Analvsis of localization of a glycosyltransferase and transfort of glycolipids by gene manipulation

从β-1，4-N-乙酰氨基半乳糖转移酶（GalNAc-T ; EC2.4.1.92）基因的克隆人cDNA推导的氨基酸序列预测了N-连接糖基化的三个潜在位点。虽然许多分离的糖基转移酶含有2至6个N-糖基化位点，但它们的重要性尚未得到充分证明。为了阐明N-糖基化在GalNAc-T功能中的作用，我们产生了一系列突变的cDNA，其中部分或全部糖基化识别位点通过聚合酶链反应（PCR）介导的定点突变被消除。使用体外转录/翻译，我们证实了所有潜在的N-糖基化位点都可以使用。虽然用突变cDNA转染的细胞系显示出与野生型相同水平的GalNAc β 1 *4（NeuAcalpha 2 *3）Galbeta 1 *4GlcCer（G_<M2>），但突变cDNA转染子的提取物显示出比野生型低的酶活性。随着去糖基化位点数量的增加，酶活性的降低更加明显，在完全去糖基化的突变体中降低约90%。酶动力学分析表明，野生型和突变型cDNA产物的Km值无明显变化。在具有野生型或突变cDNA的转染子中表达的GalNAc-T的细胞内定位也显示出类似的核周模式（高尔基模式）。这些结果表明，N-连接的碳水化合物的GalNAc-T是需要调节的酶结构的稳定性。

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T ; EC2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction( PCR)-mediated site directed mutagenesis. Using transcription/translation in vitro, we confirmed the all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAcbeta1*4 (NeuAcalpha2*3) Galbeta1*4GlcCer (G_<M2>) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of K_m among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.