Norflus F, Yamanaka S, Proia R L
Section on Biochemical Genetics, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
DNA Cell Biol. 1996 Feb;15(2):89-97. doi: 10.1089/dna.1996.15.89.
Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and HEXB mutations that could alter enzyme expression in Tay-Sachs and Sandhoff diseases, respectively.
人类溶酶体β-己糖胺酶由两个基因HEXA和HEXB编码，分别指定α亚基和β亚基。这些亚基二聚化形成β-己糖胺酶A（αβ）、β-己糖胺酶B（ββ）和β-己糖胺酶S（αα）。该酶系统有能力降解多种细胞底物：含有β-连接的N-乙酰葡糖胺基或N-半乳糖胺基残基的寡糖、糖胺聚糖和糖脂。HEXA基因或HEXB基因的突变导致GM2神经节苷脂在神经元中积累，从而导致称为GM2神经节苷脂沉积症的严重神经退行性疾病。为了鉴定负责己糖胺酶表达的DNA元件，我们将人类和小鼠己糖胺酶基因的5'侧翼序列连接到氯霉素乙酰转移酶（CAT）基因上。将所得质粒转染到NIH-3T3细胞中，并测定CAT活性作为启动子强度指标。通过5'缺失分析，发现HEXA表达的必需序列位于ATG起始密码子上游100 bp至60 bp之间的一个40 bp区域内。该区域包含两个潜在的雌激素反应元件半位点以及转录因子NF-E1和AP-2的潜在结合位点。同样，重要的HEXB启动子序列定位于ATG密码子上游150 bp至90 bp之间的一个60 bp区域。通过对150 bp的HEXB构建体中的一个60 bp区域进行扫描诱变，我们确定了一个12 bp的必需启动子元件，该元件包含两个潜在的AP-1位点。发现小鼠Hexa和Hexb基因的5'侧翼序列含有与同源人类基因中定义的必需启动子元件在序列、位置和活性上相似的区域。然而，在HEXA和HEXB基因的5'侧翼区域之间未发现序列相似性。这些必需启动子元件分别代表HEXA和HEXB突变的潜在位点，这些突变可能分别改变泰-萨克斯病和桑德霍夫病中的酶表达。
