Purification, cDNA Cloning, and Expression of GDP-l-Fuc:Asn-linked GlcNAc α1,3-Fucosyltransferase from Mung Beans*
Purification, cDNA Cloning, and Expression of GDP-l-Fuc:Asn-linked GlcNAc α1,3-Fucosyltransferase from Mung Beans*
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绿豆中 GDP-l-Fuc:Asn 连接的 GlcNAc α1,3-岩藻糖基转移酶的纯化、cDNA 克隆和表达*
DOI：
10.1074/jbc.274.31.21830
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
F. Altmann
中科院分区：
文献类型：
--
作者：
Haralt Leiter;J. Mucha;E. Staudacher;R. Grimm;J. Glössl;F. Altmann
Substitution of the asparagine-linked GlcNAc by α1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core α1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial α1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn, GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAcβ1-Asn, and GlcNAcβ1–2Manα1–3[Manα1–3(Manα1–6)Manα1–6]Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core α1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.
人类 α 1-->3 岩藻糖基转移酶的结构功能分析。
DOI：
10.1074/jbc.270.14.8145
发表时间：
1995
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Holmes,EH;Xu,Z;Sherwood,AL;Macher,BA
通讯作者：
Macher,BA