Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase
Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase
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DOI：
10.1042/0264-6021:3450365
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发表时间：
2000-01-15
影响因子：
4.1
通讯作者：
Márquez, J
中科院分区：
文献类型：
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作者：
Gómez-Fabre, PM;Aledo, JC;Márquez, J
磷酸活化的转氨酶（GA）在某些类型的肿瘤中过表达，但其在肿瘤细胞生长和增殖中的确切作用尚不清楚。在这里，我们描述了一个全长的人乳腺癌ZR 75细胞的cDNA克隆的分离，通过λ gt 10 cDNA文库筛选和cDNA末端的快速扩增（“RACE”）技术的组合。人GA cDNA全长2408 nt，开放阅读框为1806个碱基，编码602个氨基酸残基，预测分子量为66309 Da，N端含有14个氨基酸残基的线粒体输入前序列。在大肠杆菌中的异源表达和纯化产生了预期分子大小的产物，该产物通过使用针对重组人GA的抗体来识别。序列分析表明，人类GA是高度相似的大鼠肝酶。北方凝胶分析表明，该基因存在于人的肝、脑和胰腺中，其中主要转录产物为2.4kb，但在肾、心脏、骨骼肌、肺和胎盘中不存在。这些结果强烈表明，第一个人类GA克隆，GA从ZR-75乳腺癌细胞，大概是那些从人类肝脏和大脑，是肝型同工酶，与目前的观点，认为肾型的异构体中表达的所有组织与GA的活动，出生后的肝脏除外，形成鲜明对比。
Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambda gt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.