Caduff J, Sansano S, Bonnet A, Suter U, Schaeren-Wiemers N
Neurobiology Laboratory, Department of Research, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland.
Microsc Res Tech. 2001 Mar 15;52(6):645-55. doi: 10.1002/jemt.1049.
During myelin formation, membrane-associated proteins have to be sorted and transported in specified membrane regions such as compact and non-compact myelin membranes. One protein that may be involved in such a process is the Myelin and Lymphocyte protein MAL (VIP17/ MVP17). MAL was identified as a novel myelin membrane component expressed by oligodendrocytes and Schwann cells. Since MAL has been shown to be important in the apical sorting machinery of polarized cells, we have started to investigate the possible functional role of MAL in sorting myelin membrane-associated molecules. In this study, we have generated cDNA constructs with green fluorescent protein (GFP) either at the N- or C-terminus of MAL. Transfection experiments showed that GFP-MAL expression resembles that of normal MAL, whereas the MAL-GFP fusion construct was not properly transported within the cell. Furthermore, we could demonstrate that GFP-MAL is enriched in detergent insoluble glycolipid-enriched microdomains as already seen for untagged MAL. As a prerequisite for the generation of transgenic mice expressing GFP-MAL under the control of its own regulatory elements, we have generated a cDNA construct with an 8-kb MAL promotor fragment fused to GFP-MAL. Transfection experiments of the Oli-neu oligodendrocyte cell line showed that GFP-MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summary, the results confirm that the fusion protein GFP-MAL is incorporated into detergent-insoluble complexes and the 8-kb MAL promotor fragment is sufficient to be activated in oligodendrocytes.
在髓鞘形成过程中，膜相关蛋白必须在特定的膜区域进行分选和运输，如致密和非致密髓鞘膜。一种可能参与此过程的蛋白是髓鞘和淋巴细胞蛋白MAL（VIP17/MVP17）。MAL被鉴定为少突胶质细胞和施万细胞表达的一种新型髓鞘膜成分。由于MAL已被证明在极化细胞的顶端分选机制中很重要，我们开始研究MAL在分选髓鞘膜相关分子中的可能功能作用。在本研究中，我们构建了在MAL的N端或C端带有绿色荧光蛋白（GFP）的cDNA构建体。转染实验表明，GFP-MAL的表达类似于正常MAL，而MAL-GFP融合构建体在细胞内未被正确运输。此外，我们可以证明，GFP-MAL在去污剂不溶性糖脂富集微结构域中富集，这与未标记的MAL情况相同。作为在其自身调控元件控制下表达GFP-MAL的转基因小鼠产生的前提条件，我们构建了一个带有8kb MAL启动子片段与GFP-MAL融合的cDNA构建体。对Oli-neu少突胶质细胞系的转染实验表明，GFP-MAL表达，但仅在经cAMP刺激分化的细胞中表达。总之，结果证实融合蛋白GFP-MAL被整合到去污剂不溶性复合物中，并且8kb MAL启动子片段足以在少突胶质细胞中被激活。
