MAMMALIAN SIALYLTRANSFERASES

哺乳动物唾液酸转移酶

• 批准号: 2175027
• 负责人: JAMES C PAULSON
• 金额: $ 28.44万
• 依托单位: CYTEL CORPORATION
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175027
• 关键词: SDS polyacrylamide gel electrophoresis; carbohydrate biosynthesis; chemical synthesis; complementary DNA; enzyme activity; genetically modified animals; glycosylation; laboratory mouse; laboratory rat; molecular cloning; mutant; nucleic acid sequence; polymerase chain reaction; sialyltransferases; site directed mutagenesis

In the past project period cDNAs were cloned for two additional sialyltransferases, the Galbeta1,3GalNAc alpha2,3 sialyltransferase and the Galbeta1,3/4GlcNAc alpha2,3 sialyltransferase. Comparison of the coding sequences with that of the Galbeta1,4GlcNAc alpha2,6 sialyltransferase revealed a unique pattern of homology among glycosyltransferase families. There was a 55 residue stretch of high homology in the center of each enzyme (40-60% identity) with no detectable homology between any of the enzymes outside that region. We have termed this conserved region the sialylmotif. In the next project period several aims pertain to this conserved motif. 1) Additional members of the 10-12 member sialyltransferase family will be cloned using a PCR homology approach using the information contained in the sialylmotif. Preliminary efforts using this approach have been successful in pulling out another cDNA from embryonic brain that has the sialylmotif and other sequence features like the three sialyltransferases cloned to date. A 'soluble form' of this putative enzyme will be constructed and expressed to evaluate the activity and specificity in order to identify which enzyme has been cloned. Other sialyltransferase cDNAs obtained by this approach will be evaluated similarly. 2) The sialylmotif will be investigated for its functional properties by a site- directed mutagenesis expression approach. The driving force for this investigation stems from the successful crystallization of the Galbeta1,4GlcNAc alpha2,6 sialyltransferase by the group of Dr. Don Wiley at Harvard. Thus, the results regarding function will likely be interpretable from the three dimensional structure of the enzyme in the foreseeable future. Several other specific aims take advantage of the new tools resulting from the cloned sialyltransferases. 1) The long time collaboration with Dr. Jurgen Roth for localization of sialyltransferases will be extended to the newly cloned enzymes. 2) The newly expressed enzymes will be used to develop a flexible and practical route to the combined chemical and enzymatic synthesis of sialosides. 3) One of the three cloned sialyltransferases appears to be responsible for the conversion of peanut agglutinin positive (PNA+) T-cells to the PNA- phenotype during maturation in the thymus. In collaboration with Dr. Steve Hedrick and Dr. Ajit Varki at UCSD, transgenic mice are being constructed to test the function of this glycosylation change.

在过去的项目期间，将cDNA克隆了两个 siAllyltransferases，Galbeta1,3galnac alpha2,3 siAllyltransferase和 Galbeta1,3/4GlcNACα2,3siAlyltransferase。 比较 用Galbeta1,4GlcNAC alpha2,6的编码序列 辅助转移酶在 糖基转移酶家族。 有55个残留物的高度 每个酶的中心（40-60％身份）的同源性 该区域以外的任何酶之间可检测的同源性。 我们 已将这个保守的区域称为siAllylMotif。 在下一个项目中 时期的几个目标与该保守的主题有关。 1）附加 10-12成员siallyltransferase家族的成员将使用 PCR同源性方法使用包含的信息 siAlylMotif。 使用这种方法的初步努力已经 成功从胚胎大脑中取出另一个cDNA siAlylMotif和其他序列特征，例如三个siAllltransferase 克隆到迄今为止。 该推定酶的“可溶性形式”将是 构建和表达以评估的活动和特异性 为了识别已克隆哪种酶。 其他辅助转移酶 通过这种方法获得的cDNA将进行类似的评估。 2） siAllylmotif将通过位点研究其功能性能 定向诱变表达方法。 为此的动力 研究源于成功的结晶 Don Wiley博士小组的Galbeta1,4GlcNAC alpha2,6 siAllyltransferase 在哈佛。 因此，有关功能的结果可能是 可以从酶的三维结构中解释 可预见的未来。 其他几个具体目标利用了 由克隆的硫溶剂转移酶产生的新工具。 1）长时间 与尤尔根·罗斯（Jurgen Roth）博士的合作，以定位siallyltransferases 将扩展到新克隆的酶。 2）新表达 酶将用于开发一种灵活而实用的途径 唾液酸化的化学和酶促合成。 3）其中之一 三个克隆的尖升转移酶似乎是负责的 花生凝集素阳性（PNA+）T细胞转化为PNA- 胸腺成熟过程中的表型。 与博士合作 史蒂夫·赫德里克（Steve Hedrick）和加州大学（UCSD）的阿吉特·瓦基（Ajit Varki）博士，转基因小鼠正在 构建以测试这种糖基化变化的功能。