Molecular analysis on activity regulation of growth factors by a new glycosaminoglycan.

新型糖胺聚糖对生长因子活性调节的分子分析。

基本信息

批准号：
14572029
负责人：
TOIDA Toshihiko
金额：
$ 2.24万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Acharan sulfate (AS) is a new type of glycosaminoglycan from the giant African snail, Achatina fulica. AS having a primarily repeating disaccharide structure of α-D-Nacetylglucosaminyl 2-sulfoiduronate was studied as an antitumor agent using both in vivo and In vitro assays. We have found that this glycosaminoglycan binds specifically to serum ceruloplasmin. We also investigated the antitumor activity of AS in vivo as well as In vivo. To confirm the localization of exogeneous AS after s.c. injection in tumor of C57BL/6 mice implanted with Lewis lung carcinoma cells (LLC), the sectioned tissue was stained by Alcian blue/periodic acid-Schiff reagent. Exogenous AS was found to be localized mainly on the outer membrane of tissues. In vitro binding assay indicated that the binding of AS to. cells was markedly increased when 50 μg/ml of the sample was incubated for 5 hr. In the immunofluorescence assay using anti-AS antibody, AS was also detected around the cell surface membrane. Based on the fact that AS can bind the protein on cancer cell surface, the outer cell surface was biotinylated to identify any paricular binding protein. The biotinylated cells were lysed and the lysates were fractionated on AS affinity column with a stepwise salt gradient. Each fraction was visualized by SDS-PAGE and Western blotting. We focused on the protein eluted at 1 M NaCl and detected only two bands of which the molecular weights are approximately 118,000 and 52,000 Da. By ESI Q-TOF mass spectrometry, one band representing 110 kDa was determined as nucleolin, which is in a phosphorylated form on the cell surface of different cells as a cell surface receptor for various ligands, especially growth factors such as bFGF and midkines. The above results suggest that AS could show the inhibitory effects against tumor growth by. binding to specific receptors of cell surface.

Acharan 硫酸盐 (AS) 是一种来自非洲巨型蜗牛 Achatina fulica 的新型糖胺聚糖，具有 α-D-N 乙酰氨基葡萄糖基 2-磺基糖醛酸的主要重复二糖结构，通过体内和体外试验研究了其作为抗肿瘤剂的作用。我们发现这种糖胺聚糖与血清铜蓝蛋白特异性结合，我们还研究了其抗肿瘤作用。为了确认皮下注射路易斯肺癌细胞 (LLC) 的 C57BL/6 小鼠肿瘤中外源性 AS 的定位，用阿新蓝/高碘酸对切片组织进行染色。 Schiff试剂发现外源AS主要位于组织的外膜上，当50μg/ml时，AS与细胞的结合显着增加。样品孵育5小时，在使用抗AS抗体的免疫荧光检测中，在细胞表面膜周围也检测到AS。基于AS可以结合癌细胞表面的蛋白质，对细胞外表面进行生物素化以进行识别。裂解生物素化的细胞，并使用逐步盐梯度在 AS 亲和柱上分级分离每个级分，我们重点关注蛋白质。用 1 M NaCl 洗脱，仅检测到两条分子量约为 118,000 和 52,000 Da 的条带，通过 ESI Q-TOF 质谱分析，确定一条代表 110 kDa 的条带为核仁素，其在细胞表面呈磷酸化形式。不同细胞作为各种配体的细胞表面受体，特别是生长因子，例如bFGF和midkines，上述结果表明AS可以表现出不同的配体。通过与细胞表面的特定受体结合来抑制肿瘤生长。