Department of Experimental Medicine and Biochemical Sciences, University of Perugia, via del Giochetto, 06126 Perugia, Italy.
Biosci Rep. 2011 Dec;31(6):477-87. doi: 10.1042/BSR20110020.
Lysosomal α-D-mannosidase is an exoglycosidase involved in the ordered degradation of N-linked oligosaccharides. It is ubiquitously expressed, although the main transcript is more abundant in peripheral blood leucocytes. Here we report that α-D-mannosidase enzyme activity is very high in the promyelocytic leukaemia cell lines HL60 and NB4, as compared with other leukaemic cell lines or cells from different human sources. The MAN2B1 transcript level correlates with enzyme activity, indicating a transcriptional up-regulation of the α-D-mannosidase gene. The promoter was then characterized in HEK-293 cells (human embryonic kidney 293 cells) and HL60 cells; regulatory sequences crucial for its activity were determined by reporter gene assay in HEK-293 cells and located in the region -101/-71 with respect to the first ATG codon. Supershift assay demonstrated that Sp1 (specificity protein 1) bound to this sequence both in HEK-293 and HL60 cells. However, 5'-RACE (5'-rapid amplification of cDNA ends) indicated the use of multiple upstream TSSs (transcription start sites) in HL60 with respect to HEK-293 cells and gel shift analysis of the sequence -373/-269 demonstrated a specific binding by NF-κB (nuclear factor κB) transcription factor in HL60 but not in HEK-293 cells. We concluded that despite the α-D-mannosidase promoter showing typical features of housekeeping gene promoters, α-D-mannosidase transcription is specifically regulated in HL60 by NF-κB transcription factor.
溶酶体 α-D-甘露糖苷酶是一种参与 N-连接寡糖有序降解的外糖苷酶。它广泛表达，尽管主要转录本在周围血白细胞中更为丰富。在这里，我们报告说，与其他白血病细胞系或来自不同人类来源的细胞相比，早幼粒细胞白血病细胞系 HL60 和 NB4 中的 α-D-甘露糖苷酶酶活性非常高。MAN2B1 转录本水平与酶活性相关，表明 α-D-甘露糖苷酶基因的转录上调。然后在 HEK-293 细胞（人胚肾 293 细胞）和 HL60 细胞中对启动子进行了表征；通过在 HEK-293 细胞中的报告基因测定确定了其活性的关键调节序列，并将其定位在相对于第一个 ATG 密码子的-101/-71 区域。超迁移测定表明 Sp1（特异性蛋白 1）在 HEK-293 和 HL60 细胞中均与该序列结合。然而，5'-RACE（5'-快速扩增 cDNA 末端）表明相对于 HEK-293 细胞，HL60 中使用了多个上游 TSS（转录起始位点），凝胶迁移分析序列-373/-269 表明 HL60 中的 NF-κB（核因子 κB）转录因子具有特异性结合，但在 HEK-293 细胞中没有。我们得出的结论是，尽管 α-D-甘露糖苷酶启动子表现出管家基因启动子的典型特征，但 α-D-甘露糖苷酶转录在 HL60 中受 NF-κB 转录因子的特异性调节。
