STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN THE METABOLISM OF POLYSIALIC ACID

聚唾液酸代谢酶的结构和功能

• 批准号: 3748111
• 负责人: W F VANN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748111
• 关键词: CHO cells; Escherichia coli; SDS polyacrylamide gel electrophoresis; affinity chromatography; bacterial polysaccharides; bacterial proteins; carbohydrate metabolism; enzyme activity; enzyme mechanism; enzyme structure; gel filtration chromatography; mutant; phosphoenolpyruvate; protein purification; protein sequence; sialate

Polysialic acid is an important virulence factor for most of the capsular types of Escherichia coli and Neiseria meningitidis causing meningitis in infants and children. This poorly immunogenic polysaccharide plays a role in neural tissue development. The enzymes involved in the synthesis of polysialic acid in bacteria are being identified, purified and characterized. Common features between these enzymes and anologous enzymes in sialylation of mammalian glycoconjugates are being determined. Bovine pituitary CMP-sialic acid synthetase was partially purified at the time of the previous report. During this reporting period the CMP-sialic acid synthetase was purified further on a sialic acid based affinity column. The b-allyl ketoside of sialic was synthesized. This residue was coupled to activate agarose via a cysteamine linker. Enzyme eluting from this resin exhibited a major band of 60,000 on SDS. The enzyme activity eluted as 150,000 MW on gel filtration also eluted as 60,000 on SDS.A strain of CHO cells used to manufacture recombinant DNAase failed to properly sialylate glycoproteins. While this mutant strain does not produce CMP-sialic acid it does contain an active enzyme. Efforts are underway to express E. coli CMP-sialic acid synthetase in CHO mutants.Mutants in the E.coli K1 neuB gene cannot make sialic acid and thus require an addition of sugar for polysialic acid synthesis. The neuB gene was cloned into an expression vector and the enzyme purified to homogeniety. The amino terminal sequence of the purified enzyme was determed to be the same as that deduced from the nucleotide sequence of the neuB gene. The enzyme was shown to form sialic acid from N- acetylmannosamine and phosphoenol pyruvate. Efforts to further characterize this enzyme are underway and to use this enzyme to synthesize polysialic acid analogues.

聚唾液酸是大多数荚膜梭菌的重要毒力因子 引起脑膜炎的大肠杆菌和脑膜炎奈瑟菌的类型 在婴儿和儿童身上。 这种免疫原性差的多糖 在神经组织发育中的作用。 参与蛋白质合成的 在细菌中合成聚唾液酸的方法正在被鉴定、纯化 和表征了 这些酶和抗生素之间的共同特征 哺乳动物糖缀合物的唾液酸化中的酶正在被确定。 牛垂体CMP-唾液酸合成酶在200 ℃下部分纯化， 上次报告的时间。 在本报告所述期间，CMP-唾液酸 在基于唾液酸的亲和性上进一步纯化酸性合成酶 柱 合成了唾液酸的b-烯丙基酮苷。 该残基 偶联以通过半胱胺接头活化琼脂糖。 酶洗脱 来自该树脂的一条在SDS上显示60，000的主带。 酶 在凝胶过滤上洗脱为150，000 MW的活性也在凝胶过滤上洗脱为60，000 MW。 SDS.用于生产重组DNA酶的CHO细胞株失败 使糖蛋白适当地唾液酸化。 虽然这种突变株没有 产生CMP-唾液酸，它确实含有活性酶。 努力 正在进行E. CHO中的大肠杆菌CMP-唾液酸合成酶 大肠杆菌K1 neuB基因中的突变体不能产生唾液酸， 因此需要添加糖用于聚唾液酸合成。 的 将neuB基因克隆到表达载体中，并纯化该酶 同质性 纯化酶的氨基末端序列为 经测定与从以下核苷酸序列推断的序列相同： neuB基因 该酶被证明可以从N- 乙酰甘露糖胺和磷酸烯醇丙酮酸。 努力进一步 正在对这种酶进行表征，并使用这种酶来 合成聚唾液酸类似物。