Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an α1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide
Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an α1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide
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DOI：
10.1042/bj20091121
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发表时间：
2010-03-01
影响因子：
4.1
通讯作者：
Lehle, Ludwig
中科院分区：
文献类型：
--
作者：
Absmanner, Birgit;Schmeiser, Verena;Lehle, Ludwig
天冬酰胺连接的多糖的生物合成是以进化保守的方式进行的，独特的脂联低聚糖前体GLC(3)Man(9)GlcNAc-PP-Dol在内质网(ER)组装。在本研究中，我们将酵母中的海藻I描述为一种甘露糖基转移酶，催化两个α-1，2-甘露糖残基从GDP-甘露糖顺序转移到Man(3)GlcNAc(2)-PP-Dol，然后转移到Man(4)GlcNAc(2)-PP-Dol，在内质网的胞液侧形成Man(3)GlcNAc(2)-PP-Dol中间体，然后翻转到管腔侧。据预测，ALG11含有三个疏水性跨膜螺旋。使用ALG11拓扑报告融合结构，我们表明只有N-末端结构域满足这一标准。令人惊讶的是，这个结构域可以在不干扰糖基转移酶功能和膜结合的情况下被删除。这也表明另外两个疏水结构域有助于ER的定位，但不是以跨膜的方式。通过定点突变，我们研究了对转移酶活性重要的氨基酸。我们证明了EX7E基序中的第一个谷氨酸残基比第二个谷氨酸残基更为关键，并且只有当两个谷氨酸残基被交换时，或者当第一个谷氨酸残基的突变与基序中另一个氨基酸的替换相结合时，才会发生AL11功能的丧失。这表明EX7E中的扰动并不局限于第二个谷氨酸残基。此外，Gly(85)和Gly(87)作为潜在的柔性环的一部分，位于富含甘氨酸的结构域中，被发现是阿尔法11功能所必需的。类似地，保守的赖氨酸残基Lys(319)被认为对活性很重要，它可能参与糖基供体的磷酸结合。
The biosynthesis of asparagine-linked glycans Occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked olicyosaccharide precursor Glc(3)Man(9)GlcNAc-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize AlgI I from yeast as a mannosyltransferase catalysing the sequential transfer of two alpha 1,2-linked mannose residues from GDP-mannose to Man(3)GlcNAc(2)-PP-Dol and subsequently to Man(4)GlcNAc(2)-PP-Dol forming the Man(3)GlcNac(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic trans membrane-spanning helices. Using Alg11 topology reporter fusion constructs, we show that only the N-terminal domain fulfils this criterion. Surprisingly, this domain can be deleted without disturbing glycosyltransferase function and membrane association. indicating also that the other two hydrophobic domains contribute to ER localization, but in a non-trans membrane manner. By site-directed mutagenesis we investigated amino acids important for transferase activity. We demonstrate that the first glutamate residue in the EX7E motif, conserved in a variety of glycosyltransferases, is more critical than the second, and loss of Alg11 function occurs only when both glutamate residues are exchanged, or when the mutation of the first glutamate residue is combined with replacement of another amino acid in the motif. This indicates that perturbations in EX7E are not restricted to the second glutamate residue. Moreover, Gly(85) and Gly(87), within a glycine-rich domain as part of a potential flexible loop, were found to be required for Alg11 function. Similarly, a conserved lysine residue, Lys(319), was identified as being important for activity, which could be involved in the binding of the phosphate of the glycosyl donor.