Glycosaminoglycans (GAGs) are complex
polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of
proteins.
Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating
disaccharide structure alpha-D-N-acetylglucosaminyl (1-->4) 2-sulfoiduronic
acid. Exogenous AS was injected subcutaneously near the
tumor tissue in C57BL/6 mice that had been implanted with
Lewis lung carcinoma cells (LLCs). The location of AS in the
tumor was assessed by staining of sectioned tissues with
alcian blue and
periodic acid-Schiff (PAS)
reagent. In vitro assays indicated binding of cells to 50 microg/ml AS (or
heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-
binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise
salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS-PAGE with
silver staining and western blotting. We focused on the
proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight approximately 110 kDa, showed it to be
nucleolin. A phosphorylated form of
nucleolin on the surface of cells acts as a
cell surface receptor for a variety of
ligands, including
growth factors (i.e.,
basic fibroblast growth factor) and
chemokines (i.e.,
midkine). These results show that
nucleolin is one of several AS-
binding proteins and suggest that AS might demonstrate its
tumor growth inhibitory activity by binding the
nucleolin receptor
protein on the surface of
cancer cells.