Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters
Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters
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DOI：
10.1074/jbc.m011124200
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发表时间：
2001-07-13
影响因子：
4.8
通讯作者：
Gerardy-Schahn, R
中科院分区：
文献类型：
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作者：
Oelmann, S;Stanley, P;Gerardy-Schahn, R
核苷酸-糖转运蛋白（NST）是真核生物糖基化途径的重要组成部分，鉴定NST功能的结构元件是一项重要的任务。中国仓鼠卵巢糖基化突变体的核苷酸糖转运缺陷提供了访问非活性转运蛋白，可以定义这样的结构/功能关系。在这项研究中，我们克隆了仓鼠UDP-半乳糖转运蛋白（UGT），并确定了缺陷的UGT基因转录本从9个独立的中国仓鼠卵巢突变体，属于Lec 8互补组。逆转录聚合酶链反应的引物，跨越UGT开放阅读框显示，3个Lec 8突变体表达全长开放阅读框，而6个Lec 8突变体主要表达截短的UGT基因转录本。测序鉴定了来自三个突变体的全长UGT转录物中不同的单个或三联体核苷酸变化。这些突变在所有已知的哺乳动物NST中高度保守的位置处翻译成三种不同的氨基酸变化。编码突变Δ丝氨酸213或G281 D的cDNA的转染未能纠正Lec 8转染子中的UDP-半乳糖转运缺陷。最重要的是，将这些相同的突变引入小鼠CMP-唾液酸转运蛋白的同源区域导致该转运蛋白失活。因此，鉴定Lec 8突变体中抑制UGT的点突变导致发现了对UGT和CST（两种最不同的哺乳动物NST）的活性都至关重要的氨基酸。
Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes, The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.