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

阿查兰硫酸盐(AS)是从非洲巨型蜗牛Achat ina fulica中提取的一种新型糖胺多糖。由于具有α-D-N-乙酰氨基葡萄糖-2-磺酸二糖酯的主要重复二糖结构，因此在体内和体外实验中对其作为抗肿瘤药物进行了研究。我们发现这种糖胺多聚糖能与血清铜蓝蛋白特异性结合。我们还考察了AS在体内和体内的抗肿瘤活性。确定S.C.后外源AS的定位。将Lewis肺癌细胞(LLC)接种于C57BL/6小鼠的肿瘤内，用Alcian蓝/高碘酸-Schiff试剂对切片进行染色。外源As主要定位于组织外膜。体外结合实验表明，AS与TO的结合。当50μg/ml的样品孵育5小时后，细胞数显著增加。在免疫荧光检测中使用抗AS抗体，AS也在细胞表面膜周围检测到。基于AS可以结合癌细胞表面的蛋白质的事实，细胞外表面被生物素化以识别任何颗粒结合蛋白。生物素化的细胞被裂解，裂解产物在AS亲和层析柱上以分级盐梯度进行分级。每一组分均经SDS-PAGE和Western blotting检测。我们将重点放在1M氯化钠洗脱的蛋白质上，只检测到两条相对分子质量约为118,000和52,000 Da的条带。经ESI Q-TOF质谱仪鉴定，其中一条约110 kDa的谱带为核仁素，它以磷酸化形式存在于不同细胞的细胞表面，作为多种配体的细胞表面受体，尤其是生长因子如碱性成纤维细胞生长因子和中间体。上述结果提示，AS可能通过以下途径抑制肿瘤生长。与细胞表面的特定受体结合。

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.