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

硫酸Acharan（AS）是从非洲大蜗牛Achatinafulica中提取的一种新型糖胺聚糖。用体内和体外试验研究了具有α-D-N乙酰葡糖胺基2-磺基艾杜糖醛酸酯（α-D-N acetylglucosaminyl 2-sulfiduronate）的主要重复二糖结构的AS作为抗肿瘤剂。我们已经发现，这种糖胺聚糖特异性结合血清铜蓝蛋白。我们还研究了AS在体内以及在体内的抗肿瘤活性。为了证实s.c.接种刘易斯肺癌细胞（LLC）的C57 BL/6小鼠的肿瘤内注射，切片组织用阿辛蓝/高碘酸-希夫试剂染色。外源性AS主要定位于组织外膜。体外结合实验表明AS与. 50 μg/ml的样品孵育5小时后，细胞内AS含量明显增加，用抗AS抗体进行免疫荧光检测，在细胞膜表面也检测到AS。基于AS可以结合癌细胞表面蛋白的事实，将细胞外表面生物素化以鉴定任何特定的结合蛋白。将生物素化的细胞裂解，并将裂解物在AS亲和柱上用逐步盐梯度分级。通过SDS-PAGE和蛋白质印迹法观察每个级分。我们聚焦于在1 M NaCl洗脱的蛋白质，仅检测到分子量约为118，000和52，000 Da的两条带。通过ESI Q-TOF质谱，代表110 kDa的一条带被确定为核仁素，其在不同细胞的细胞表面上以磷酸化形式作为各种配体的细胞表面受体，特别是生长因子如bFGF和中期因子。以上结果提示AS对肿瘤生长有抑制作用。与细胞表面的特异性受体结合。

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.