PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES IN INSECT CELLS - N-ACETYLGLUCOSAMINYLTRANSFERASE I AND II ACTIVITIES IN CULTURED LEPIDOPTERAN CELLS
PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES IN INSECT CELLS - N-ACETYLGLUCOSAMINYLTRANSFERASE I AND II ACTIVITIES IN CULTURED LEPIDOPTERAN CELLS
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DOI：
10.1093/glycob/3.6.619
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发表时间：
1993-12-01
期刊：
影响因子：
4.3
通讯作者：
GLOSSL, J
中科院分区：
文献类型：
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作者：
ALTMANN, F;KORNFELD, G;GLOSSL, J
研究了家蚕（Bm-N）、芸苔螟（IZD-Mb-0503）和frugiperda Spodoptera （Sf-9和Sf-21）培养细胞中β - 1,2- n -乙酰氨基葡萄糖转移酶（GlcNAc-T） I和II的活性水平。除了以Man α -3(Man α 1-6)-Man β 1-O(CH2)(8)COOH3和h -3标记的UDP-GlcNAc为底物的初始实验外，我们还以pyridylaminated Man(3)-GlcNAc(2)和Man(5)GlcNAc(2)为受体低聚糖，采用非放射性高效液相色谱法测定了GlcNAc- t I的活性。通过反相高效液相色谱、外糖苷酶酶切和甲基化分析表明，Man(3)GlcNAc(2)的产物含有末端GlcNAc残基，将β 1,2连接到受体的α 1,3臂上。与来自人肝癌细胞系HepG2的酶相比，昆虫细胞GlcNAc-T I对Man(5)底物表现出更高的偏好。来自Mb-0503细胞的GlcNAc- t I对pyridylaminated Man(3)-和Man(5)GlcNAc(2)的K-m和V-max分别为2.15和0.21 mM， 3.4和11.4 nmol/h/mg细胞蛋白。当Man(5)GlcNAc(2)作为受体底物时，四种昆虫细胞系的GlcNAc- t I活性水平在7.5 ~ 14.7 nmol/h/mg细胞蛋白之间，与HepG2细胞相当。证据表明鳞翅目聚焦转移酶依赖于末端n -乙酰氨基葡萄糖的存在。以GlcNAc β 1-2Man α 1-3(Man α 1-6)Man β 1-4GlcNAc β 1-4GlcNAc-pyridylamine为受体，6-乙酰氨基-6-脱氧castanospermine作为β - n -乙酰氨基葡萄糖苷酶抑制剂存在时，GlcNAc- t II活性可以通过高效液相色谱法证明。然而，昆虫细胞中GlcNAc-T II的特异性活性仅为0.02-0.11 nmol/h/mg细胞蛋白，远低于HepG2细胞。
The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)(8)COOH3 and H-3-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a nonradioactive HPLC method using pyridylaminated Man(3)-GlcNAc(2) and Man(5)GlcNAc(2) as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man(3)GlcNAc(2) contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor. Compared to the enzyme from the human hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man(5) substrate. The GlcNAc-T I from Mb-0503 cells had apparent K-m and V-max values for pyridylaminated Man(3)- and Man(5)GlcNAc(2) of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man(5)GlcNAc(2) was used as the acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine. GlcNAc-T II activity could be demonstrated by HPLC using GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc-pyridylamine as the acceptor in the presence of 6-acetamido-6-deoxycastanospermine as an inhibitor of beta-N-acetylglucosaminidase. However, the insect cells exhibited specific activities of GlcNAc-T II of only 0.02-0.11 nmol/h/mg of cell protein, much less than HepG2 cells.