HETEROTYPIC BINDING BETWEEN NEURONAL MEMBRANE-VESICLES AND GLIAL-CELLS IS MEDIATED BY A SPECIFIC CELL-ADHESION MOLECULE
HETEROTYPIC BINDING BETWEEN NEURONAL MEMBRANE-VESICLES AND GLIAL-CELLS IS MEDIATED BY A SPECIFIC CELL-ADHESION MOLECULE
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DOI：
10.1083/jcb.98.5.1746
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发表时间：
1984-01-01
影响因子：
7.8
通讯作者：
EDELMAN, GM
中科院分区：
文献类型：
--
作者：
GRUMET, M;EDELMAN, GM
通过多阶段定量分析，在鸡胚神经元细胞上鉴定出了一种新型细胞粘附分子（CAM），该分子参与神经胶质细胞的粘附。用于识别结合成分（称为神经元-胶质细胞 CAM 或 Ng-CAM）的测定旨在区分同型结合（例如神经元与神经元）和异型结合（例如神经元与神经胶质）。这种区别至关重要，因为单个神经元可能同时携带不同的 CAM，分别介导这些相互作用。体外神经元细胞与神经胶质细胞的粘附先前被由针对神经元膜的抗血清制备的Fab''片段抑制，但不被针对神经细胞粘附分子N-CAM的Fab''片段抑制。神经元-胶质细胞粘附是由不同于先前分离的 CAM 的特定细胞表面分子介导的。为了验证这一点，神经元膜囊泡内部用 6-羧基荧光素标记，外部用 125I 标记的 N-CAM 抗体标记，以阻断它们的同型结合。在 30 分钟的孵育期内，标记的囊泡与神经胶质细胞结合，但不与成纤维细胞结合。通过荧光显微镜和γ测量神经元囊泡与神经胶质细胞的特异性结合。 125I 标记的光谱。结合随着神经胶质细胞和神经元囊泡浓度的增加而增加。从抑制神经元和神经胶质细胞之间结合的抗神经元膜血清制备的Fab''片段抑制神经元囊泡与神经胶质细胞的结合。 Fab'' 片段的抑制活性因与神经元细胞预孵育而被耗尽，但与神经胶质细胞预孵育则没有。胰蛋白酶处理神经元膜囊泡释放出中和 Fab'' 片段抑制的物质；层析后，中和活性增强了 50 倍。将该组分注射到小鼠体内以产生单克隆抗体；获得了一种与神经元相互作用的抗体，抑制神经元膜囊泡与神经胶质细胞的结合，并在鸡胚脑膜的免疫印迹中识别出 MW = 135,000 的条带。该分子存在于神经元表面，它直接或间接介导神经元和神经胶质细胞之间的粘附。由于在测定中具有活性的单克隆抗体和原始多特异性抗体不与神经胶质细胞结合，因此神经元-神经胶质相互作用是异嗜性的，即它发生在神经元上的 Ng-CAM 和神经胶质细胞上存在的尚未识别的 CAM 之间。
By means of a multistage quantitative assay, a new kind of cell adhesion molecule (CAM) was identified on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which is named neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAM separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously inhibited by Fab'' fragments prepared from antisera against neuronal membranes but not by Fab'' fragments against N-CAM, the neural cell adhesion molecule. Neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAM. To verify this, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and .gamma. spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab'' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells inhibited neuronal vesicle binding to glial cells. The inhibitory activity of the Fab'' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab'' fragment inhibition; after chromatography, neutralizing activity was enriched 50-fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an MW = 135,000 band in immunoblots of embryonic chick brain membranes. This molecule is present on the surfaces of neurons and it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody and the original polyspecific antibodies that were active in the assay did not bind to glial cells, neuron-glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.