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Synthesis of photo-affinity oligosaccharide probe and LacdiNAc sequence specific for secretory glycoproteins in MDCK cells

MDCK 细胞中分泌糖蛋白特异性光亲和寡糖探针和 LacdiNAc 序列的合成

基本信息

批准号：
11680618
负责人：
OHKURA Takashi
金额：
$ 2.37万
依托单位：
SASAKI INSTITUTE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680618/
关键词：
N-linked sugar chain MDCK cell lectin N-acetylalactosamine secretion photoaffinity labeling VIP36 細胞内輸送

项目摘要

As the first step to identify new intracellular lectins binding to complex type of glycans, photo-affinity oligosaccharide probe was synthesized using sulfo-SBDE.Then, to determine the target glycan for photo-affinity probe, secretory and surface glycoproteins of apical and basolateral sides were separated from metabolically labeled MDCK cells ([^3H]-glucosamine), and the respective N-linked glycans were analyzed. The [^3H]-oligosaccharides released from secretory and surface glycoproteins of apical side had about twice radioactivity than those of basolateral side, but their structures of both sides were equal to each other. Their predominant structures consisted of high mannose type glycans and biantennary complex type glycans with or without bisecting GlcNAc. The most interesting carbohydrate features were LacdiNAc sequences on the non-reducing terminal. Over 20% of oligosaccharides derived from secretory glycoproteins of both sides contained LacdiNAc, in contrast oligosaccharides from both surface glycoproteins did not contain this moiety. Moreover, the LacdiNAc could not be observed in oligosaccharides containing bisecting GlcNAc. These results indicate that the LacdiNAc is synthesized under strict substrate specificities of various glycosyl-transferases and this moiety might play an important role for secretory pathway of MDCK cells.

作为鉴定新的细胞内凝集素与复合型聚糖结合的第一步，我们利用硫代sbde合成了光亲和寡糖探针。然后，为了确定光亲和探针的目标聚糖，从代谢标记的MDCK细胞（[^3H]-氨基葡萄糖）中分离出顶端和基底外侧的分泌糖蛋白和表面糖蛋白，并对各自的n -链聚糖进行分析。根尖侧分泌的[^3H]-寡糖和表面糖蛋白释放的[^3H]-寡糖的放射性约为基底侧的两倍，但其两侧结构相同。它们的主要结构是高甘露糖型聚糖和双触角复合物型聚糖，有或没有分割GlcNAc。最有趣的碳水化合物特征是位于非还原末端的LacdiNAc序列。两侧分泌糖蛋白衍生的低聚糖中超过20%含有LacdiNAc，而表面糖蛋白衍生的低聚糖则不含该片段。此外，在含有分割GlcNAc的低聚糖中没有观察到LacdiNAc。这些结果表明，LacdiNAc是在多种糖基转移酶的严格底物特异性下合成的，该片段可能在MDCK细胞的分泌途径中发挥重要作用。