Cheng Fang, Fransson Lars-Åke, Mani Katrin
Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84, Lund, Sweden.
Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84, Lund, Sweden.
Exp Cell Res. 2017 Nov 15;360(2):171-179. doi: 10.1016/j.yexcr.2017.09.003. Epub 2017 Sep 9.
Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein.
抗坏血酸盐诱导硫酸乙酰肝素从S-亚硝基化硫酸乙酰肝素蛋白聚糖磷脂酰肌醇蛋白聚糖-1中释放发生在内体中。硫酸乙酰肝素穿透膜并被转运到细胞核。这个过程依赖于铜以及淀粉样前体蛋白的表达和加工。目前尚不清楚外源性提供的抗坏血酸盐如何在内体中产生HS-anMan以及如何促进其穿过膜。在这里，我们通过使用去卷积免疫荧光显微镜、siRNA技术和[S]硫酸盐标记、囊泡分离和凝胶色谱法，研究了野生型、阿尔茨海默病Tg2576和淀粉样前体蛋白(-/-)小鼠成纤维细胞以及人胎儿和成纤维细胞系尼曼-皮克C1成纤维细胞。我们发现抗坏血酸盐诱导的硫酸乙酰肝素释放依赖于内体细胞色素b561的表达。淀粉样前体蛋白的β加工抑制可抑制硫酸乙酰肝素的形成和核转运，而铜(I)离子可恢复其形成。膜穿透不依赖于淀粉样β通道的形成。内体出口的抑制导致硫酸乙酰肝素在囊泡中积累，这些囊泡将淀粉样前体蛋白的C末端暴露在外部。内体到细胞核的转运也依赖于尼曼-皮克C1蛋白的表达。我们提出，抗坏血酸盐从培养基中摄取并被细胞色素b561氧化，细胞色素b561进而将存在于淀粉样前体蛋白N末端β裂解结构域中的铜(II)还原为铜(I)。铜(I)的再氧化与硫酸乙酰肝素从磷脂酰肌醇蛋白聚糖-1的还原性脱氨释放相偶联。淀粉样前体蛋白的C末端β裂解片段和尼曼-皮克C1蛋白可能促进其穿过膜。
