Boggs Joan M, Wang Huimin
Department of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, ON, Canada.
J Neurosci Res. 2004 May 1;76(3):342-55. doi: 10.1002/jnr.20080.
We have shown previously that addition of liposomes containing the two major glycosphingolipids of myelin, galactosylceramide (GalC) and cerebroside sulfate (CBS), to cultured oligodendrocytes (OLs) caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface to the same membrane domains. It also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the liposomes with the OL surface induces transmembrane signal transmission. We show that a multivalent form of galactose conjugated to bovine serum albumin has a similar effect as the multivalent GalC/CBS-containing liposomes. Because GalC and CBS can interact with each other across apposed membranes and because anti-GalC and anti-CBS antibodies also cause redistribution of GalC/CBS and depolymerization of microtubules, we believe that the multivalent carbohydrate interacts with GalC and CBS in the OL membrane. Several myelin-specific transmembrane proteins could be involved in this transmembrane signal transmission from GalC/CBS. We looked at co-clustering of several myelin constituents by confocal microscopy to determine if they are located in or redistribute to GalC/MBP-containing domains. Myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), MAPK, and some phosphotyrosine-containing proteins were found to co-cluster with GalC and MBP, but myelin-associated glycoprotein (MAG) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) did not. These results suggest that MOG and PLP, but probably not MAG, are possible candidates for transmembrane transmission of the signal received by GalC/CBS. To determine if depolymerization of actin microfilaments was required for co-clustering, or was secondary to clustering, we stabilized F-actin with jasplakinolide. This also prevented depolymerization of the microtubules and prevented clustering of all constituents, including GalC. The prevention of clustering or redistribution of these glycolipids and proteins by an intact cytoskeleton is consistent with the picket fence model.
我们之前已经表明，将含有髓鞘的两种主要糖鞘脂，即半乳糖神经酰胺（GalC）和硫酸脑苷脂（CBS）的脂质体添加到培养的少突胶质细胞（OLs）中，会导致细胞外表面的GalC和胞质表面的髓鞘碱性蛋白（MBP）聚集到相同的膜结构域。这也会导致肌动蛋白微丝和微管解聚，表明脂质体与OL表面的相互作用诱导跨膜信号传递。我们发现与牛血清白蛋白偶联的多价形式的半乳糖具有与含多价GalC/CBS的脂质体类似的作用。由于GalC和CBS可以跨相对的膜相互作用，并且由于抗GalC和抗CBS抗体也会导致GalC/CBS的重新分布和微管解聚，我们认为多价碳水化合物与OL膜中的GalC和CBS相互作用。几种髓鞘特异性跨膜蛋白可能参与了这种从GalC/CBS的跨膜信号传递。我们通过共聚焦显微镜观察了几种髓鞘成分的共聚集，以确定它们是否位于含GalC/MBP的结构域中或重新分布到该结构域。发现髓鞘少突胶质细胞糖蛋白（MOG）、蛋白脂蛋白（PLP）、丝裂原活化蛋白激酶（MAPK）以及一些含磷酸酪氨酸的蛋白与GalC和MBP共聚集，但髓鞘相关糖蛋白（MAG）和磷脂酰肌醇-4,5-二磷酸（PIP(2)）没有。这些结果表明，MOG和PLP，但可能不是MAG，是GalC/CBS接收的信号跨膜传递的可能候选者。为了确定肌动蛋白微丝的解聚是共聚集所必需的，还是聚集的继发结果，我们用茉莉酮酸甲酯稳定F-肌动蛋白。这也阻止了微管的解聚，并阻止了包括GalC在内的所有成分的聚集。完整的细胞骨架对这些糖脂和蛋白聚集或重新分布的阻止与栅栏模型一致。
