Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.
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中国仓鼠卵巢细胞中高尔基定位酶 α-甘露糖苷酶 IIx 的过度表达导致 N-聚糖加工途径中的六甘露糖基-N-乙酰壳二糖转化为四甘露糖基-N-乙酰壳二糖。

DOI:
10.1046/j.1432-1327.2001.01992.x
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发表时间:
2001
期刊:
European journal of biochemistry
影响因子:
--
通讯作者:
Fukuda,MN
Fukuda,MN
中科院分区:
--
文献类型:
--
作者:
Oh-Eda,M;Nakagawa,H;Akama,TO;Lowitz,K;Misago,M;Moremen,KW;Fukuda,MN

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高尔基体α-甘露糖苷酶II是一种在N-聚糖生物合成过程中将中间寡糖Gn1M5Gn2加工成Gn1M3Gn2的酶。此前,我们分离了编码与α-甘露糖苷酶II同源的蛋白质的cDNA,并将其命名为α-甘露糖苷酶 IIx。在这里,我们通过免疫细胞化学证明 α-甘露糖苷酶 IIx 存在于 HeLa 细胞的高尔基体中。当与 α-甘露糖苷酶 II 共表达时,α-甘露糖苷酶 IIx 与 α-甘露糖苷酶 II 在 COS 细胞中共定位。 α-甘露糖苷酶 IIx 催化结构域的蛋白质 A 融合物会水解合成底物 4-伞形基-α-d-甘露糖苷,并且这种活性会被苦马豆素抑制。 [3H]葡萄糖胺标记的过表达α-甘露糖苷酶IIx的中国仓鼠卵巢细胞显示M6Gn2减少和M4Gn2积累。结构分析确定M4Gn2为Manα1→6(Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc。结果表明,在 N-聚糖加工过程中,α-甘露糖苷酶 IIx 水解 [(Manα1→6)(Manα1→3)Manα1→6](Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc 的两个外周 Manα1→6 和 Manα1→3 残基。
Golgi α‐mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn1M5Gn2to Gn1M3Gn2during biosynthesis of N‐glycans. Previously, we isolated a cDNA encoding a protein homologous to α‐mannosidase II and designated it α‐mannosidase IIx. Here, we show by immunocytochemistry that α‐mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with α‐mannosidase II, α‐mannosidase IIx colocalizes with α‐mannosidase II in COS cells. A protein A fusion of the catalytic domain of α‐mannosidase IIx hydrolyzes a synthetic substrate, 4‐umbelliferyl‐α‐d‐mannoside, and this activity is inhibited by swainsonine. [3H]glucosamine‐labeled Chinese hamster ovary cells overexpressing α‐mannosidase IIx show a reduction of M6Gn2and an accumulation of M4Gn2.Structural analysis identified M4Gn2to be Manα1→6(Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc. The results suggest that α‐mannosidase IIx hydrolyzes two peripheral Manα1→6 and Manα1→3 residues from [(Manα1→6)(Manα1→3)Manα1→6](Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, during N‐glycan processing.
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