Functions of chondroitin sulfate/dermatan sulfate chains in brain development -: Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7
Functions of chondroitin sulfate/dermatan sulfate chains in brain development -: Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7
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DOI：
10.1074/jbc.m700630200
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发表时间：
2007-07-06
影响因子：
4.8
通讯作者：
Sugahara, Kazuyuki
中科院分区：
文献类型：
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作者：
Purushothaman, Anurag;Fukuda, Junko;Sugahara, Kazuyuki
硫酸软骨素(CS)和硫酸皮肤素(DS)参与了脑神经发育过程。在这项研究中，我们使用噬菌体展示技术产生的单链抗体GD3G7来表征发育调节的脑CS/DS链。GD3G7对各种糖胺多聚糖制剂的特异性评价表明，该抗体可与鱿鱼CS-E(富含GlcUAβ1-3GalNAc(4，6-O-硫酸盐)二糖单元)、八角鳗CS-H(富含IdoUAα1-3GalNAc(4，6-O硫酸酯)单元)和鲨鱼皮肤DS(富含E和Ie单元)发生特异性反应。对参与CS/DS-E生物合成的N-乙酰半乳糖胺-4-硫酸盐6-O-磺酸转移酶在出生后小鼠脑内的表达进行了原位杂交，结果表明，该转录物在发育中的小鼠脑中广泛表达，但在出生后第7天，该转录物在小脑的外颗粒细胞层有较强的表达。随着发育，表达由外颗粒细胞层向内颗粒细胞层转移。GD3G7在小鼠脑内的免疫组织化学定位显示，GD3G7表位在小脑、海马体和嗅球中相对丰富。GD3G7抑制CS-E介导的胚胎海马神经元突起的生长，提示该表位嵌入在CS-E的突起生长促进基序中。此外，还发现CS-E脱糖组分是GD3G7识别所需的关键最小结构。确定了四个离散的脱糖表位序列。GD3G7抗体在中枢神经系统发育和各种病理条件下CS/DS链的研究中有着广泛的应用。
Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the specificity of GD3G7 toward various glycosaminoglycan preparations showed that this antibody specifically reacted with squid CS-E (rich in the GlcUA beta 1-3GalNAc(4,6-O-sulfate) disaccharide unit E), hagfish CS-H (rich in the IdoUA alpha 1-3GalNAc(4,6-Osulfate) unit iE), and shark skin DS (rich in both E and iE units). In situ hybridization for the expression of N-acetylgalactosamine-4-sulfate 6-O-sulfotransferase in the postnatal mouse brain, which is involved in the biosynthesis of CS/DS-E, showed a widespread expression of the transcript in the developing brain except at postnatal day 7, where strong expression was observed in the external granule cell layer in the cerebellum. The expression switched from the external to internal granule cell layer with development. Immunohistochemical localization of GD3G7 in the mouse brain showed that the epitope was relatively abundant in the cerebellum, hippocampus, and olfactory bulb. GD3G7 suppressed the growth of neurites in embryonic hippocampal neurons mediated by CS-E, suggesting that the epitope is embedded in the neurite outgrowth- promoting motif of CS-E. In addition, a CS-E decasaccharide fraction was found to be the critical minimal structure needed for recognition by GD3G7. Four discrete decasaccharide epitopic sequences were identified. The antibody GD3G7 has broad applications in investigations of CS/DS chains during the central nervous system's development and under various pathological conditions.