Cloning and expression of a brain-specific putative UDP-GalNAc:: Polypeptide N-acetylgalactosaminyltransferase gene
Cloning and expression of a brain-specific putative UDP-GalNAc:: Polypeptide N-acetylgalactosaminyltransferase gene
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DOI：
10.1248/bpb.28.429
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发表时间：
2005-03-01
影响因子：
2
通讯作者：
Kurosaka, A
中科院分区：
文献类型：
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作者：
Nakamura, N;Toba, S;Kurosaka, A
我们分离了大鼠cDNA克隆和它的人类直系同源物，这是最同源的UDP-GaINAe：多肽N-乙酰氨基半乳糖转移酶9，同源性为基础的PCR从大脑。这些推定的GaINAe-转移酶（命名为pt-GaINAe-T）的核苷酸序列分析表明，它们含有GaINAe-转移酶家族的特征性结构特征。还发现人pt-GaINAe-T与基因WBSCR 17相同，据报道，WBSCR 17位于威廉斯-博伦综合征（一种神经发育障碍）患者的关键区域，并且主要在脑和心脏中表达。为了更详细地研究pt-GaINAc-T在脑中的表达，我们首先通过北方印迹分析检测了人pt-GaINAc-T的表达，发现5.0-kb mRNA在大脑皮层中的表达最丰富，而在小脑中的表达稍少。更广泛地研究了大鼠pt-GaINAc-T的表达。通过北方印迹分析证实了2.0-kb和5.0-kb转录物的脑特异性表达。在成人大脑中的原位杂交显示高水平的表达在小脑，海马，丘脑和大脑皮层。此外，在高倍镜下观察显示，表达与神经元有关，但与胶质细胞无关。对大鼠胚胎的分析还表明，大鼠pt-GaINAc-T在神经系统中表达，包括间脑、小脑原基和背根神经节。然而，在昆虫细胞中表达的重组人pt-GaINAc-T不糖基化来自哺乳动物粘蛋白的几个肽，这表明它可能具有严格的底物特异性。pt-GaINAc-T的脑特异性表达表明其通过神经元中蛋白质的O-糖基化参与脑发育。
We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GaINAe: polypeptide N-acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GaINAe-transferases (designated pt-GaINAe-T) showed that they contained structural features characteristic of the GaINAc-transferase family. It was also found that human pt-GaINAe-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmentall disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GaINAc-T in brain in more detail, we first examined that of human pt-GaINAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GaINAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GaINAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GaINAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GaINAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.