The effects of N-glycosylation sites and the N-terminal region on the biological function of β1,3-N-acetylglucosaminyltransferase 2 and its secretion
The effects of N-glycosylation sites and the N-terminal region on the biological function of β1,3-N-acetylglucosaminyltransferase 2 and its secretion
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DOI：
10.1016/j.bbrc.2005.02.033
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发表时间：
2005-04-08
影响因子：
3.1
通讯作者：
Park, EY
中科院分区：
文献类型：
--
作者：
Kato, T;Suzuki, M;Park, EY
人β 1，3-N-乙酰葡糖胺转移酶2（β 3GnT 2）被认为是一种延伸聚乳糖胺受体链的酶，但其功能和结构分析尚不清楚。为了深入了解β 3GnT 2的结构，使用添加抑制剂、潜在N-糖基化位点的定点诱变以及在杆状病毒表达系统中使用与GFP（uv）的融合蛋白缺失其N-末端区域来评估N-糖基化对其生物学功能的影响。发现5个潜在的N-糖基化位点中有4个被占据，用N-糖基化抑制剂衣霉素处理后，其生物学功能和分泌受到抑制。Asn 219的N-糖基化是β 3GnT活性所必需的;此外，Asn 127和Asn 219的N-糖基化对于有效的蛋白分泌是至关重要的。当Ser 221被Thr取代时，融合蛋白表达为单一条带，表明表达的融合蛋白的双带是由于Asn 219处糖基化的异质性。由氨基酸82-397（GFP（uv）-β 3GnT 2 Delta 83）组成的截短蛋白质（其在Asn 79处缺少一个N-糖基化位点和糖基转移酶的茎区）仅表达为小形式并且不显示β 3GnT活性。这些结果表明，在Asn 219的N-糖基化位点，这是保守的整个β 1，3-糖基转移酶家族，是必不可少的，不仅就其生物学功能，而且其分泌。N-末端区域属于糖基转移酶的茎区，可能对活性蛋白质结构也很重要。(c)2005年爱思唯尔公司All rights reserved.
Human beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta 3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta 3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta 3GnT2 Delta 83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta 3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta 1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure. (c) 2005 Elsevier Inc. All rights reserved.