Patnaik S K, Zhang A, Shi S, Stanley P
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York, 10461, USA.
Arch Biochem Biophys. 2000 Mar 15;375(2):322-32. doi: 10.1006/abbi.1999.1693.
Gain-of-function glycosylation mutants provide access to glycosylation pathways, glycosylation genes, and mechanisms that regulate expression of a glycotype. Previous studies have shown that the gain-of-function Chinese hamster ovary (CHO) mutants LEC12, LEC29, and LEC30 express an N-ethylmaleimide-resistant alpha(1, 3)fucosyltransferase (alpha(1,3)Fuc-T) activity that is not detected in CHO cells and that generates the Lewis(X) but not the sialyl-Lewis(X) determinant. The three mutants differ, however, in lectin resistance properties, expression of fucosylated antigens, and in vitro alpha(1,3)Fuc-T activities. In this paper we show that each mutant expresses Fuc-TIX, but only LEC30 cells express Fuc-TIV. Using genomic PCR and reverse-transcriptase (RT)-PCR strategies, we isolated coding portions of the CHO Fut4 and Fut9 genes. Each gene is present in a single copy in the CHO and mutant genomes. The Fut4 gene is expressed only in LEC30 cells, while all three mutants express the Fut9 gene. Interestingly, the fucosylation phenotypes of LEC12 and LEC29 cells do not correlate with the relative abundance of their Fut9 gene transcripts (LEC29 >> LEC12). Compared to LEC29 cells, LEC12 cells have an approximately 40-fold higher in vitro alpha(1,3)Fuc-T activity and bind the VIM-2 monoclonal antibody, whereas LEC29 cells do not bind VIM-2. Mixing experiments did not detect Fuc-TIX inhibitory activity in LEC29 cell extracts, and CHO cells expressing a transfected Fut9 gene behaved like LEC12 cells. Therefore, it seems that LEC29 cells may not translate their more abundant Fut9 gene transcripts efficiently or may not synthesize appropriate acceptors for internal alpha(1,3)fucosylation. Alternatively, LEC12 cells may possess, in addition to Fuc-TIX, a novel alpha(1,3)Fuc-T activity.
功能获得性糖基化突变体为研究糖基化途径、糖基化基因以及调控糖型表达的机制提供了途径。先前的研究表明，功能获得性中国仓鼠卵巢（CHO）突变体LEC12、LEC29和LEC30表达一种对N - 乙基马来酰亚胺有抗性的α(1, 3)岩藻糖基转移酶（α(1,3)Fuc - T）活性，这种活性在CHO细胞中未被检测到，并且能产生Lewis(X)决定簇而非唾液酸化Lewis(X)决定簇。然而，这三个突变体在凝集素抗性特性、岩藻糖基化抗原的表达以及体外α(1,3)Fuc - T活性方面存在差异。在本文中，我们表明每个突变体都表达Fuc - TIX，但只有LEC30细胞表达Fuc - TIV。使用基因组PCR和逆转录酶（RT）-PCR策略，我们分离出了CHO Fut4和Fut9基因的编码部分。每个基因在CHO和突变体基因组中均以单拷贝形式存在。Fut4基因仅在LEC30细胞中表达，而所有三个突变体都表达Fut9基因。有趣的是，LEC12和LEC29细胞的岩藻糖基化表型与其Fut9基因转录本的相对丰度不相关（LEC29 >> LEC12）。与LEC29细胞相比，LEC12细胞具有大约高40倍的体外α(1,3)Fuc - T活性，并能结合VIM - 2单克隆抗体，而LEC29细胞不结合VIM - 2。混合实验未在LEC29细胞提取物中检测到Fuc - TIX抑制活性，并且表达转染Fut9基因的CHO细胞表现得与LEC12细胞相似。因此，似乎LEC29细胞可能无法有效地翻译其更丰富的Fut9基因转录本，或者可能无法合成用于内部α(1,3)岩藻糖基化的合适受体。或者，LEC12细胞除了Fuc - TIX之外，可能还具有一种新的α(1,3)Fuc - T活性。
