Oligosaccharide substrate interactions with beta-1,4-Gal

寡糖底物与 β-1,4-Gal 的相互作用

• 批准号: 7291793
• 负责人: Pradman K Qasba
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7291793
• 关键词: Oligosaccharide; substrate; interactions; beta; Gal

The oligosaccharide moieties of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. For the biosynthesis of these complex oligosaccharides intricate machineries exists in a cell. Defective glycan synthesis has serious pathological consequences and results in several human diseases. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetro-dimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. Although all beta-4Gal-T-family members, that are responsible for the synthesis of beta-linked Gal moieties in different oligosaccharides, transfer Gal to GlcNAc, each recognizes differently the remaining monosaccharide units of the oligosaccharide to which GlcNAc is attached. The sequence comparison of the human b4Gal-T family members and the structural homology models based on the 3D structure of b4Gal-T1 reveals only a little or no variation in the GlcNAc binding site among the family members, where as the extended oligosaccharide binding region shows significant variations. This indicates that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site we have carried out the crystal structure analysis of the b4Gal-T1-oligosaccharide complexes, enzyme kinetic analysis and MD simulations. Defining the oligosaccharide binding site of b4Gal-T1 by crystal structure investigations of the complexes with the oligosaccharides : By molecular modeling and docking studies we have previously defined the oligosaccharide binding site of b4Gal-T1, the 3D-structure of which has been determined in our laboratory, either in complex with UDP-galactose and Mn2+ion, or in complex with alpha-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009304), or of the mutant Met344His-b4Gal-T1 in complex with chitobiose (see Project # Z01 BC 009305). Examination of the GlcNAc binding site in b4Gal-T1 from the crystal structure reveals an "open canal shaped" extended sugar binding site that lay behind the GlcNAc binding site. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. In the crystal structure of b4Gal-T1-LA-complex, LA binds to this region and therefore LA is expected to compete with the GlcNAc containing oligosaccharides, such as chitobiose. Crystallization of the wild type b4Gal-T1with the acceptor either in the presence or absence of UDP has not been successful. This is mainly due to the absence of the acceptor binding-site in the apo-b4Gal-T1 that exists in the open conformation. The enzyme has been crystallized in the closed conformation, where the acceptor site is present, only when UDP-Gal is bound. Although UDP or the acceptor molecules can induce the essential conformational changes, such complexes have been crystallized thus far only in the presence of LA. Since LA binds to the extended sugar binding site it is not possible to crystallize b4Gal-T1 with the oligosaccharide acceptors in the presence of LA.

糖缀合物的寡糖部分在细胞的几个生物过程中起重要作用，包括糖蛋白的折叠和跨细胞区室的运输。对于这些复杂寡糖的生物合成，细胞中存在复杂的机制。聚糖合成缺陷具有严重的病理后果，并导致几种人类疾病。寡糖部分以高特异性与细胞蛋白结合，并调节糖蛋白的同源和异源二聚化。由于寡糖的构象灵活性，二糖单元的扭转角，特别是围绕1 -6-键的扭转角，以这样的方式进行调整，即寡糖的侧基以促进与蛋白质的结合残基的有利相互作用的方式进行定向。分支寡糖交联蛋白质并产生蛋白质-碳水化合物复合物的无限网络，从而调节各种细胞反应。 尽管负责不同寡糖中β-连接的Gal部分合成的所有β-4Gal-T-家族成员将Gal转移至GlcNAc，但各自不同地识别与GlcNAc连接的寡糖的剩余单糖单元。人b4 Gal-T家族成员的序列比较和基于b4 Gal-T1的3D结构的结构同源性模型揭示了家族成员之间的GlcNAc结合位点只有很少或没有变化，而延伸的寡糖结合区显示出显著的变化。这表明这些酶可能偏好不同的含GlcNAc的寡糖作为其优选的糖受体。为了确定结合位点中寡糖的确切结合模式，我们进行了b4 Gal-T1-寡糖复合物的晶体结构分析、酶动力学分析和MD模拟。 通过与寡糖的复合物的晶体结构研究确定b4 Gal-T1的寡糖结合位点：通过分子模拟和对接研究，我们先前已经确定了b4 Gal-T1的寡糖结合位点，其三维结构已经在我们的实验室中确定，或者与UDP-半乳糖和Mn 2+离子复合，或与α-乳白蛋白和N-乙酰葡糖胺复合（参见项目#Z01 BC 009304），或与壳二糖复合的突变体Met 344 His-b4 Gal-T1（参见项目#Z01 BC 009305）。从晶体结构中检查b4 Gal-T1中的GlcNAc结合位点揭示了位于GlcNAc结合位点后面的“开口管形”延伸的糖结合位点。该位点由来自三个区域的残基形成;残基280至289、残基319至325和残基359至368。在b4 Gal-T1-LA-复合物的晶体结构中，LA结合到该区域，因此预期LA与含有GlcNAc的寡糖（例如壳二糖）竞争。在存在或不存在UDP的情况下，野生型b4 Gal-T1与受体的结晶尚未成功。这主要是由于apo-b4 Gal-T1中不存在以开放构象存在的受体结合位点。只有当UDP-Gal结合时，酶才以封闭构象结晶，其中存在受体位点。虽然UDP或受体分子可以诱导基本的构象变化，这种复合物已结晶到目前为止，只有在LA的存在下。由于LA与延伸的糖结合位点结合，因此在LA存在下不可能用寡糖受体使b4 Gal-T1结晶。