GAL-ALPHA-1-3GALGLYCOCONJUGATES--BIOCHEMISTRY/EVOLUTION

GAL-ALPHA-1-3GALG糖缀合物--生物化学/进化

• 批准号: 3297599
• 负责人: Bruce A. Macher
• 金额: $ 22.67万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-20 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297599
• 关键词: Ceboidea; Cercopithecidae; animal tissue; antigens; beta galactosidase; biochemical evolution; carbohydrate biosynthesis; carbohydrate sequence; carbohydrate structure; chemical conjugate; complementary DNA; computer assisted sequence analysis; enzyme linked immunosorbent assay; enzyme mechanism; enzyme substrate; galactose; galactosyltransferases; gangliosides; gene expression; gene mutation; genetic polymorphism; genetic transcription; glycosphingolipids; kidney; laboratory rabbit; mass spectrometry; molecular cloning; northern blottings; nuclear magnetic resonance spectroscopy; nucleic acid hybridization; polymerase chain reaction; sheep; site directed mutagenesis; swine; thymus

Our studies have focused on an analysis of the structure and biosynthesis of glycoconjugates expressing the carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc. We have established that glycoconjugates bearing this epitope are abundantly expressed n nonprimate mammals and New World monkeys. In contrast, it isnot expressed by Old World monkeys (OWM), apes or man. However, these latter species produce a large quantity of a naturally ocurring antibody, anti-Gal, which has a strict binding specificity for the Galalpha-3Galbeta1-4GlcNAc eiptope on mammalian glycoconjugates. We have determined that the suppression of Galalpha1-3Galbeta1-4GlcNAc epitope expression in OWM results from a diminution of the activity of the enzyme alpha1-3 galactosyltransferase (alpha1-3GT) which catalyzes the following reaction. Galbeta1-4GlcNAc-R + UDP-Gal Galalpha1-3Galbeta1-4GlcNAc-R +UDP Our overall objective is to study this evolutionarily unique enzyme and its biosynthetic products using a combination of molecular biology, enzymology and carbohydrate structural analysis. Cloning studies of the cDNAfor this enzyme have established that the gene for alpha1-3GT has been conserved in Old World primates in a nonexpressed form. Through molecular biology approaches we propose to study several issues concerning this gene including its evolution in mammals, its differential expression in various tissues, the identity of its catalytic domain and the molecular basis for its evolutionary suppression in OWM. It has also been established that there are different patterns of Galalpha1-3Galbeta1-4GlcNAc glycoconjugate expression among various mammalian species. To understand the biosynthetic factors that result in different patterns of expression of glycoconjugates with the Galapha1-3Galbeta1-4GlcNAc epitope, we propose to carry out carbohydrate structural analyses and enzyme acceptor specificity studies. Proton NMR, FAB-MS and antibody immunostaining methods will be used to elucidate carbohydrate structures, and a novel ELISA based glycosyltransferase assay will be used for the enzyme studies. The combination of molecular biology and biochemical approaches will enable the elucidation of the mechanism(s) which results in the differential expression of the Galalpha1-3Galbeta1-4GlcNAc epitope.

我们的研究重点是对结构和 表达碳水化合物表位的糖缀合物的生物合成 galalpha1-3galbeta1-4glcnac。 我们已经确定了糖缀合物 带有此表位的表现大量表达了非哺乳动物， 新世界猴子。 相比之下，它不是由旧世界猴子表达的 （OWM），猿类或男人。 但是，这些后一种物种产生了一个大的 天然抗体抗体的量，抗-gal，具有严格 galalpha-3galbeta1-4glcnac eiptope的结合特异性 哺乳动物糖缀合物。 我们已经确定抑制 galalpha1-3galbeta1-4glcnac表位表达在OWM中是由A 酶Alpha1-3半乳糖基转移酶活性的减少 （α1-3GT）催化以下反应。 galbeta1-4glcnac-r + udp-gal galalpha1-3galbeta1-4glcnac-r + udp 我们的总体目标是研究这种进化上独特的酶和 它的生物合成产物结合了分子生物学， 酶学和碳水化合物结构分析。 克隆研究 该酶已经确定alpha1-3gt的基因具有 在旧世界的灵长类动物中保守了不受欢迎的形式。 通过 分子生物学方法我们建议研究几个问题 关于该基因，包括其在哺乳动物中的进化， 在各种组织中的差异表达，其身份 催化域及其进化的分子基础 镇压。 还已经确定有不同的模式 galalpha1-3galbeta1-4glcnac糖缀合物表达在各种 哺乳动物。 了解导致的生物合成因子 在不同的糖缀合物表达方式中 galapha1-3galbeta1-4glcnac表位，我们建议携带碳水化合物 结构分析和酶受体特异性研究。 质子 NMR，FAB-MS和抗体免疫染色方法将用于 阐明碳水化合物结构和一种新型ELISA 糖基转移酶测定法将用于酶研究。 这 分子生物学和生化方法的结合将实现 阐明机制导致差异的机理 galalpha1-3galbeta1-4glcnac表位的表达。