Glycobiology Advance Access published online on August 25, 2004
Glycobiology, doi:10.1093/glycob/cwh132
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1 Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 110-460, Korea
* To whom correspondence should be addressed. E-mail: kims{at}plaza.snu.ac.kr.
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
Revised July 19, 2004
Accepted July 22, 2004
ORIGINAL ARTICLES
Nucleolin: acharan sulfate-binding protein on the surface of cancer cells
2 Department of Biochemistry, NCMLS, UMC Nijmegen, 6500 HB Nijmegen, The Netherlands
3 Graduate School of Pharmaceutical Science, Chiba University, Chiba 263-8522, Japan
4 Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA; Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA; Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA
Abstract 
-D-N-acetylglucosaminyl (1
4) 2-sulfoiduronic acid. Exogenous AS was injected s.c. 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 tissue with alcian blue and periodic acid-Schiff (PAS) reagent. In vitro assays indicated that the binding of cells to 50 µg/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 was 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 sodium dodecyl sulfate (SDS) polyacrylamide gel-electrophoresis (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. Electrospray ionization quadrapole time-of-flight mass spectral (ESI Q-TOF MS) analysis of one of these bands, MW 
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). The above 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.
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