SYNTHESES APPLICATIONS OF ALPHA GALACTOSYL EPITOPES

描述：(申请人摘要)主要的异种活性抗原 对超急性排斥反应的作用最近被确认为碳水化合物 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αGal1-3βGal1-4βGlcNAc(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上大量表达，除了 人类、猿类和东半球的猴子。相反，天然抗体与 α-Gal表位(抗-Gal)的特异性仅存在于人类和其他老年人 世界灵长类动物。α-半乳糖与抗半乳糖相互作用的发现 导致了克服超急性排斥反应的试验性尝试 通过含有亲和力的α-半乳糖耗尽受体的抗半乳糖 柱(抗Gal免疫吸附方法)或通过以下方法拮抗抗Gal 注入可溶性合成α-半乳糖寡糖(抗半乳糖中和 方法)。此外，α-半乳糖-抗原结合物被证明能增强抗原。 天然人抗半乳糖抗体和α-半乳糖免疫毒素的呈递 糖偶联物被用来靶向产生抗Gal的B细胞 抗体。所有这些生物医学应用都需要获得大量的 α-半乳糖寡糖及其合成产物的含量 对抗Gal抗体具有高亲和力的类似物和模拟物。因此，这一点 研究项目的目标是开发合成技术 α-半乳寡糖的规模化生产及寻找 强大的阿尔法-半乳糖模拟物。 1)α-Gal表位及其多价表位合成方法的改进 衍生品。第三个必需的糖基转移酶，β-1，3-GlcNAc 转移酶，将被克隆并高效表达。然后是一锅酶系统 将开发利用糖的原位再生来合成4 核苷酸UDP-Gal和UDP-GlcNAc通过多个酶循环。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-半乳糖齐聚物和α-半乳糖树枝状大分子。装订 这些α-半乳糖衍生物中的人抗半乳糖将通过以下方法测量 竞争酶联免疫吸附试验、流式细胞仪和免疫组织化学分析。 2)α-半乳糖/反半乳糖相互作用的结构研究。统一的C-13 标记α-半乳糖三糖αGal1-3βGal1-4βGlc，5 构象受限的α-半乳糖类似物和7个单脱氧α-半乳糖 二糖衍生物将被合成并用于核磁共振实验 α-半乳糖与单抗-半乳糖结合活性构象测定 抗体，并研究α-半乳糖的柔韧性和受控刚性。 表位。 3)Alpha-Gal模拟文库和高通量筛选。一项新技术 平台(从裂解合成到流式细胞仪筛选再到MS结构 分析)将被建立以生成和筛选大量(更大的 比10的6次方)的糖肽来鉴定α-半乳糖的模拟物 序列。天然和非天然氨基酸将被用来构建两个 筛选不同表型抗Gal抗体的组合文库 对于具有共识约束力的结构。 总之，这项研究计划的成功将使阿尔法-伽罗 低聚糖易于获取，将提供有关α-半乳糖的基本数据 /反Gal相互作用，并将发现有效的α-Gal模拟铅 作为免疫诊断剂有用的结构，碳水化合物 治疗学，或疫苗。

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.