Pal S, Bigbee J W, Saito M, Ariga T, Yu R K
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614, USA.
Neurochem Res. 1996 Apr;21(4):403-9. doi: 10.1007/BF02527703.
Previous studied from the laboratory demonstrated the presence of a UDP-galactose:Gb3Cer alpha 1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Gal alpha 1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the alpha-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Gal alpha 1-3Gb3Cer in DRGN, we examined the expression of Gal alpha 1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Gal alpha 1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cells bodies and neurites. The expression of Gal alpha 1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Gal alpha 1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.
该实验室先前的研究表明，在培养的PC12嗜铬细胞瘤细胞中存在一种UDP-半乳糖：Gb3Cerα1-3半乳糖基转移酶活性，负责合成一种独特的糖鞘脂（GSL），即Galα1-3Gb3Cer（21）。在本研究中，我们检测了分离的大鼠胚胎背根神经节神经元（DRGN）中这种酶活性的存在，DRGN与嗜铬细胞瘤细胞一样，起源于神经嵴细胞。DRGN表现出α-半乳糖基转移酶活性，且该活性与PC12细胞的活性相当，而其他几种大鼠组织，除肾脏外，活性极低。为了确定Galα1-3Gb3Cer在DRGN中的时空表达，我们检测了源自胚胎第16天大鼠胚胎的培养DRGN中Galα1-3Gb3Cer的表达。使用针对Galα1-3Gb3Cer产生的多克隆抗体，我们检测了培养5、8、12和15天后该糖脂在DRGN细胞中的定位。免疫染色仅限于神经元，而施万细胞呈阴性。在第5天，免疫染色较弱，局限于DRGN的细胞体，尽管此时已有神经突。第5天至第15天期间是神经突快速生长和细胞体持续增大的时期。到第8天，细胞体中的免疫反应性显著增加。到第12天，神经突中出现免疫反应性，到第15天，细胞体和神经突中的免疫反应性都很强。背根神经节冰冻切片的免疫染色证实了Galα1-3Gb3Cer在体内的表达。我们目前的研究表明，Galα1-3Gb3Cer在神经发生过程中具有神经元特异性表达，结合之前在PC12细胞中对其表达的观察结果，强烈提示这种GSL在神经元发育中起作用。
