the cytoskeleton and clustering of membrane domains, suggesting that GalC and SGC could participate in glycosynapses between apposed OL membranes or extracellular surfaces of mature myelin. Such glycosynapses may be important for myelination and/or myelin function.
    FEBS letters 11/2009; 584(9):1771-8. · 3.54 Impact Factor
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    ABSTRACT: Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et al. (2008) Biochemistry 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P(2) was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge. This study suggests that changes in membrane negative surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus could regulate the activity of Fyn at the oligodendrocyte membrane surface.
    Biochemistry 02/2009; 48(11):2385-93. · 3.38 Impact Factor
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    ABSTRACT: The 18.5 kDa isoform of myelin basic protein (MBP) has recently been shown to sequester phosphatidylinositol-(4,5)-bis-phosphate (PI(4,5)P2) in vesicular membranes in vitro, as do domains of other membrane- and cytoskeleton-associated proteins such as MARCKS (myristoylated alanine-rich C kinase substrate) and GAP-43 (growth-associated protein of 43 kDa), known collectively as “PI(4,5)P2-modulins” [Musse et al., Biochemistry, 47 (2008) 10372–10382 (doi:10.1021/bi801302b)]. Here, we demonstrate co-localisation of MBP and MARCKS in primary rat oligodendrocytes, and co-distribution of MBP, MARCKS, and GAP-43 in lipid raft fractions recovered from Triton X-100 detergent-extracted isolated myelin and brain homogenates. The results lend further support to MBP's multifunctionality, particularly as an additional modulator of PI(4,5)P2 availability in myelin.
    Neuroscience Letters 01/2009; · 2.03 Impact Factor
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    ABSTRACT: The 18.5 kDa isoform of myelin basic protein (MBP) is multifunctional and has previously been shown to have structural and phenomenological similarities with domains of other membrane- and cytoskeleton-associated proteins such as MARCKS (myristoylated alanine-rich C kinase substrate). Here, we have investigated whether 18.5 kDa MBP can sequester phosphatidylinositol-(4,5)-bis-phosphate (PI(4,5)P 2) in membranes, like MARCKS and other "PIPmodulins" do. Using fluorescence-quenching and electron paramagnetic resonance (EPR) spectroscopy, and model membranes containing BODIPY-FL- or proxyl-labeled PI(4,5)P 2, respectively, we have demonstrated that MBP laterally sequesters PI(4,5)P 2. The MBP-PI(4,5)P 2 interactions are electrostatic, partially cholesterol-dependent, and sensitive to phosphorylation, deimination, and Ca (2+)-CaM binding. Confocal microscopy of cultured oligodendrocytes also revealed patched colocalization of MBP and PI(4,5)P 2, indicating the spatial clustering of PI(4,5)P 2 in the plasma membrane. On the basis of these findings as well as the overwhelming convergence of functional properties, modifying enzymes, and interaction partners, we propose that MBP is mechanistically related to GAP-43, MARCKS, and CAP-23. During myelinogenesis, it may mediate calcium and phosphorylation-sensitive plasma membrane availability of PI(4,5)P 2. This regulation of PI(4,5)P 2 availability at the cell cortex may be coupled to the elaboration and outgrowth of the membranous cellular processes by oligodendrocytes.
    Biochemistry 10/2008; 47(39):10372-82. · 3.38 Impact Factor
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    ABSTRACT: There is general acceptance that the estrogen receptor can act as a transcription factor. However, estrogens can also bind to receptors that are located at the plasma membrane and stimulate rapid intracellular signaling processes. We recently showed that a membrane-associated estrogen receptor (mER) is present within myelin and at the oligodendrocyte (OL) plasma membrane. To understand the physiological function of mER in OLs, we investigated its cellular localization and involvement in rapid signaling in CG4 cells and OL primary cultures. An ERalpha was expressed along the lacy network of veins in the membrane sheets and in the perikaryon and nucleus in OLs. ERbeta was located in the nucleus, and to a lesser extent along the veins. The expression of ERalpha and ERbeta in OL membranes was confirmed by Western analysis of isolated membranes. OL membranes mainly had truncated forms of ERalpha, 53 and 50 kDa, in addition to some 65 kDa form, whereas ERbeta was a 54 kDa form. CG4 cells and OLs were pulsed with 17alpha- and 17beta-estradiol for various times and the total lysates were analyzed for phosphorylated kinases. Both 17alpha- and 17beta-estradiol elicited rapid phosphorylation of p42/44MAPK, Akt, and GSK-3beta within 8 min. This rapid signaling is consistent with estradiol ligation of a membrane form of ER. Since 17alpha-estradiol is produced at higher concentrations than 17beta-estradiol in the brain of both sexes, signaling via 17alpha-estradiol-liganded mER may have an important function in OLs.
    Glia 09/2008; 57(2):153-65. · 5.07 Impact Factor
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    ABSTRACT: We showed previously that the addition to cultured oligodendrocytes (OLs) of multivalent carbohydrate in the form of liposomes containing the two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and cerebroside sulfate (Sulf), or galactose conjugated to bovine serum albumin caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface. Multivalent carbohydrate also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the carbohydrate with the OL surface transmits a transmembrane signal to the cytoskeleton. In the present study we show that inhibition of GSL synthesis with fumonisin B1 prevents clustering of MBP in GalC/Sulf-negative oligodendrocytes, suggesting that GSLs are required for the effect. Because the effects of multivalent carbohydrate resemble those caused by the addition of anti-GalC/Sulf antibodies to OLs and because GalC and Sulf can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes, these results support the conclusion that the OL receptor for GalC/Sulf in liposomes is GalC/Sulf in the OL membrane. Inhibition of MBP expression using MBP siRNA inhibited GalC clustering, suggesting that MBP is required for the effect. We also investigate the signal transduction pathways involved using a number of enzyme inhibitors. These indicated that the Akt and p42/p44 MAPK pathways, Rho GTPases, and GSK-3beta are involved, consistent with their known involvement in regulation of the cytoskeleton. These interactions between GalC/Sulf-containing liposomes and the OL membrane may mimic interactions between GalC/Sulf-enriched signaling domains when OL cell membranes or the extracellular surfaces of compact myelin come into contact.
    Journal of Neuroscience Research 06/2008; 86(7):1448-58. · 2.97 Impact Factor
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    ABSTRACT: Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate-carbohydrate interactions across apposed membranes, resulting in cell-cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate-carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.
    Biochimica et Biophysica Acta 04/2008; 1780(3):445-55. · 4.66 Impact Factor
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    ABSTRACT: Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to bind it to a negatively charged membrane.
    Biochemistry 02/2006; 45(2):391-401. · 3.38 Impact Factor
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    ABSTRACT: Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I3-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication. Published in 2003.
    Glycoconjugate Journal 01/2004; 21. · 1.88 Impact Factor