Brodsky G, Barnes T, Bleskan J, Becker L, Cox M, Patterson D
Eleanor Roosevelt Institute, 1899 Gaylord Street, Denver, CO 80206, USA.
Hum Mol Genet. 1997 Nov;6(12):2043-50. doi: 10.1093/hmg/6.12.2043.
Purines are critical for energy metabolism, cell signalling and cell reproduction. Nevertheless, little is known about the regulation of this essential biochemical pathway during mammalian development. In humans, the second, third and fifth steps of de novo purine biosynthesis are catalyzed by a trifunctional protein with glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART) enzymatic activities. The gene encoding this trifunctional protein is located on chromosome 21. The enzyme catalyzing the intervening fourth step of de novo purine biosynthesis, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17. To investigate the regulation of these proteins, we have generated monoclonal and/or polyclonal antibodies specific to each of these enzymatic domains. Using these antibodies on western blots of Chinese hamster ovary (CHO) cells transfected with the human GARS-AIRS-GART gene, we show that this gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa. This carboxy-truncated human GARS protein is produced by alternative splicing resulting in the use of a polyadenylation site in the intron between the terminal GARS and the first AIRS exons. The expression of both the GARS and GARS-AIRS-GART proteins are regulated during development of the human cerebellum, while the expression of FGARAT appears to be constitutive. All three proteins are expressed at high levels during normal prenatal cerebellum development while the GARS and GARS-AIRS-GART proteins become undetectable in this tissue shortly after birth. In contrast, the GARS and GARS-AIRS-GART proteins continue to be expressed during the postnatal development of the cerebellum in individuals with Down syndrome.
嘌呤对于能量代谢、细胞信号传导和细胞繁殖至关重要。然而，关于这一重要生化途径在哺乳动物发育过程中的调控，我们所知甚少。在人类中，从头合成嘌呤的第二步、第三步和第五步由具有甘氨酰胺核糖核苷酸合成酶（GARS）、氨基咪唑核糖核苷酸合成酶（AIRS）和甘氨酰胺核糖核苷酸甲酰基转移酶（GART）酶活性的三功能蛋白催化。编码这种三功能蛋白的基因位于21号染色体上。催化从头合成嘌呤中间第四步的酶，磷酸核糖甲酰甘氨酰胺转酰胺酶（FGARAT），由17号染色体上的一个单独基因编码。为了研究这些蛋白质的调控，我们已经产生了针对这些酶结构域中每一个的单克隆和/或多克隆抗体。在转染了人GARS-AIRS-GART基因的中国仓鼠卵巢（CHO）细胞的蛋白质免疫印迹中使用这些抗体，我们表明该基因不仅编码110 kDa的三功能蛋白，还编码50 kDa的单功能GARS蛋白。这种羧基末端截短的人GARS蛋白是通过可变剪接产生的，导致在末端GARS和第一个AIRS外显子之间的内含子中使用了一个聚腺苷酸化位点。GARS和GARS-AIRS-GART蛋白的表达在人类小脑发育过程中受到调控，而FGARAT的表达似乎是组成性的。在正常产前小脑发育过程中，所有这三种蛋白都高水平表达，而GARS和GARS-AIRS-GART蛋白在出生后不久在该组织中就检测不到了。相比之下，在唐氏综合征个体的小产后发育过程中，GARS和GARS-AIRS-GART蛋白继续表达。
