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

克隆的人β-1，4-N-乙酰半乳糖胺转移酶(GalNAc-T；EC2.4.1.92)基因的氨基酸序列预测了三个潜在的N-连接糖基化位点。虽然许多分离的糖基转移酶含有2到6个N-糖基化位点，但它们的意义还没有得到充分的证明。为了阐明N-糖基化在GalNAc-T功能中的作用，我们构建了一系列突变的cDNA，通过聚合酶链式反应(PCR)介导的定点突变消除了其中的部分或全部糖基化识别位点。通过体外转录/翻译，我们证实了所有可能的N-糖基化位点都可以使用。尽管转导突变型cDNAs的细胞株的GalNAcbeta1*4(NeuAcalpha2*3)Galbeta1*4GlcCer(G&lt；M2&gt；)的表达水平与野生型相当，但转染型突变型cDNAs提取液的酶活性低于野生型。随着脱糖位点数目的增加，酶活性的下降更加明显，在完全脱糖的突变体中，酶活性下降约90%。酶动力学分析表明，野生型和突变型基因产物的Km没有明显变化。野生型和突变型表达的GalNAc-T在细胞内的定位也显示出类似的核周模式(高尔基模式)。这些结果表明，GalNAc-T上的N-连接碳水化合物是调节酶结构稳定性所必需的。

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.