Structure-Function Studies and Design of Novel Glycosyltransferases

糖基转移酶的结构和功能：迄今为止，我们实验室对糖基转移酶的详细结构和功能研究，特别是对beta1,4-半乳糖转移酶-1 （b4Gal-T1）的研究表明：(1)糖基转移酶在其催化袋附近具有柔性环，这些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：为了使催化活性多样化，针对不太理想的底物，如糖受体或蛋白质、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。同样属于这一类的还有α -1,6- focusyltransferase (FUT8)，其催化结构域的c端连接着一个SH3结构域。在本年度10-11中，我们确定了果蝇b4Gal-T7的晶体结构，它将Gal从UDP-Gal转移到附着在蛋白聚糖的丝氨酸/苏氨酸残基侧链羟基上的受体β -木糖（bXyl）上，合成了Gal- β -1- 4xyl双糖部分。晶体结构表明果蝇b4Gal-T7也有一个柔性环，在金属离子和供体底物结合时发生构象变化，为蛋白质的木化肽段产生受体结合位点。（IV）催化口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催化口袋中的突变改变了其供体糖特异性；基于结构信息，我们之前已经证明，b4Gal-T1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-叠氮-半乳糖。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，将该酶转化为b4Gal-T1，使其n -乙酰半乳糖氨基转移酶活性降低近1000倍，而将其半乳糖氨基转移酶活性提高80倍。(b)牛α -1,3-半乳糖基转移酶（a3Gal-T）催化区域的少量突变扩大了供体特异性：我们突变了牛α -1,3-半乳糖基转移酶（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衍生物转移到LacNAc受体。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（如癌症和类风湿性关节炎）中糖基化模式的改变，以及(2)用于生物活性药物靶向药物递送的纳米颗粒的糖缀合和组装。(V)供体糖的n -乙酰基通常嵌入酶的疏水袋中。在这两种突变酶Y289L-b4Gal-T1和SGG-a3Gal-T中，供体糖GalNAc的n -乙酰基片段嵌入疏水口袋中，允许该片段被CH2-CO-CH3基团取代。我们现在已经证明，n -乙酰氨基葡萄糖转移酶和n -乙酰半乳糖氨基转移酶的供体糖GlcNAc和GalNAc的n -乙酰基通常嵌入在一个空腔或疏水口袋中，这些空腔或疏水口袋也可以容纳n -乙酰基结合口袋中的酮基，从而可以附着在化学处理亲和探针上，用于检测、分离和表征产物和连接生物分子。半乳糖凝集素-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 -半乳糖转移酶- t7：半乳糖凝集素-1作为融合伙伴已被用于大肠杆菌中可溶性折叠和活性形式的重组蛋白的表达。果蝇β -1,4-半乳糖转移酶- t7的催化结构域晶体结构：对果蝇的基因敲除研究表明，b4Gal-T7是b4Gal-T家族成员之一，可将Gal转移到蛋白聚糖上的木糖，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b4Gal-T7催化结构域与人b4Gal-T1催化结构域的氨基酸序列相似性为39%，与果蝇b4Gal-T7催化结构域的氨基酸序列相似性为68%。在建立了b4Gal-T1的晶体结构后，我们将晶体结构研究扩展到b4Gal-T7，并从果蝇中求解了b4Gal-T7催化域的晶体结构。人类β -1,4-半乳糖转移酶- t7催化结构域的晶体结构：在建立果蝇b4Gal-T7晶体结构后，我们正在对人类b4Gal-T7晶体结构的研究进行延伸。由于果蝇b4Gal-T7的催化结构域与人类b4Gal-T7的相应区域具有73%的蛋白质序列相似性，因此它为突变A186D， L206P和R270C的影响提供了基于结构的解释，这些突变与Ehlers-Danlos综合征（一组遗传性结缔组织疾病）有关。然而，在cys255和Cys310之间的c端存在二硫键，仅存在于来自飞虫的b4Gal-T7蛋白中。为了解释果蝇b4Gal-T7中这个额外的c端序列和额外的二硫键的功能，我们目前正在确定人类b4Gal-T7的晶体结构。

Structure and Function of Glycosyltransferases: To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown following: (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: 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. In this FY 10-11, we have now determined the crystal structure of Drosophila b4Gal-T7, which transfers Gal from UDP-Gal to an acceptor beta-xylose (bXyl) attached to side chain hydroxyl group of the Ser/Thr residue of proteoglycans synthesizing a Gal-beta-1-4Xyl disaccharide moiety. The crystal structure shows that the Drosophila b4Gal-T7 has also a flexible loop that undergoes conformational change upon binding of metal ion and donor substrate creating an accepter binding site for xylated peptide segment of a protein. (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: 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. 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.(b) Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) broadens the donor specificity: 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. (V) The N-acetyl group of the donor sugar is generally embedded in a hydrophobic pocket of the enzyme. 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. We have now shown that the N-acetyl groups of the donor sugars GlcNAc and GalNAc of the N-acetylglucosaminyl- and N-acetylgalactosaminyl-transferases are generally embedded in a cavity or a hydrophobic pocket which can also accommodate a ketone group in the N-acetyl-binding pocket, making it possible to attach to the chemical handle affinity probes for detection, isolation, and characterization of the product and linking biomolecules. Galectin -1 as a fusion partner for the production of soluble and folded beta-1, 4- Galactosyltransferase-T7 in E. coli: Galectin-1 as fusion partner has been used for the expression of recombinant proteins in soluble folded and active form in E. coli. Crystal structure of the catalytic domain of Drosophila beta-1,4-galactosyltransferase-T7:Gene knockout studies in Drosophila have shown that that the b4Gal-T7, one member of the b4Gal-T family that transfers Gal to Xylose on proteoglycans, 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-T7 from Drosophila. Having established crystal structure of b4Gal-T1, we have extended our crystal structure studies to include b4Gal-T7 and solved the crystal structure of the catalytic domain of b4Gal-T7 from Drosophila. Crystal structure of the catalytic domain of human beta-1,4-galactosyltransferase-T7: Having established the crystal structure of Drosophila b4Gal-T7, we are now extending our crystal structure studies on human b4Gal-T7. Since the Drosophila catalytic domain of b4Gal-T7 has a 73% protein sequence similarity to the corresponding region of human b4Gal-T7, it has offered a structure-based explanation on the effect of mutations, A186D, L206P, and R270C, the have been linked to the Ehlers-Danlos syndrome, a group of inherited connective tissue disorders. However, a disulfide bond present at the C-terminal end between Cys 255 and Cys310 is present only in the b4Gal-T7 protein from flying insects. To explain the function of this extra C-terminal sequence and the extra disulphide bond in Drosophila b4Gal-T7 we are at present determining the crystal structure of human b4Gal-T7.