Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9.
Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9.
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来自鳞翅目昆虫细胞系 Sf9 的 α1,2-甘露糖苷酶 cDNA 的分离和表征。
DOI：
10.1093/glycob/7.3.433
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发表时间：
1997
期刊：
影响因子：
4.3
通讯作者：
Jarvis,DL
中科院分区：
文献类型：
--
作者：
Kawar,Z;Herscovics,A;Jarvis,DL
作为我们持续努力表征鳞翅目昆虫细胞 N-糖基化途径的一部分，我们从 Sf9 cDNA 文库中分离出了 α1,2-甘露糖苷酶同源物。该 cDNA 包含一个开放阅读框，编码 670 个氨基酸的蛋白质，计算分子量为 75,225 Da。该蛋白质具有两个潜在的 N-糖基化位点、两个共有的钙结合序列，预计是具有 22 个氨基酸跨膜结构域（残基 31-52）的 II 型整合膜蛋白。该蛋白质的氨基酸序列与果蝇、人类、小鼠和酵母 α1,2-甘露糖苷酶有 35-47% 的同一性。通过 Sf9 mRNA 的 Northern 印迹分析检测到大约 6 KB 的转录物。用 α1,2-甘露糖苷酶基因的无内含子片段探测的基因组 Southern 印迹表明，Sf9 基因组中至少有两个该基因的拷贝或交叉杂交变体。使用重组杆状病毒体内表达 cDNA 产生了从 [3H]Man9GlcNAc 释放 [3H] 甘露糖的蛋白质。该活性需要钙，但不需要镁，并且被 1-脱氧甘露尻霉素抑制。这些结果表明Sf9细胞编码并表达α1,2-甘露糖苷酶，其性质与其他真核加工α1,2-甘露糖苷酶的性质相似。
As part of our ongoing efforts to characterize the N-glycosylation pathway of lepidopteran insect cells, we have isolated an α1,2-mannosidase homolog from an Sf9 cDNA library. This cDNA contains an open reading frame which encodes a 670 amino acid protein with a calculated molecular weight of 75,225 Da This protein has two potential N-giycosylation sites, two consensus calcium binding sequences, and is predicted to be a type II integral membrane protein with a 22 amino acid transmembrane domain (residues 31–52). The amino acid sequence of this protein is 35–47% identical toDrosophila, human, murine, and yeast α1,2-mannosidases. A transcript of approximately 6 kilobases was detected by Northern blot analysis of Sf9 mRNA. Genormic Southern blots probed with an intron-free fragment of the α1,2-mannosidase gene indicated that there are at least two copies or cross-hybridizing variants of this gene in the Sf9 genome.In vivoexpression of the cDNA using a recombinant baculovirus produced a protein that released [3H]mannose from [3H]Man9GlcNAc. This activity required calcium, but not magnesium, and was inhibited by 1-deoxymannojirimycin. These results indicate that Sf9 cells encode and express an α1,2-mannosidase with properties similar to those of other eukaryotic processhg α1,2-mannosidases.