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，关于糖基转移酶的详细结构 - 功能研究，特别是在我们实验室的beta1,4-乳糖基转移酶-1（b4gal-t1）上，我们的实验室中显示了以下：</p> <p> <i> <i>在其催化口袋附近的环中的循环在供体底物结合并创建受体结合位点上经历构象变化，并在金属离子依赖性酶中：金属离子结合位点通常是在氨基的末端铰链区域在柔性循环的氨基末端铰链区域与柔性循环的相互作用。倾向于较少首选的底物，例如糖受体，蛋白质或脂质或aglycons，糖基转移酶的催化域与其他蛋白质相互作用（1），或者在N-末端获得了C-terminus或获得的附加结构域，或者在N-末端获得了附加域。例如，在乳糖合酶酶中，B4GAL-T1在柔性环的构象变化为封闭构象后，与乳腺特异性蛋白Alpha-lactalbumin（LA）在其羧基末端末端与乳腺特异性蛋白质相互作用，从而改变了Enzyme的受体特异性，将其转换为较少受众的受体。 LA蛋白虽然与B4GAL-T1无关，但它充当附加域。已经显示或建议其他几种糖基转移酶需要激活蛋白。与两种相互作用的蛋白质相反，多肽A-N-乙酰乳糖酰氨基氨基转移酶（PPGALNAC-TS）的催化结构域具有凝集素结构域，该结构域具有与催化域的C-末端相关的凝集素结构域，并通过接头区域确定肽或玻璃纤维的特异性。这些酶的催化结构域中的环在金属离子和糖供体的结合后也会发生构象变化，而凝集素结构域则移动，将催化袋中的结合糖肽受体带入催化袋中，以便在糖肽上合成O-A-Galnac Moitie。 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酶对半乳糖的2substituant的底物特异性，即galnac或2-酮 - 半乳糖或2-氮杂 - 半乳糖。 （请参阅项目＃Z01 BC 010742）。在B4GAL-T同源物中的无脊椎动物中，Tyr的相应位置有一个Ile残基，它们是B4Galnac-T酶。在果蝇B4galnac-T1中，​​静脉残基的突变通过将其N-乙酰基乳糖酰氨基氨基转移酶的活性降低，将酶转化为B4GAL-T1，同时将其半乳糖基转移酶活性增强在80倍的情况下，以增强其半乳糖基转移酶的活性。 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位置为280至282位的供体结合残基。将HIS280的突变与Leu/Thr/thr/ser/ala或Gly以及Ala281和Ala282的突变导致突变体A3GAL-T酶的GALNAC转移酶的活性，至其原始Gal-T活性的5-19％。 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.这使得使用这些突变体是可能的，（1）用于检测许多病理状态中糖基化模式的改变，例如癌症和类风湿关节炎，以及（2）在糖结合和组装纳米粒子的纳米粒子的靶向靶向药物的靶向药物均匀糖的nano颗粒中。突变酶，Y289L-B4GAL-T1和SGG-A3GAL-T的疏水口袋。这是一种化学手柄，允许与链接分子的氨基氧基共轭。</p> <p> <p> <i> <b> lectectin -1作为融合伴侣，用于生产可溶性和折叠β-1、4-甲基乳糖基转移酶-T7在E. coli中的Coli：</i> </i> </i> </i> </> </> </> </> </> </> </> </> </> </> </> </>聚集的蛋白质称为包容体。修改细菌生长条件有时可以解决聚集问题。尽管我们已经开发了一种体外折叠程序，在许多情况下，它有助于从包含体中折叠蛋白质，例如B4GAL-T1或PPGALNAC-TS，但它仍然无法与所有蛋白质一起使用。迄今为止，最好的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，增强了重组蛋白的溶解度。 但是，这些融合标签都与每个合作伙伴蛋白质都普遍起作用。在这里，我们首次表明另一种碳水化合物结合蛋白lectectin-1可以充当融合伴侣，在大肠杆菌中产生可溶性折叠的重组蛋白。我们已经从PET-23A设计了一种新的向量构建体PLGALS1，其中包括Galectin-1的序列和一个多键位点，其中插入了克隆的基因。独特的蛋白酶裂解位点使感兴趣的蛋白质在乳糖亲和柱纯化后从lectectin-1裂解。在这里，我们表明，融合到Galectin-1的人β1，4-半乳糖基转移酶T7（β4GAL-T7）作为大肠杆菌中的可溶性，折叠的酶活性蛋白产生。 </p> <P><i><b>Crystal structure of the catalytic domain of Drosophila &#946;-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蛋白聚糖的Ser/Thr残基，合成Gal-Beta-1-4xyl二糖部分。果蝇中的基因敲除研究表明，B4GAL-T7对于物种存活至关重要，而缺乏B4GAL-T1基因导致多种疾病。然而，已知人类B4GAL-T7中的突变会导致Ehlers-Danlos综合征的皮肤成纤维细胞。人B4GAL-T7的催化结构域与人B4GAL-T1的催化结构域表现出39％的氨基酸序列相似性，而它显示出与B4GAL-的催化结构域的68％序列相似性，[汇总以7800个字符截断为7800个字符]]