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

聚唾液酸是大多数衣壳细胞的重要致病因子。 引起脑膜炎的大肠埃希菌和脑膜炎奈瑟氏菌 在婴儿和儿童身上。这种免疫原性很差的多糖会 在神经组织发育中的作用。参与这一过程的酶 细菌中合成聚唾液酸的鉴定、纯化 并以此为特征。这些酶和同源酶之间的共同特征 哺乳动物糖偶联物唾液酸化中的酶正在测定中。 牛脑下垂体细胞色素P-唾液酸合成酶在 上次报告的时间。在本报告所述期间，《议定书》--唾液酸 唾液酸亲和层析进一步纯化了酸性合成酶 纵队。合成了唾液酸烯丙基酮苷。这个残留物 偶联后通过半胱胺连接物激活琼脂糖凝胶。酶洗脱 该树脂在十二烷基硫酸钠上显示了一条60,000的主带。这种酶 15万mW的凝胶过滤洗脱的活性也被洗脱为6万mW的 一株用于生产重组DNA酶的CHO细胞失败 以适当地分析糖蛋白。而这种突变菌株不会 产生CMP-唾液酸它确实含有一种活性酶。努力是 大肠杆菌cMP-唾液酸合成酶在CHO中的高效表达 突变株。E.ColiK1Neub基因突变株不能产生唾液酸和 因此需要添加糖来合成聚唾液酸。这个 将Neub基因克隆到表达载体中，并对其进行纯化 为了同质化。该酶的氨基末端序列为 被确定为与根据该病毒的核苷酸序列推导的序列相同 Neub基因。该酶能从N-生成唾液酸。 乙酰甘露糖胺和磷酸烯醇丙酮酸。努力进一步 鉴定这种酶正在进行中，并使用这种酶来 合成聚唾液酸类似物。

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.