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着重要作用，包括糖蛋白在跨细胞室中的折叠和转运。对于这些复杂的寡糖，复杂的机器存在于细胞中。有缺陷的聚糖合成具有严重的病理后果，并导致多种人类疾病。寡糖部分与具有高特异性的细胞蛋白结合，并调节糖蛋白的同型和乙酸二聚体。由于寡糖的构象柔韧性，二糖单元的扭转角，尤其是在1-6-链接附近，以这样的方式进行调整，以使寡糖的侧基团体以一种促进与蛋白质结合残基的有利相互作用的方式进行了定向。分支的寡糖交联蛋白，并产生无限的蛋白质 - 碳水化合物复合物网络，从而调节各种细胞反应。 尽管所有负责在不同的寡糖中合成β-链接的gal部分的β-4GAL-T-family成员，都将GAL转移到GlcNAC中，但每个人都以不同的方式识别寡糖的剩余单糖单位单位，而GlcNAC与GlcNAC相连。人类B4GAL-T家族成员和基于B4GAL-T1的3D结构的结构同源模型的序列比较显示，家族成员之间GlcNAC结合位点的差异或没有差异，在这些位置中，由于扩展的寡糖结合区域显示出显着的变化。这表明这些酶可能偏爱含有寡糖的GlcNAC作为其首选糖受体。为了确定在结合位点寡糖的确切结合模式，我们已经对B4GAL-T1-寡糖复合物，酶动力学分析和MD模拟进行了晶体结构分析。 通过与寡糖的晶体结构研究来定义B4GAL-T1的寡糖结合位点：通过分子建模和对接研究，我们以前已经定义了B4GAL-T1的寡糖结合位点，该结构在我们的实验室中与UDP-GALSCARTOSE和MN的3D结构一起确定α-乳糖蛋白和N-乙酰基葡萄糖胺（请参见项目＃Z01 BC 009304），或与Chitobiose的复合物中的突变体MET344HIS-B4GAL-T1（请参阅Project＃Z01 BC 009305）。从晶体结构中检查B4GAL-T1中的GlcNAC结合位点，发现一个“开放的管形”扩展的糖结合位点，该位点位于GlcNAC结合位点后面。该地点由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B4GAL-T1-LA-COMPERX的晶体结构中，LA与该区域结合，因此LA有望与含有GlcNAC的含寡糖竞争，例如Chitobiose。在存在或不存在UDP的情况下，野生型B4GAL-T1的结晶尚未成功。这主要是由于在开放构象中存在的APO-B4GAL-T1中没有受体结合位置。仅当UDP-GAL结合时，该酶已经在存在受体位点的闭合构象中结晶。尽管UDP或受体分子可以诱导必要的构象变化，但到目前为止，此类复合物仅在LA存在下才结晶。由于LA与扩展的糖结合位点结合，因此在LA存在的情况下，不可能用寡糖受体结晶B4GAL-T1。