Schepers U, Lemm T, Herzog V, Sandhoff K
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
Biol Chem. 2000 Jul;381(7):531-44. doi: 10.1515/BC.2000.069.
Lysosomal degradation of the ganglioside GM2 by human beta-hexosaminidase A requires the presence of the GM2 activator protein as an essential cofactor. Here we demonstrate that GM2 activator mRNA is differentially expressed and mainly localized to the apical part of the epithelial cells of distal renal tubules and the collecting duct. In order to understand the mechanism underlying the regulation of the GM2 activator gene, we analyzed the genomic organization upstream exon 2 as well as the 5'-flanking region. The GM2 activator gene spans about 16.8 kb with a first intron of 6.5 kb, and the transcription start is located at position -96 upstream from the ATG. DNA elements responsible for GM2 activator expression were identified in a PCR-based method of long-distance DNA walking. Sequence analysis revealed a 2.9 kb region upstream of the ATG that contained regulatory elements like CAAT boxes, Sp1 binding sites as well as AP1, and AP2 sites. Transfection experiments in COS-1 cells with a series of chimeras of 5'-stepwise deletion mutants of the GM2 activator gene 5'-flanking region and the secretory alkaline phosphatase (SEAP)-reporter gene indicated that a genomic fragment encompassing -323 to +1 bp had significant promoter activity. EMSA experiments showed that Sp1 and other transcription factors like AP1, AP2 and CCAAT-Box binding proteins are involved in GM2 activator gene regulation.
人β-己糖胺酶A对神经节苷脂GM2的溶酶体降解需要GM2激活蛋白作为必需的辅助因子。在此我们证明，GM2激活蛋白mRNA存在差异表达，且主要定位于远端肾小管和集合管上皮细胞的顶端部分。为了解GM2激活蛋白基因调控的潜在机制，我们分析了外显子2上游的基因组结构以及5'侧翼区域。GM2激活蛋白基因跨度约16.8 kb，有一个6.5 kb的第一内含子，转录起始位点位于ATG上游-96位。通过基于PCR的长距离DNA步移法鉴定了负责GM2激活蛋白表达的DNA元件。序列分析显示，在ATG上游2.9 kb区域含有CAAT盒、Sp1结合位点以及AP1和AP2位点等调控元件。用GM2激活蛋白基因5'侧翼区域的一系列5'逐步缺失突变体与分泌性碱性磷酸酶（SEAP）报告基因构建的嵌合体在COS-1细胞中进行转染实验，结果表明，包含-323至+1 bp的基因组片段具有显著的启动子活性。电泳迁移率变动分析（EMSA）实验表明，Sp1以及AP1、AP2和CCAAT框结合蛋白等其他转录因子参与GM2激活蛋白基因的调控。
