Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein
Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein
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DOI：
10.1016/j.bbapap.2011.04.004
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发表时间：
2011-07-01
影响因子：
3.2
通讯作者：
Cheung, Marian C.
中科院分区：
文献类型：
--
作者：
Albers, John J.;Day, Joseph R.;Cheung, Marian C.
血浆磷脂转运蛋白（PLTP）在脂质和脂蛋白代谢中起关键作用。它有六个潜在的N-糖基化位点。为了研究这些位点对PLTP分泌和活性的影响，通过定点诱变制备了六种含有丝氨酸至丙氨酸点突变的变体，并在中国仓鼠卵巢Flp-In细胞中表达。通过蛋白质印迹，六个PLTP突变体中的每一个的表观大小略小于野生型的表观大小，表明所有六个位点都被糖基化或利用。在每个N-糖基化位点的碳水化合物的大小范围为3.14至4.2 kDa。位点特异性N-糖基化去除对PLTP分泌的影响从适度增强（15%和60%）或基本无影响到分泌减少（8%、14%和32%）不等。与野生型相比，在六个糖基化位点中的任何一个处去除N-糖基化导致PLTP活性显著降低35-78%，并且PLTP比活性显著降低29-80%。这些数据表明，虽然没有单一的N-连接的碳水化合物链是分泌或活性的要求，但碳水化合物链的去除对PLTP的细胞分泌及其磷脂转移活性具有定量影响。(C)2011 Elsevier B. V.保留所有权利。
The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2 kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity. (C) 2011 Elsevier B.V. All rights reserved.