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.
Glycoconj J. 2015 Dec;32(9):675-84. doi: 10.1007/s10719-015-9616-4. Epub 2015 Aug 29.
Heparan sulfate (HS)-containing, S-nitrosylated (SNO) glypican-1 (Gpc-1) releases anhydromannose-containing HS (anMan-HS) by SNO-catalyzed autodegradation in endosomes. Transport of anMan-HS to the nucleus requires processing of the amyloid precursor protein (APP) to amyloid beta peptides (Aβ). To further examine the relationship between APP and Gpc-1 processing in normal fibroblasts we have suppressed Gpc-1 autodegradation by aminoguanidine inhibition of NO synthesis and prevented lysosomal degradation of anMan-HS by using chloroquine. Deconvolution immunofluorescence microscopy and SDS-PAGE using anMan- and APP/Aβ-specific antibodies and markers for nuclei and autophagosomes were used to identify subcellular localization of Aβ and its oligomeric state. Wild-type mouse embryonic fibroblasts (WT MEF) grown during NO-deprivation accumulated 95-98% of Aβ as oligomers in the nucleus. WT MEF treated with chloroquine accumulated both anMan-HS and Aβ, first in the nucleus then in autophagosomes. Maximal nuclear anMan-HS and Aβ accumulation was obtained after 4 and 7 h of growth, respectively. Both yielded similar banding patterns on SDS-PAGE which were also similar to the Aβ oligomers obtained after NO-deprivation. Nuclear Aβ accumulation was marginally increased (from 54 to 58%) by suppression of both release and degradation of anMan-HS. Nuclear exit of Aβ, accumulated during growth in aminoguanidine, was enhanced by ascorbate-induced reactivation of anMan-HS production. Transgenic Alzheimer disease mouse (Tg2576) MEF, which produces excess amount of Aβ was used for comparison. Overall, nuclear Aβ exit and lysosomal degradation was compromised by inhibition of the autophagosome-lysosome pathway in both WT and Tg2576 MEF, while only WT MEF was sensitive to suppression of Gpc-1 autodegradation.
含硫酸乙酰肝素（HS）的S-亚硝基化（SNO）磷脂酰肌醇蛋白聚糖-1（Gpc-1）在内体中通过SNO催化的自降解释放含脱水甘露糖的HS（anMan-HS）。anMan-HS向细胞核的转运需要将淀粉样前体蛋白（APP）加工成淀粉样β肽（Aβ）。为了进一步研究正常成纤维细胞中APP与Gpc-1加工之间的关系，我们通过氨基胍抑制NO合成来抑制Gpc-1自降解，并使用氯喹防止anMan-HS的溶酶体降解。使用anMan和APP/Aβ特异性抗体以及细胞核和自噬体标记物的去卷积免疫荧光显微镜和SDS-PAGE来鉴定Aβ的亚细胞定位及其寡聚状态。在无NO条件下生长的野生型小鼠胚胎成纤维细胞（WT MEF）在细胞核中积累了95-98%的Aβ作为寡聚体。用氯喹处理的WT MEF首先在细胞核中积累anMan-HS和Aβ，然后在自噬体中积累。生长4小时和7小时后分别获得最大的细胞核anMan-HS和Aβ积累。两者在SDS-PAGE上产生相似的条带模式，这也与无NO条件下获得的Aβ寡聚体相似。通过抑制anMan-HS的释放和降解略微增加了细胞核Aβ的积累（从54%增加到58%）。在氨基胍中生长期间积累的Aβ从细胞核中排出，通过抗坏血酸诱导的anMan-HS产生的重新激活而增强。产生过量Aβ的转基因阿尔茨海默病小鼠（Tg2576）MEF用于比较。总体而言，WT和Tg2576 MEF中自噬体-溶酶体途径的抑制损害了细胞核Aβ的排出和溶酶体降解，而只有WT MEF对Gpc-1自降解的抑制敏感。
