Oligosaccharide substrate interactions with beta-1,4-Ga

寡糖底物与 beta-1,4-Ga 的相互作用

基本信息

项目摘要

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. In a previous study we showed (see Project # Z01 BC 009305) that when residue Met344 in bovine b4Gal-T1 is mutated to histidine, the mutant M344H in the presence of Mn2+ and UDP-hexanolamine readily changes to the closed conformation that creates the acceptor binding site, thereby facilitating the structural analysis of the enzyme with various oligosaccharide acceptors. The branch specificity of human b4-Gal-T1 was investigated with the mutant human Met344His-b4Gal-T1 that was crystallized in complex with UDP-hexanolamine, Mn2+, and different trisaccharides. The following trisaccharides of the N-Glycan moiety were used: GlcNAcb1-2Mana1-6Man (1-6-arm), GlcNAc b1-2Mana1-2Man (1-2-arm), GlcNAcb1-4Mana1-3Man (triantennary), GlcNAcb1-2Mana1-3Man (1-3-arm), and GlcNAcb1-4GlcNAcb1-4GlcNAc (chitotriose). Crystal data was collected at 2.0 for all the complexes, except for the complex with triantennary trisaccharide (1.9 ). In all the structures the mutant human b4Gal-T1-Met344His was found to be in the closed conformation with the trisaccharides bound to it. The electron density for the core Man residue (the third residue from the non-reducing end) among the trisaccharides bound to b4Gal-T1 was clearly observed in the 1-6-arm trisaccharide where the core mannose rests over Tyr286 making hydrophobic interactions. In contrast, the core Man residue of the 1-3-arm trisaccharide possesses vague electron density with partial occupancy. The enzyme kinetic analysis also indicated a preferential binding of the 1-6-arm trisaccharide to the mutant enzyme. The Km of GlcNAcb1-2Mana1-6Man is approximately 10-fold lower than the Km for GlcNAcb1-2Mana1-3Man and GlcNAcb1-4Mana1-3Man, and 22-fold lower than the Km for GlcNAc b1-2Mana1-2Man and chitotriose. However, the catalytic activity of the Met344His mutant, in the presence of Mn2+, with the trisaccharide GlcNAcb1,2Mana1-6Man was inhibited above 0.1 mM concentrations, whereas much higher concentrations (1-2 mM) for the other four trisaccharides where needed to inhibit the catalytic activity. Since b4Gal-T1 binds strongly to GlcNAcb1-2Mana1-6Man and the turnover number, kcat, is hardly reduced, the catalytic efficiency (kcat/Km) of the enzyme with this trisaccharide is high compared to the other trisaccharides used in this study. Based on the structural and kinetic analysis it is proposed that b4Gal-T1 may prefer to transfer Gal to 1-3-arm in a bi- or tri- or tetra-antennary oligosaccharide while as it may act as lectin when concentration of glycan with 1-6 arm is higher than the glycan with 1-3 arm.
糖复合物的寡糖部分在细胞的多个生物过程中发挥重要作用,包括糖蛋白跨细胞区室的折叠和运输。为了生物合成这些复杂的寡糖,细胞中存在着复杂的机器。聚糖合成缺陷会产生严重的病理后果,并导致多种人类疾病。寡糖部分以高特异性结合细胞蛋白质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,尤其是1-6-键周围的扭转角,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自行定向。支链寡糖交联蛋白质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。 尽管负责不同寡糖中β-连接Gal部分合成的所有β-4Gal-T-家族成员将Gal转移至GlcNAc,但每个成员对GlcNAc所附着的寡糖的剩余单糖单元的识别不同。人类b4Gal-T家族成员和基于b4Gal-T1 3D结构的结构同源性模型的序列比较显示,家族成员之间的GlcNAc结合位点只有很小的变化或没有变化,而延伸的寡糖结合区则显示出显着的变化。这表明这些酶可能更喜欢不同的含有寡糖的 GlcNAc 作为它们的首选糖受体。为了确定寡糖在结合位点的确切结合模式,我们对 b4Gal-T1-寡糖复合物进行了晶体结构分析、酶动力学分析和 MD 模拟。 通过与寡糖复合物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,我们之前已经定义了 b4Gal-T1 的寡糖结合位点,其 3D 结构已在我们的实验室中确定,无论是与 UDP-半乳糖和 Mn2+ 离子复合,还是与 UDP-半乳糖和 Mn2+ 离子复合。 α-乳清蛋白和 N-乙酰氨基葡萄糖(参见项目号 Z01 BC 009304),或与壳二糖复合的突变体 Met344His-b4Gal-T1(参见项目号 Z01 BC 009305)。从晶体结构检查 b4Gal-T1 中的 GlcNAc 结合位点揭示了位于 GlcNAc 结合位点后面的“开放运河形”延伸糖结合位点。该位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-复合物的晶体结构中,LA与该区域结合,因此预计LA会与含有GlcNAc的寡糖(例如壳二糖)竞争。无论存在或不存在UDP,野生型b4Gal-T1与受体的结晶均未成功。这主要是由于开放构象中存在的apo-b4Gal-T1 中缺乏受体结合位点。仅当 UDP-Gal 结合时,酶才会以闭合构象结晶,其中存在受体位点。尽管 UDP 或受体分子可以诱导必要的构象变化,但迄今为止,此类复合物仅在 LA 存在的情况下才能结晶。由于 LA 与延伸的糖结合位点结合,因此在 LA 存在的情况下不可能使 b4Gal-T1 与寡糖受体结晶。在之前的研究中,我们表明(参见项目号 Z01 BC 009305),当牛 b4Gal-T1 中的残基 Met344 突变为组氨酸时,突变体 M344H 在 Mn2+ 和 UDP-己醇胺存在下很容易转变为创建受体结合位点的闭合构象,从而促进酶与各种寡糖的结构分析 接受者。使用与 UDP-己醇胺、Mn2+ 和不同三糖形成复合物结晶的突变型人 Met344His-b4Gal-T1 研究了人 b4-Gal-T1 的分支特异性。使用以下 N-聚糖部分的三糖:GlcNAcb1-2Mana1-6Man(1-6 臂)、GlcNAc b1-2Mana1-2Man(1-2 臂)、GlcNAcb1-4Mana1-3Man(三触角)、GlcNAcb1-2Mana1-3Man(1-3 臂)和 GlcNAcb1-4GlcNAcb1-4GlcNAc(壳三糖)。除具有三触角三糖的复合物(1.9)外,所有复合物的晶体数据均在 2.0 处收集。在所有结构中,发现突变型人b4Gal-T1-Met344His 均处于与其结合的三糖的闭合构象。在 1-6 臂三糖中可以清楚地观察到与 b4Gal-T1 结合的三糖中核心 Man 残基(非还原端的第三个残基)的电子密度,其中核心甘露糖位于 Tyr286 上,形成疏水相互作用。相反,1-3臂三糖的核心Man残基具有模糊的电子密度和部分占据。酶动力学分析还表明1-6臂三糖优先结合突变酶。 GlcNAcb1-2Mana1-6Man 的 Km 比 GlcNAcb1-2Mana1-3Man 和 GlcNAcb1-4Mana1-3Man 的 Km 低约 10 倍,比 GlcNAc b1-2Mana1-2Man 和壳三糖的 Km 低 22 倍。然而,在 Mn2+ 存在的情况下,三糖 GlcNAcb1,2Mana1-6Man 的 Met344His 突变体的催化活性在浓度高于 0.1 mM 时受到抑制,而其他四种三糖则需要更高的浓度 (1-2 mM) 来抑制催化活性。由于 b4Gal-T1 与 GlcNAcb1-2Mana1-6Man 强烈结合,并且周转数 kcat 几乎不降低,因此与本研究中使用的其他三糖相比,使用该三糖的酶的催化效率 (kcat/Km) 较高。基于结构和动力学分析,推测b4Gal-T1可能更愿意将Gal转移到双触角、三触角或四触角寡糖中的1-3臂,而当1-6臂聚糖浓度高于1-3臂聚糖浓度时,b4Gal-T1可能充当凝集素。

项目成果

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Pradman K Qasba其他文献

Pradman K Qasba的其他文献

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{{ truncateString('Pradman K Qasba', 18)}}的其他基金

Structural Studies and 3D Structure Determination of Recombinant <FONT FACE=symb
重组体的结构研究和 3D 结构测定 <FONT FACE=symb
  • 批准号:
    6433157
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Oligosaccharide Interactions with Proteins
低聚糖与蛋白质的相互作用
  • 批准号:
    6559116
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Oligosaccharide substrate interactions with beta-1,4-Ga
寡糖底物与 beta-1,4-Ga 的相互作用
  • 批准号:
    6944635
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Utilizing Glycosyltransferases for Bioconjugation
利用糖基转移酶进行生物共轭
  • 批准号:
    8552799
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Detection of Specific Glycan Moieties on the Cell Surface
细胞表面特定聚糖部分的检测
  • 批准号:
    8349512
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Oligosaccharide Substrate and Inhibitor Interactions with beta-1,4-Gal-T1
寡糖底物和抑制剂与 β-1,4-Gal-T1 的相互作用
  • 批准号:
    7965207
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Oligosaccharide Substrate and Inhibitor Interactions with beta-1,4-Gal-T1
寡糖底物和抑制剂与 β-1,4-Gal-T1 的相互作用
  • 批准号:
    7732974
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Using Glycosyltransferases for Conjugation of Single-Chain Antibodies and Lipids
使用糖基转移酶缀合单链抗体和脂质
  • 批准号:
    8157471
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
PRINCIPALS OF CONFORMATIONAL ANALYSIS OF CARBOHYDRATES - A TEXT BOOK
碳水化合物构象分析原理 - 教科书
  • 批准号:
    6289310
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:

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