Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM.
Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM.
复制标题
牛 NCAM 中含聚唾液酸的 N 连接聚糖的定位和表征。
作者：
M. von der Ohe;S. Wheeler;M. Wuhrer;D. Harvey;S. Liedtke;M. Mühlenhoff;R. Gerardy;H. Geyer;R. Dwek;R. Geyer;D. R. Wing;M. Schachner
神经细胞粘附分子（NCAM）在成人神经系统的发育、可塑性和再生过程中起着重要作用。其功能受到不寻常的α 2-8-连接的聚唾液酸（PSA）的附着的强烈影响。在这里，我们分析了来自新生小牛脑的聚唾液酸化NCAM的N-糖基化模式。纯化的PSA-NCAM糖蛋白用胰蛋白酶消化，并通过免疫亲和层析分离PSA-糖肽。为了确定N-糖基化位点，通过Edman降解和基质辅助激光解吸/电离质谱法（MALDI-MS）分析PNGase F处理的糖肽。发现它们仅与NCAM的第五免疫球蛋白样结构域中的第五（Asn 439）和第六（Asn 468）N-糖基化位点相连。阴离子交换层析结果表明，PSA的链长至少由30个唾液酸残基组成。为了分析核心结构，通过SDS-PAGE分离经神经内磷酸酶N处理的PSA-NCAM，并用PNGase F消化。采用MALDI-MS结合糖苷酶外切酶双链反应和色谱分离对聚唾液酸聚糖的核心结构进行了表征。它们包括杂合、二-、三-和少量的四触角碳水化合物，这些碳水化合物都在最里面的N-乙酰葡糖胺处被岩藻糖基化。对于三触角聚糖，“2，6”臂在聚唾液酸化结构中是优选的。在多聚唾液酸化结构上发现了高水平的硫酸化基团，在较低程度上也在非多聚唾液酸化聚糖上发现了硫酸化基团。此外，在PSA-NCAM糖型上可检测到高甘露糖型聚糖，范围为（GlcNAc）（2）（Man）（5）至（GlcNAc）（2）（Man）（9）。总之，我们观察到连接到NCAM分子的PSA-聚糖的结构变异性和高区域选择性，这最有可能影响其生物学功能。
The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.
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DOI：
10.1073/pnas.81.7.1971
发表时间：
1984
影响因子：
11.1
作者：
Vimr,ER;McCoy,RD;Vollger,HF;Wilkison,NC;Troy,FA
通讯作者：
Troy,FA
DOI：
10.1016/s0021-9258(19)76560-0
发表时间：
1988-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
B D Livingston;J. Jacobs;M C Glick;F. A. Troy
通讯作者：
B D Livingston;J. Jacobs;M C Glick;F. A. Troy
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DOI：
--
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Iwasaki,M;Inoue,S;Troy,FA
通讯作者：
Troy,FA
采用脉冲电流分析法的高性能阴离子交换色谱法分离 5-N-乙酰神经氨酸、5-N-羟乙酰神经氨酸和 2-酮-3-脱氧-D-甘油-D-半乳糖壬酸的低聚物/聚合物
影响因子：
2.9
作者：
Zhang,Y;Inoue,Y;Inoue,S;Lee,YC
通讯作者：
Lee,YC