Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans.
Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans.
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第二种番茄 α1,3/4-岩藻糖苷酶 (α-Fucase Sl-2) 的分子特征，该酶是糖基水解酶家族 29 的成员，对植物 N-聚糖中的核心 α1,3-岩藻糖基残基具有活性。
DOI：
10.1093/jb/mvy029
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Kimura Y.
中科院分区：
文献类型：
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作者：
Rahman Z;Tsujimori Y;Maeda M;Hossain A;Ishimizu T;Kimura Y.
在先前的研究中，我们对番茄（Solyc 03 g 006980）基因编码的α1，3/4-岩藻糖苷酶（α-Fuc'ase Sl-1）进行了分子鉴定，表明α-Fuc'ase Sl-1参与了含Lea表位的N-聚糖的周转。在本研究中，我们鉴定了另一个编码α1，3/4-岩藻糖苷酶（α-Fuc'ase S1 -2）的番茄基因（Solyc 11 g 069010），该基因也对含有Lea表位的复杂N型聚糖具有活性。利用杆状病毒-昆虫细胞表达系统表达了带有抗FLAG标签的α-Fuc'ase S1 -2，SDS-PAGE显示表达产物（rFuc'ase S1 -2）为65 kDa蛋白，最适pH为5.0左右。与rFuc'ase Sl-1类似，rFuc'ase Sl-2水解LNFP III上的非还原末端α1，3-岩藻糖残基和植物复合N型聚糖上Lea表位的α1，4-岩藻糖残基，但不水解Manβ1-4GlcNAcβ1-4（Fucα1-3）GlcNAc或Fucα1-3GlcNAc上的核心α1，3-岩藻糖残基。然而，我们发现α-Fuc'酶Sl-1和Sl-2都对GlcNAcβ1-4（Fucα1-3）GlcNAc上的α1，3-岩藻糖残基具有特异性活性，表明与植物特异性N-聚糖的核心三糖部分的近端GlcNAc残基连接的未取代的β-GlcNAc必须是α-Fuc'酶活性的先决条件。α-Fuc'ase Sl-2催化位点的3D建模结构表明Asp 192和Glu 236可能对于结合α1，3/4岩藻糖残基很重要。
In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1, 3/4-fucosidase (α-Fuc’ase Sl-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc’ase Sl-1 is involved in the turnover of Leaepitope-containingN-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1, 3/4-fucosidase (α-Fuc’ase Sl-2), which is also active toward the complex typeN-glycans containing Leaepitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc’ase Sl-2 with anti-FLAG tag, and the expression product (rFuc’ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc’ase Sl-1, rFuc’ase Sl-2 hydrolyzed the non-reducing terminal α1, 3-fucose residue on LNFP III and α1, 4-fucose residues of Leaepitopes on plant complex typeN-glycans, but not the core α1, 3-fucose residue on Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc’ases Sl-1 and Sl-2 were specifically active toward α1, 3-fucose residue on GlcNAcβ1-4(Fucα1-3)GlcNAc, indicating that the non-substituted β-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specificN-glycans must be a pre-requisite for α-Fuc’ase activity. A 3 D modelled structure of the catalytic sites of α-Fuc’ase Sl-2 suggested that Asp192and Glu236may be important for binding to the α1, 3/4 fucose residue.