Structure-Function Studies and Design of Novel Glycosyltransferases

新型糖基转移酶的结构功能研究和设计

• 批准号: 7965164
• 负责人: Pradman K Qasba
• 金额: $ 25.38万
• 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/7965164
• 关键词: Affinity; Amino Acid Sequence; Amino Acids; Binding; Binding Sites; Biochemical; Biological Process; C-terminal; Carbohydrates; Carbon; Catalytic Domain; Categories; Cattle; Cell Adhesion; Cell Communication; Cells; Cellular biology; Chemicals; Cleaved cell; Complex; Cytoplasmic Tail; Detection; Dimerization; Disaccharides; Disease; Drosophila genus; Drug Delivery Systems; Ehlers-Danlos Syndrome; Enzymes; Escherichia coli; Eukaryotic Cell; Exhibits; Face; Family; Fibroblasts; Galactose; Galactosyltransferases; Galectin 1; Genes; Glucose; Glycoconjugates; Glycolipids; Glycopeptides; Glycoprotein 6-alpha-L-fucosyltransferase; Glycoproteins; Golgi Apparatus; Growth; Homo; Homologous Gene; Human; Immune; In Vitro; Inclusion Bodies; Invertebrates; Ions; Laboratories; Lactose; Lactose Synthase; Lead; Lectin; Link; Lipids; Malignant Neoplasms; Mammary gland; Membrane; Membrane Proteins; Metal Ion Binding; Metals; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Monosaccharides; Mutate; Mutation; N-Acetylgalactosaminyltransferases; N-terminal; Neutrophil Infiltration; Nucleotides; Oligosaccharides; Pattern; Peptide Hydrolases; Peptides; Play; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides; Positioning Attribute; Procedures; Production; Proteins; Proteoglycan; Reaction; Recombinant Proteins; Research Design; Rheumatoid Arthritis; Role; SH3 Domains; Side; Site; Skin; Solubility; Specificity; Structure; Substrate Specificity; Time; Tissues; Uridine Diphosphate Sugars; Work; Xylose; acquired immunity; alpha Lactalbumin; base; carbohydrate binding protein; cellular development; cofactor; crosslink; design; flexibility; gene cloning; glycosylation; glycosyltransferase; human disease; hydroxyl group; in vivo; inhibitor/antagonist; interest; knockout gene; link protein; member; mutant; nanoparticle; novel; pathogen; polypeptide; protein aggregate; protein folding; protein protein interaction; response; stem; sugar; sugar nucleotide; tool; tumorigenesis; vector

<P><i><b> Structure and Function of Glycosyltransferases: </i></b> To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown following:</p><P><i><b> (I) Glycosyltransferases have flexible loop(s) in the vicinity of their catalytic pocket which undergo conformational changes upon donor substrate binding and create the acceptor binding site: (II) In the metal-ion dependent enzymes, the metal ion binding site is generally at the amino terminal hinge region of the flexible loop: (III) Glycosyltransferases interact with the add-on domains: </i></b> To diversify the catalytic activity towards less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of glycosyltransferases either interact (1) with an additional protein, or have acquired add-on domains at the C-terminus or acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme, the b4Gal-T1, after conformational changes in the flexible loops to a closed conformation, interacts with a mammary gland-specific protein, alpha-lactalbumin (LA) at its carboxyl terminal end, changing the acceptor specificity of the enzyme towards less preferred acceptor glucose. LA protein, although not linked to b4Gal-T1, acts as an add-on domain. Several other glycosyltransferases have been shown or suggested to require an activating protein. In contrast to two interacting proteins, the catalytic domains of polypeptide a-N-Acetylgalactosaminyltransferases (ppGalNAc-Ts) have a lectin domain that is linked to at the C-terminus of the catalytic domain via a linker region and determines the specificity towards a peptide or a glycopeptide. The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, while the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize O-a-GalNAc moiety on the glycopeptide. Also in this category is the alpha-1,6-Fucosyltransferase (FUT8), where an SH3 domain has been identified that is linked at the C-terminus of the catalytic domain.</p> <P><i><b> (IV) A few residues in the catalytic pocket determine the donor sugar specificity of glycosyltransferases: Role of a single amino acid in the evolutionary divergence of invertebrate and vertebrate glycoconjugates: (a) Mutations in catalytic pocket of b4Gal-T1 change its donor specificity: </i></b> Based on the structural information, we have previously shown, that the residue Tyr/Phe289 in the catalytic pocket of b4Gal-T1, which is conserved among all vertebrate homologs, when mutated to Leu or Ile broadens the donor substrate specificity of the enzyme to 2substituants of galactose i.e., GalNAc or 2-keto-galactose or 2-azido-galactose. (see Project # Z01 BC 010742). In invertebrates in the b4Gal-T homologs there is an Ile residue at the corresponding position of Tyr and they are b4GalNAc-T enzymes. Mutation of the Ile residue to Tyr in Drosophila b4GalNAc-T1 converts the enzyme to a b4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold.<i><b>(b) Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) broadens the donor specificity: </i></b> We have mutated bovine a1,3-galactosyltransferse (a3Gal-T) enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives by mutating the sugar donor-binding residues at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant a3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants 280SGG282 and 280AGG282 with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of glycoprotein, thus enabling the detection of LacNAc moiety by a chemiluminescence method. This makes it possible to use these mutants, (1) for the detection of alterations in the glycosylation patterns in many pathological states, such as cancers and rheumatoid arthritis, and (2) in the glycoconjugation and assembly of nano-particles for the targeted drug delivery of bioactive-agents.<i><b> (V) The N-acetyl group of the donor sugar is generally embedded in a hydrophobic pocket of the enzyme.</i></b> In both mutant enzymes,Y289L-b4Gal-T1 and SGG-a3Gal-T, the N-acetyl moiety of the donor sugar GalNAc, is embedded in a hydrophobic pocket that allows the substitution of this moiety by CH2-CO-CH3 group. This acts as a chemical handle allowing conjugating with an amino-oxy group of a linking molecule.</p><P><i><b> Galectin -1 as a fusion partner for the production of soluble and folded beta-1, 4- Galactosyltransferase-T7 in E. coli: </i></b> The expression of recombinant proteins in soluble and active form in E. coli often leads to aggregated proteins known as inclusion bodies. Modifying the bacterial growth conditions can sometimes solve the aggregation problem. Although we have developed an in vitro folding procedure that in many cases helps to fold the proteins from inclusion bodies, e.g., b4Gal-T1 or ppGalNAc-Ts, it nevertheless does not work with all the proteins. To date, the best available tool has been the use of several different fusion tags including the carbohydrate-binding protein, MBP that enhance the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. Here we show for the first time that another carbohydrate-binding protein galectin-1 can function as a fusion partner to produce soluble folded recombinant protein in E. coli. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for galectin-1, and a multi-cloning site where a cloned gene is inserted. The unique protease cleavage site allows the protein of interest to be cleaved from galectin-1 after lactose affinity column purification. Here we show that human beta1, 4-Galactosyltransferase-T7 (beta 4Gal-T7) fused to galectin-1 is produced as soluble, folded enzymatically active protein in E. coli. </p> <P><i><b>Crystal structure of the catalytic domain of Drosophila β-1,4-galactosyltransferase-T7:</i></b>Among the seven members of b4Gal-T family, the b4Gal-T7 transfers Gal from UDP-Gal to an acceptor, beta-xylose (bXyl), which is attached to side chain hydroxyl group of the Ser/Thr residue of proteoglycans, synthesizing a Gal-beta-1-4Xyl disaccharide moiety. Gene knockout studies in Drosophila have shown that b4Gal-T7 is essential for species survival while lack of b4Gal-T1 gene led to multiple disorders. However, mutations in the human b4Gal-T7 are known to cause skin fibroblasts of an Ehlers-Danlos syndrome. The catalytic domain of human b4Gal-T7 exhibits a 39% amino acid sequence similarity with the catalytic domain of human b4Gal-T1, while it shows a 68% sequence similarity with the catalytic domain of b4Gal- [summary truncated at 7800 characters]

<P><i><b>糖基转移酶的结构和功能：</i></b>迄今为止，我们实验室对糖基转移酶，特别是β1,4-半乳糖基转移酶-1（b4Gal-T1）的详细结构功能研究表明：</p><P><i><b>（一）糖基转移酶具有灵活的结构和功能。 催化袋附近的环在供体底物结合时发生构象变化并产生受体结合位点：(II) 在金属离子依赖性酶中，金属离子结合位点通常位于柔性环的氨基末端铰链区：(III) 糖基转移酶与附加结构域相互作用：</i></b> 使催化活性多样化 对于不太优选的底物，例如糖受体或蛋白质或脂质或苷元，糖基转移酶的催化结构域要么与额外的蛋白质相互作用(1)，要么在C末端获得附加结构域或在N末端获得附加结构域。例如，在乳糖合成酶中，b4Gal-T1 在柔性环中构象变化为闭合构象后，与乳腺特异性蛋白、α-乳白蛋白 (LA) 在其羧基末端相互作用，将酶的受体特异性改变为不太优选的受体葡萄糖。 LA 蛋白虽然不与 b4Gal-T1 连接，但充当附加结构域。几种其他糖基转移酶已被证明或暗示需要激活蛋白。与两个相互作用的蛋白质相比，多肽α-N-乙酰半乳糖胺基转移酶 (ppGalNAc-Ts) 的催化结构域具有凝集素结构域，该凝集素结构域通过接头区域连接到催化结构域的 C 末端，并决定对肽或糖肽的特异性。这些酶的催化结构域中的环在金属离子和糖供体结合时也会发生构象变化，同时凝集素结构域移动，将结合的糖肽受体带入催化口袋中，以便在糖肽上合成O-a-GalNAc部分。同样属于这一类的还有 α-1,6-岩藻糖基转移酶 (FUT8)，其中已鉴定出一个 SH3 结构域，该结构域连接在催化结构域的 C 末端。</p> <P><i><b> (IV) 催化口袋中的一些残基决定了糖基转移酶的供体糖特异性：单个氨基酸在进化趋异中的作用 无脊椎动物和脊椎动物糖复合物：（a）b4Gal-T1催化口袋中的突变改变了其供体特异性：</i></b>基于结构信息，我们之前已经表明，b4Gal-T1催化口袋中的残基Tyr/Phe289在所有脊椎动物同源物中是保守的，当突变为Leu或Ile时，拓宽了供体特异性 该酶对半乳糖的 2 个取代基（即 GalNAc 或 2-酮基半乳糖或 2-叠氮基半乳糖）的底物特异性。 （参见项目# Z01 BC 010742）。在无脊椎动物中，b4Gal-T同源物中的Tyr相应位置有一个Ile残基，它们是b4GalNAc-T酶。果蝇 b4GalNAc-T1 中 Ile 残基突变为 Tyr，将该酶转化为 b4Gal-T1，其 N-乙酰半乳糖胺基转移酶活性降低近 1000 倍，同时其半乳糖基转移酶活性增强 80 倍。<i><b>(b) 牛催化结构域中的少量突变 α-1,3-半乳糖基转移酶 (a3Gal-T) 拓宽了供体特异性：</i></b>我们已经突变了牛 a1,3-半乳糖基转移酶 (a3Gal-T) 酶，该酶通常将 Gal 从 UDP-Gal 转移到 LacNAc 受体，通过突变糖从其 UDP 衍生物转移 GalNAc 或 C2 修饰的半乳糖 供体结合残基位于位置 280 至 282。His280 突变为 Leu/Thr/Ser/Ala 或 Gly，Ala281 和 Ala282 突变为 Gly，导致突变型 a3Gal-T 酶的 GalNAc 转移酶活性降至其原始 Gal-T 活性的 5-19%。我们发现，具有最高 GalNAc-T 活性的突变体 280SGG282 和 280AGG282 也可以将修饰糖（例如 2-酮半乳糖或 GalNAz）从各自的 UDP-糖衍生物转移到存在于糖蛋白聚糖非还原端的 LacNAc 部分，从而能够通过化学发光方法检测 LacNAc 部分。这使得使用这些突变体成为可能，(1) 用于检测许多病理状态下糖基化模式的变化，例如癌症和类风湿性关节炎，以及 (2) 用于生物活性剂的靶向药物递送的纳米颗粒的糖缀合和组装。<i><b> (V) 供体糖的 N-乙酰基通常嵌入 </i></b> 在两种突变酶 Y289L-b4Gal-T1 和 SGG-a3Gal-T 中，供体糖 GalNAc 的 N-乙酰基部分嵌入疏水性口袋中，允许用 CH2-CO-CH3 基团取代该部分。它充当化学手柄，允许与连接分子的氨基氧基团缀合。</p><P><i><b> 半乳糖凝集素 -1 作为融合配偶体，用于在大肠杆菌中产生可溶性和折叠的 β-1, 4- 半乳糖基转移酶-T7：</i></b> 在大肠杆菌中以可溶性和活性形式表达重组蛋白通常会导致 聚集的蛋白质称为包涵体。改变细菌生长条件有时可以解决聚集问题。尽管我们开发了一种体外折叠程序，在许多情况下有助于折叠包涵体中的蛋白质，例如 b4Gal-T1 或 ppGalNAc-Ts，但它并不适用于所有蛋白质。迄今为止，最好的可用工具是使用几种不同的融合标签，包括碳水化合物结合蛋白、MBP，可增强重组蛋白的溶解度。 然而，这些融合标签中没有一个对所有伙伴蛋白都通用。在这里，我们首次证明另一种碳水化合物结合蛋白半乳糖凝集素-1可以作为融合伴侣在大肠杆菌中产生可溶性折叠重组蛋白。我们从 pET-23a 中设计了一种新的载体构建体 pLgals1，其中包含 galectin-1 的序列以及插入克隆基因的多克隆位点。独特的蛋白酶切割位点允许在乳糖亲和柱纯化后将目标蛋白从 galectin-1 上切割下来。在这里，我们展示了与半乳糖凝集素-1 融合的人β1, 4-半乳糖基转移酶-T7 (β 4Gal-T7) 在大肠杆菌中作为可溶性、折叠的酶活性蛋白产生。 </p> <P><i><b>果蝇&#946;-1,4-半乳糖基转移酶-T7催化结构域的晶体结构：</i></b>b4Gal-T家族的七个成员中，b4Gal-T7将Gal从UDP-Gal转移到受体β-木糖(bXyl)，其连接到侧链羟基上 蛋白聚糖的 Ser/Thr 残基，合成 Gal-beta-1-4Xyl 二糖部分。果蝇基因敲除研究表明，b4Gal-T7 对于物种生存至关重要，而 b4Gal-T1 基因的缺乏会导致多种疾病。然而，已知人类 b4Gal-T7 的突变会导致皮肤成纤维细胞出现埃勒斯-当洛斯综合征。人b4Gal-T7的催化结构域与人b4Gal-T1的催化结构域具有39%的氨基酸序列相似性，而与b4Gal-的催化结构域具有68%的序列相似性[摘要截断为7800个字符]