II GLYCOLIPID BIOSYNTHESIS IN MOUSE T- AND B-LYMPHOMAS

II 小鼠 T 和 B 淋巴瘤中的糖脂生物合成

• 批准号: 3171542
• 负责人: MANJU BASU
• 金额: $ 10.46万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1989-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3171542
• 关键词: B lymphocyte; T lymphocyte; affinity chromatography; carbohydrate biosynthesis; cell membrane; chemical structure; complementary DNA; enzyme structure; galactosyltransferases; gangliosides; genetic library; genetic manipulation; glycolipids; glycosphingolipids; glycosyltransferase; ion exchange chromatography; lipid biosynthesis; lipid structure; lymphoma; mass spectrometry; monoclonal antibody; nucleic acid sequence

The immediate objectives of the research are fivefold: 1. Separation, characterization, and purification of G1cNAcT-2 (UDP-G1cNAc:nLcOse4Cer-(Beta1-3)-N-acetylglucosaminyltransferase) and G1cNAcT-3 (UDP-G1cNAc:i-core(Beta1-6)N-acetylglucosaminyltransferase) present in the neutral, detergent-solubilized super natant from p-1798 mouse T-lymphoma (estrogen-induced). 2. Purification and kinetic studies of Ga1T-4 (UDP-Gal:LcOse3Cer(Beta1-4)galactosyltransferase) from the 0.1% Triton CF-54-solubilized supernatant fraction of p-1798 mouse T-lymphoma. 3. Completion of the biosynthesis in vitro of the I/i biantennary structure from previously synthesized I/i core glycolipid, using purified GalT-4 and G1cNAcT-2. 4. Characterization of FucT-2 (GDP-Fuc:nLcOse4Cer(Alpha1-2)fucosyltransferase) and FucT-3 (GDP-Fuc:LcOse3Cer(Alpha1-3)fucosyltransferase) from Beta-lymphoma (TEPC-15 alkane-induced, and ABLS-140 Abelson virus-induced). Determination of exact chemical linkages in the biosynthetic products (FucAlpha-Gal-G1cNAc-Gal-G1c-Cer and FucAlpha-G1cNAc-Gal-G1c-Cer) obtained from TEPC-15 and ABLS-140. 5. Screening of the mouse genomic library, prepared from a partial Eco RI digestion of AJ mouse genomic DNA (Shotgun in Charon 4a) with a 32P-labeled cDNA insert of GalT-4. Indirect assays for the translation products for particular GalT-4 DNA sequences, using radioiodinated monoclonal antibodies against GalT-4. Our overall goal is to develop an understanding of the specificities of glycosyltransferases in mouse lymphoreticular systems using one T-lymphoma (P-1798) and two B-lymphomas (TEPC-15 and ABLS-140) as model systems.

研究的近期目标有五个方面： 1. G1cNAcT-2的分离、表征和纯化 （UDP-G1cNAc：nLcOse4Cer-（Beta1 - 3）-N-乙酰葡糖胺基转移酶）和 G1cNAcT-3（UDP-G1cNAc：i-核心（Beta1 - 6）N-乙酰葡糖胺基转移酶） 存在于p-1798的中性、洗涤剂溶解的上清液中 小鼠T淋巴瘤（雌激素诱导）。 2. Ga1T-4的纯化及动力学研究 （UDP-Gal：LcOse3Cer（β 1 - 4）半乳糖基转移酶），来自0.1% Triton p-1798小鼠T淋巴瘤的CF-54溶解的上清液部分。 3.完成I/i双触角结构的体外生物合成 从先前合成的I/i核心糖脂，使用纯化的GalT-4和 G1cNAcT-2。 4. FucT-2的表征 （GDP-Fuc：nLcOse4Cer（Alpha1 - 2）岩藻糖基转移酶）和FucT-3 （GDP-Fuc：LcOse3Cer（Alpha1 - 3）岩藻糖基转移酶），来自β-淋巴瘤（TEPC-15 烷烃诱导的和ABLS-140 Abelson病毒诱导的）。 测定 生物合成产物中的精确化学键 获得的（FucAlpha-Gal-G1cNAc-Gal-G1c-Cer和FucAlpha-G1cNAc-Gal-G1c-Cer） 从TEPC-15和ABLS-140。 5.筛选从部分EcoRI制备的小鼠基因组文库 用32P标记的酶消化AJ小鼠基因组DNA（Shotgun in Charon 4a） GalT-4的cDNA插入片段。 翻译产物的间接测定 特别是GalT-4 DNA序列，使用放射性碘标记的单克隆抗体， 对抗GalT-4 我们的总体目标是了解 用一种T淋巴瘤研究小鼠淋巴网状系统中的糖基转移酶 （P-1798）和两种B淋巴瘤（TEPC-15和ABLS-140）作为模型系统。