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

GAL-ALPHA-1-3GALG糖缀合物--生物化学/进化

• 批准号: 3297603
• 负责人: Bruce A. Macher
• 金额: $ 21.51万
• 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/3297603
• 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; nucleic acid sequence; 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.

我们的研究集中在结构分析上， 表达糖表位的糖缀合物的生物合成 Galalpha1-3Galbeta1-4GlcNAc。 我们已经确定糖缀合物 在非灵长类哺乳动物中大量表达， 新世界的猴子。 相反，旧大陆的猴子不表达相反，旧大陆的猴子不表达 (OWM)，猿或人。然而，这些后一种物种产生了大量的 量的天然抗体，抗Gal，其具有严格的 Gal α-3Galbeta1-4GlcNAc表位的结合特异性 哺乳动物糖缀合物。 我们已经确定， OWM中的Galalpha 1 - 3Galbeta 1 -4GlcNAc表位表达是由于 α 1 -3半乳糖基转移酶活性的降低 （α 1 -3GT），其催化以下反应。 Galalpha 1 - 3Galbeta 1 - 4GlcNAc-R +UDP 我们的总体目标是研究这种进化上独特的酶， 它的生物合成产品使用分子生物学， 酶学和糖结构分析。 克隆研究 这种酶的cDNA已经确定，α 1 -3GT的基因具有 在旧大陆灵长类动物中以非表达形式保存下来。 通过 分子生物学的方法，我们建议研究几个问题 关于这个基因，包括它在哺乳动物中的进化， 在不同组织中的差异表达， 催化结构域及其进化的分子基础 抑制OWM。 也已经确定，有不同的模式， 在不同的细胞中Galalpha 1 - 3Galbeta 1 -4GlcNAc糖缀合物的表达 哺乳动物物种。 为了了解生物合成因素， 在糖缀合物的不同表达模式中， Galapha 1 - 3Galbeta 1 -4GlcNAc表位，我们建议进行碳水化合物 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 阐明碳水化合物的结构，和一种新的基于ELISA的 糖基转移酶测定将用于酶研究。 的 结合分子生物学和生物化学方法， 阐明导致差异的机制 Galalpha 1 - 3Galbeta 1 -4GlcNAc表位的表达。