Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells.
Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells.
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DOI：
10.1016/s0021-9258(19)76560-0
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发表时间：
1988-07
期刊：
影响因子：
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通讯作者：
B D Livingston;J. Jacobs;M C Glick;F. A. Troy
中科院分区：
文献类型：
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作者：
B D Livingston;J. Jacobs;M C Glick;F. A. Troy
由至少55个唾液酸残基的延伸链组成的含聚唾液酸的糖蛋白（DP 55，其中DP代表聚合度）在人神经母细胞瘤细胞CHP-134上表达。用于检测这些独特的碳水化合物结构的策略是基于使用两种高度特异性的原核衍生的酶系统和抗聚唾液酸抗体（H.46）。这些探针被开发用于检测神经细胞粘附分子上的聚唾液酸（Troy，F.一、哈伦贝克，宾夕法尼亚州，麦考伊河D、和Vimr，E. R.（1987）Methods Enzymol. 138，169-185）。唾液酸长链多聚体存在的证据基于两种类型的实验，使用：1）CHP-134细胞的糖肽部分，用D-[3 H]GlcN进行代谢标记，2）CHP-134细胞的膜部分，在大肠杆菌K1唾液酸转移酶试验中作为[14 C] NeuNAc残基的外源受体。在体外，这种酶CMP-NeuNAc：聚α-2，8-唾液酸基唾液酸转移酶催化[14 C]NeuNAc从CMP-[14 C] NeuNAc转移至含有至少3个唾液酸残基的外源性受体。在第一系列实验中，使用内切-N-乙酰神经氨酸酶（Endo-N），其是一种对水解含有最少5个唾液酸残基的聚-α-2，8-唾液酸链具有特异性的噬菌体衍生酶。来自[3 H] GlcN标记细胞的3 H-糖肽的限制性Endo-N消化释放短[3 H]唾液酸寡聚物[（3 H] DP 1 -6），其被外唾液酸酶降解为[3 H]NeuNAc。部分Endo-N消化释放了一系列延伸至DP 55的[3 H]唾液酸寡聚物。分离较长（DP 20 -55）和中等大小（DP 10 -20）的寡聚体，并通过用Endo-N再处理转化为短寡聚体（（3 H）DP 1 -6），从而确认其为α-2，8-连接的[3 H]NeuNAc残基的同源寡聚体。在第二系列实验中，通过E. coliK 1唾液酸转移酶。膜部分具有高Mr和多分散性（Mr 100，000 - 250，000）的放射性的主要部分，如十二烷基硫酸钠-聚丙烯酰胺凝胶中所示。使用蛋白质印迹，显示类似大小的预先存在的材料与抗体H. 46反应。（400字处删节）
Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [(3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)