SYNTHESES APPLICATIONS OF ALPHA GALACTOSYL EPITOPES

α半乳糖基表位的合成应用

• 批准号: 6532762
• 负责人: Peng George Wang
• 金额: $ 17.15万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6532762
• 关键词: antigens; bioreactors; carbohydrate structure; chemical synthesis; drug design /synthesis /production; galactose; oligosaccharides; transplant rejection; xenotransplantation

DESCRIPTION: (Applicant's Abstract) The major xenoactive antigens responsible for hyperacute rejection have recently been identified as carbohydrate structures containing terminal alpha Gal1-3Gal sequence (alpha-Gal epitopes). The most common alpha-Gal epitopes are disaccharide alpha Gal1-3Gal1 (1), trisaccharides alphaGal1-3betaGal1-4betaGlcNAc (2) and alphaGal1-3betaGal1-4betaGlc (3), and pentasaccharide alphaGal1-3betaGal1-4betaGlcNAc1-3betaGal1-4betaGlc (4). Alpha-Gal epitopes are abundantly expressed on the cells of most mammals, with the exception of humans, apes and Old World monkeys. Conversely, the natural antibody with specificity to alpha-Gal epitope (anti-Gal) exists only in humans and other Old World primates. The discovery of the interaction of alpha-Gal and anti-Gal has led to experimental attempts to overcome hyperacute rejection by either depleting the recipient's anti-Gal through an alpha-Gal containing affinity column (anti-Gal immunoadsorption approach) or antagonizing anti-Gal by infusing soluble synthetic alpha-Gal oligosaccharides (anti-Gal neutralization approach). Moreover, alpha-Gal-antigen conjugates were shown to enhance antigen presentation by the natural human anti-Gal antibody, and alpha-Gal-immunotoxin glycoconjugates are being used to target B cells which generate anti-Gal antibody. All these biomedical applications require access to a substantial amount of alpha-Gal oligosaccharides as well as synthetically derived alpha-Gal analogs and mimetics with high-affinity to anti-Gal antibodies. Thus, this research program is aimed at development of synthetic technology for large-scale production of alpha-Gal oligosaccharides and at searching for potent alpha-Gal mimetics. 1) Improvement on the synthesis of alpha-Gal epitopes and its multivalent derivatives. The third necessary glycosyltranferase, beta1,3 GlcNAc transferase, will be cloned and overexpressed. Then a one-pot enzymatic system will be developed to synthesize 4 with in situ regeneration of sugar nucleotides UDP-Gal and UDP-GlcNAc through multiple enzyme cycles. To further explore the polyvalent effect of alpha-Gal epitope, structurally defined alpha-Gal oligomers and alpha-Gal dendrimers will be synthesized. The binding of these alpha-Gal derivatives to human anti-Gal will be measured by competition ELISA, flow cytometry, and immunohischemistry assays. 2) Structural studies on alpha-Gal / anti-Gal interaction. Uniformly C-13 labeled alpha-Gal trisaccharide alphaGal1-3betaGal1-4betaGlc, five conformationally constrained alpha-Gal analogs and seven monodeoxy alpha-Gal disaccharide derivatives will be synthesized and used in NMR experiments to determine the active conformation of alpha-Gal bound to monoclonal anti-Gal antibody, and to study the flexibility and controlled rigidity of alpha-Gal epitopes. 3) Alpha-Gal mimetic library and high-throughput screening. A new technology platform (from split synthesis to flow cytometry screening and to MS structural analysis) will be established to generate and screen a large number (greater than ten to the six power) of glycopeptides to identify lead alpha-Gal mimetic sequences. Both natural and unnatural amino acids will be used to construct two combinatorial libraries for screening against different phenotype of anti-Gal for consensus binding structures. In summary, success of this research program will make alpha-Gal oligosaccharides easily accessible, will provide fundamental data on alpha-Gal / anti-Gal interaction and will discover lead potent alpha-Gal mimetic structures that are useful as immunodiagnostic agents, carbohydrate therapeutics, or vaccines.

描述：（申请人的摘要）主要的异种活性抗原 用于超急性排斥反应的物质最近被确定为碳水化合物 含有末端 α Gal1-3Gal 序列（α-Gal 表位）的结构。 最常见的 α-Gal 表位是二糖 α Gal1-3Gal1 (1)， 三糖 alphaGal1-3betaGal1-4betaGlcNAc (2) 和 alphaGal1-3betaGal1-4betaGlc (3) 和五糖 alphaGal1-3betaGal1-4betaGlcNAc1-3betaGal1-4betaGlc (4)。 Alpha-Gal 表位是 在大多数哺乳动物的细胞中大量表达，除了 人类、猿类和旧大陆猴子。相反，天然抗体 α-Gal 表位（抗 Gal）的特异性仅存在于人类和其他古老生物中。 世界灵长类动物。 α-Gal 和抗-Gal 相互作用的发现 导致了克服超急性排斥反应的实验尝试 通过含有 α-Gal 的亲和力耗尽接受者的抗 Gal 柱（抗 Gal 免疫吸附法）或拮抗抗 Gal 注入可溶性合成 α-Gal 寡糖（抗 Gal 中和 方法）。此外，α-Gal-抗原缀合物被证明可以增强抗原 由天然人抗 Gal 抗体和 α-Gal 免疫毒素呈递 糖复合物被用于靶向产生抗 Gal 抗体的 B 细胞 抗体。所有这些生物医学应用都需要获得大量的资源 α-Gal 寡糖以及合成衍生的 α-Gal 的量 与抗 Gal 抗体具有高亲和力的类似物和模拟物。因此，这个 研究计划旨在开发合成技术 α-半乳糖寡糖的大规模生产并寻找 有效的 α-Gal 模拟物。 1) α-Gal表位及其多价合成的改进 衍生物。第三种必需的糖基转移酶，beta1,3 GlcNAc 转移酶将被克隆并过表达。然后是一锅酶解系统 将开发通过糖原位再生合成4 通过多个酶循环产生核苷酸 UDP-Gal 和 UDP-GlcNAc。为了进一步 探索结构定义的 α-Gal 表位的多价效应 将合成α-Gal寡聚物和α-Gal树枝状聚合物。绑定 这些 α-Gal 衍生物对人抗 Gal 的影响将通过以下方法测量 竞争 ELISA、流式细胞术和免疫组织化学测定。 2) α-Gal/抗-Gal相互作用的结构研究。统一C-13 标记的α-Gal三糖αGal1-3betaGal1-4betaGlc，五 构象受限的 α-Gal 类似物和七种单脱氧 α-Gal 二糖衍生物将被合成并用于核磁共振实验 确定与单克隆抗 Gal 结合的 α-Gal 的活性构象 抗体，并研究α-Gal的灵活性和受控刚性 表位。 3）Alpha-Gal模拟文库及高通量筛选。一项新技术 平台（从分割合成到流式细胞术筛选再到 MS 结构 分析）将被建立来生成和筛选大量（更大 超过十的六次方）的糖肽来鉴定先导α-Gal模拟物 序列。天然和非天然氨基酸都将用于构建两种 用于筛选抗 Gal 不同表型的组合文库 用于共识绑定结构。 总之，该研究计划的成功将使α-Gal 易于获取的寡糖，将提供 α-Gal 的基础数据 / 抗 Gal 相互作用，并将发现领先的有效 α-Gal 模拟物 可用作免疫诊断剂、碳水化合物的结构 疗法或疫苗。

项目成果

Efficient synthesis of globoside and isogloboside tetrasaccharides by using beta(1-->3) N-acetylgalactosaminyltransferase/UDP-N-acetylglucosamine C4 epimerase fusion protein.

使用 β(1-->3) N-乙酰半乳糖胺基转移酶/UDP-N-乙酰葡糖胺 C4 差向异构酶融合蛋白高效合成球糖苷和异球糖苷四糖。

• DOI: 10.1039/b300831b
• 发表时间: 2003
• 期刊: Chemical communications (Cambridge, England)
• 作者: Shao,Jun;Zhang,Jianbo;Kowal,Przemyslaw;Lu,Yuquan;Wang,PengGeorge
• 通讯作者: Wang,PengGeorge

Investigation of the conformational states of Wzz and the Wzz.O-antigen complex under near-physiological conditions.

在接近生理条件下研究 Wzz 和 Wzz.O-抗原复合物的构象状态。

• DOI: 10.1021/bi701181r
• 发表时间: 2007
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Tang,Kuo-Hsiang;Guo,Hongjie;Yi,Wen;Tsai,Ming-Daw;Wang,PengGeorge
• 通讯作者: Wang,PengGeorge

The wbnH gene of Escherichia coli O86:H2 encodes an alpha-1,3-N-acetylgalactosaminyl transferase involved in the O-repeating unit biosynthesis.

• DOI: 10.1016/j.bbrc.2006.03.181
• 发表时间: 2006-06
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: W. Yi;Q. Yao;Yalong Zhang;Edwin Motari;Steven Lin;P. Wang

Two different O-polysaccharides from Escherichia coli O86 are produced by different polymerization of the same O-repeating unit.

• DOI: 10.1016/j.carres.2005.11.001
• 发表时间: 2006-01
• 期刊: Carbohydrate research
• 影响因子: 3.1
• 作者: W. Yi;P. Bystrický;Q. Yao;Hongjie Guo;Lizhi Zhu;Hanfen Li;Jie Shen;Mei Li;Soumya Ganguly;C. A. Bushb;Peng G. Wanga

Creatine phosphate--creatine kinase in enzymatic synthesis of glycoconjugates.

磷酸肌酸 - 糖复合物酶促合成中的肌酸激酶。

• DOI: 10.1021/ol034319a
• 发表时间: 2003
• 期刊: Organic letters.
• 作者: Zhang,Jianbo;Wu,Bingyuan;Zhang,Yingxin;Kowal,Przemyslaw;Wang,PengGeorge
• 通讯作者: Wang,PengGeorge