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首页> 外文期刊>The FEBS journal >Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells
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Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

机译：爪蟾中间垂体细胞中ion病毒蛋白的细胞类型特异性转基因表达

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The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPC in vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.
机译：form病毒蛋白（PrPC）的细胞形式锚定在细胞的质膜上，并在大多数组织中表达，但主要在脑（包括垂体）中表达。迄今为止，仅在培养的（转染的）肿瘤细胞系中而不在原代细胞中研究了PrPC的生物合成。在这里，我们通过使用南非爪趾青蛙非洲爪蟾的神经内分泌中间垂体黑素体细胞作为模型系统研究了体内PrPC的细胞内命运。这些细胞参与动物的背景适应，并在动物适应黑色时产生高水平的主要分泌性货物黑皮皮质素（POMC）。稳定的非洲爪蟾转基因技术与POMC基因启动子相结合，被用作表达在黑色素细胞中特异性表达绿色荧光蛋白（GFP-PrPC）氨基末端标记的非洲爪蟾PrPC的工具。 GFP-PrPC融合蛋白从25级t开始向幼蛙表达，在黑色背景上诱导表达，并且融合蛋白主要位于高尔基体和质膜的亚细胞内。脉冲追踪代谢细胞标记研究表明，GFP-PrPC最初合成为45-kDa蛋白，随后逐步糖基化为48-，51-和最终55-kDa形式。此外，我们发现成熟的55 kDa GFP-PrPC蛋白被硫酸化，锚定在质膜上并裂解为33 kDa的产物。尽管高水平的转基因表达，亚细胞结构以及POMC的合成和加工，以及POMC衍生产物的分泌在转基因黑素体细胞中仍然不受影响。因此，我们在神经内分泌细胞中以及在定义明确的生理环境中研究了PrPC。

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