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

我们的研究集中在对结构的分析和 表达碳水化合物表位的糖结合物的生物合成 Galalpha1-3Galbeta1-4GlcNAc。我们已经确定了糖偶联物 携带这一表位的非灵长类哺乳动物和 新大陆的猴子。相比之下，东半球的猴子并不表达这种基因 (OWM)，猿或人。然而，这些后一种物种产生了大量的 一种天然产生的抗体，抗Gal，它具有严格的 Galpha-3Galbeta1-4GlcNAc表位的结合特异性 哺乳动物的糖结合物。我们已经确定，对叙利亚的镇压 OWM中Galalpha1-3Galbeta1-4GlcNAc表位的表达 α1-3半乳糖基转移酶活性降低 (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 它的生物合成产品使用了分子生物学的组合， 酶学和碳水化合物结构分析。克隆人的研究 这种酶的cDNA已经确定alpha1-3GT的基因已经 在东半球灵长类动物中以未表达的形式保存。穿过 我们建议研究的几个问题的分子生物学方法 关于这个基因，包括它在哺乳动物中的进化，它的 在不同组织中的差异表达，其同源性 催化结构域及其进化的分子基础 OWM中的抑制。 也已经确定，有不同的模式 Galalpha1-3Galbeta1-4GlcNAc糖共轭在不同组织中的表达 哺乳动物物种。要了解导致生物合成的因素 在不同的糖偶联物的表达模式中 Galapha1-3Galbeta1-4GlcNAc表位，我们建议进行碳水化合物 结构分析和酶受体专一性研究。质子 核磁共振、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酶联免疫吸附试验的方法 糖基转移酶试验将用于该酶的研究。这个 分子生物学和生化方法的结合将使 对导致这种差异的机制的解释(S) Galalpha1-3Galbeta1-4GlcNAc表位的表达

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.