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

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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.