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>< &gt；</ I ></b&gt；到目前为止，我们实验室对糖基转移酶，特别是对β - 1,4-半乳糖基转移酶-1 （b4Gal-T1）的详细结构-功能研究表明：&lt；/ >< >< >< I >< < > (I)糖基转移酶在其催化袋附近具有柔性环，在供体底物结合时发生构象变化并产生受体结合位点；（II）在金属离子依赖酶中，金属离子结合位点一般位于柔性环的氨基末端铰区；（III）糖基转移酶与附加结构域相互作用：/i>& gt; /b> /b&gt；为了使催化活性多样化，针对不太优选的底物，如糖受体或蛋白质或脂质或糖酰子，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端获得附加结构域或在n端获得附加结构域。例如，在乳糖合成酶中，b4Gal-T1在柔性环构象改变为封闭构象后，与乳腺特异性蛋白α -乳蛋白（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蛋白虽然没有与b4Gal-T1连接，但它是一个附加结构域。其他几种糖基转移酶已被证明或建议需要激活蛋白。与两种相互作用的蛋白质相比，多肽a- n -乙酰半乳糖氨基转移酶（ppGalNAc-Ts）的催化结构域具有凝集素结构域，该结构域通过连接子区域连接到催化结构域的c端，并决定了对肽或糖肽的特异性。当金属离子与糖供体结合时，这些酶的催化结构域中的环也发生构象变化，而凝集素结构域移动，将结合的糖肽受体带入催化口袋中，以合成糖肽上的O-a-GalNAc片段。α -1,6- focusyltransferase （FUT8）也属于这一类，其中已鉴定出在催化结构域c端连接的SH3结构域。</ >< >< >& gt;& gt;& gt; （IV）催化袋中的几个残基决定了糖基转移酶的供体糖特异性：单个氨基酸在无脊椎动物和脊椎动物糖缀合物进化分化中的作用：(a) b4Gal-T1的催化口袋突变改变了它的供体特异性：&lt；/i></b&gt；基于结构信息，我们之前已经证明，b4Gal-T1的催化口袋中的残基Tyr/Phe289在所有脊椎动物同源物中都是保守的，当突变为Leu或Ile时，该酶的供体底物特异性扩大到2个半乳糖取代基，即GalNAc或2-酮-半乳糖或2-氮杂-半乳糖。（见项目# Z01 BC 010742）。在无脊椎动物中，b4galac - t的同源物在Tyr的相应位置有一个Ile残基，它们是b4galac - t酶。果蝇b4galac - t1的Ile残基突变为Tyr，通过将其n -乙酰半乳糖氨基转移酶活性降低近1000倍，将其转化为b4Gal-T1，同时将其半乳糖氨基转移酶活性提高80倍。<i>< >(b)牛α -1,3-半乳糖氨基转移酶（a3Gal-T）催化结构域的少量突变拓宽了供体特异性。我们突变了牛a1,3-半乳糖转移酶（a3Gal-T），该酶通常将Gal从UDP-Gal转移到LacNAc受体，通过突变糖供体结合残基280至282位，将GalNAc或c2修饰的半乳糖从其UDP衍生物转移。His280突变为Leu/Thr/Ser/Ala或Gly， Ala281和Ala282突变为Gly导致突变体a3Gal-T酶的GalNAc转移酶活性降低到原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还可以将修饰的糖（如2-酮-半乳糖或GalNAz）从其各自的udp糖衍生物转移到糖蛋白聚糖非还原端存在的LacNAc片段，从而可以通过化学发光方法检测LacNAc片段。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中糖基化模式的改变，例如癌症和类风湿性关节炎；(V)供体糖的n -乙酰基通常嵌入酶的疏水袋中。</i></b&gt；在Y289L-b4Gal-T1和SGG-a3Gal-T这两种突变酶中，供体糖GalNAc的n -乙酰基部分嵌入疏水袋中，允许该部分被CH2-CO-CH3基团取代。这就像一个化学柄，允许与连接分子的氨基氧基结合。</p>< >< >< >& gt;& gt;& gt;& gt;& gt;& gt；半乳糖基转移酶-1作为融合伙伴，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&gt；重组蛋白在大肠杆菌中以可溶性和活性形式的表达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。尽管我们已经开发出一种体外折叠程序，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，以提高重组蛋白的溶解度。然而，没有一种融合标签可以普遍地与每一个伴侣蛋白一起工作。本研究首次发现另一种碳水化合物结合蛋白半乳糖凝集素-1可以作为融合伙伴在大肠杆菌中产生可溶性折叠重组蛋白。我们从pET-23a中设计了一个新的载体pLgals1，其中包括半乳糖凝集素-1的序列，以及一个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从半乳糖凝集素-1中切割出感兴趣的蛋白质。在这里，我们展示了人类β - 1,4 -半乳糖基转移酶- t7 （β - 4Gal-T7）与半乳糖凝集素-1融合在大肠杆菌中作为可溶性的折叠酶活性蛋白产生。</p> <i>< i>< & b&gt；果蝇&amp；#946;-1,4-半乳糖基转移酶- t7:&lt；/i>< & b&gt；在b4Gal-T家族的7个成员中，b4Gal-T7将Gal从UDP-Gal转移到受体β -木糖（bXyl）上，该受体连接在蛋白聚糖的Ser/Thr残基侧链羟基上，合成Gal- β -1- 4xyl双糖部分。果蝇基因敲除研究表明，b4Gal-T7对物种生存至关重要，而缺乏b4Gal-T1基因会导致多种疾病。然而，已知人类b4Gal-T7的突变会导致埃勒斯-丹洛斯综合征的皮肤成纤维细胞。人类b4Gal- t7催化结构域与人类b4Gal- t1催化结构域的氨基酸序列相似性为39%，与b4Gal-催化结构域的氨基酸序列相似性为68%[摘要截断为7800个字符]