Wälti Martin Andreas, Walser Piers Jamie, Thore Stéphane, Grünler Anke, Bednar Michaela, Künzler Markus, Aebi Markus
Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Str. 10, CH-8093 Zürich, Switzerland.
J Mol Biol. 2008 May 23;379(1):146-59. doi: 10.1016/j.jmb.2008.03.062. Epub 2008 Apr 3.
Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.
基因组测序工作的最新进展揭示了大量新的假定凝集素。其中，许多半乳糖凝集素相关蛋白在真核生物超界的各个角落都有发现，其特点是有许多保守残基，但有趣的是缺少关键氨基酸。在此，我们对一种具有代表性的蛋白进行了结构和生化分析，该蛋白是在墨汁鬼伞（Coprinopsis cinerea）中发现的半乳糖凝集素相关凝集素CGL3。除了一个已知参与半乳糖凝集素中β-半乳糖苷结合的保守残基外，该蛋白包含其他所有保守残基。在半乳糖凝集素中严格保守的一个色氨酸残基在CGL3中变为精氨酸（R81）。因此，该半乳糖凝集素相关蛋白不能结合乳糖。聚糖阵列筛选显示，CGL3对β1-4连接的N-乙酰葡糖胺（壳寡糖）和GalNAcβ1-4GlcNAc（LacdiNAc）的寡聚物表现出偏好。使用等温滴定量热法对这种新型凝集素的碳水化合物结合亲和力进行了定量，并通过X射线晶体学研究了其不涉及任何芳香族氨基酸残基的壳寡糖配位模式。利用结构信息改变了CGL3的碳水化合物结合特异性和底物亲和力。通过将这个精氨酸替换为色氨酸残基（R81W），证明了残基R81在决定碳水化合物结合特异性方面的重要性。这一单氨基酸变化导致一种凝集素不能结合壳寡糖，但获得了乳糖结合能力。我们的结果表明，与豆科凝集素折叠类似，半乳糖凝集素折叠代表了一个保守的结构框架，在该框架上通过结合位点的少量改变就可以嫁接出显著改变的特异性，因此，许多后生动物半乳糖凝集素相关蛋白可能代表具有新型碳水化合物结合特异性的凝集素。
