Abstract
Tumor-derived adhesion factor/angiomodulin (AGM) is accumulated in tumor blood vessels and on the endothelial cell surface (Akaogi, K., Okabe, Y., Sato, J., Nagashima, Y., Yasumitsu, H., Sugahara, K., and Miyazaki, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8384-8389). In cell culture, it promotes cell adhesion and morphological changes to form cord-like structures of the human bladder carcinoma cell line ECV-304. The cord formation is prevented by heparin, which inhibits the binding of AGM to ECV-304 cells. This observation suggests that AGM interacts with cell surface heparan sulfate (HS) proteoglycans. In this study, HS glycosaminoglycans and core proteins of integral transmembrane proteoglycans, syndecan-1 and -4, were identified by immunocytochemistry on ECV-304 cells, and the structural requirements for the interaction of HS with AGM were characterized. Inhibition experiments with sulfated polysaccharides and chemically modified heparin derivatives indicated that sulfate groups were essential for both AGM-HS binding and cord-like structure formation and that the rank order of the different sulfate groups in terms of their contribution was N-sulfate > 6-O-sulfate > 2-O-sulfate. The minimum size of heparin, a chemical analog of HS, required for the binding to AGM was a dodecasaccharide as determined by competition experiments using size-defined heparin oligosaccharides. Thus, a specific sulfation pattern in the HS of cell surface syndecans of ECV-304 cells is required for AGM binding and the morphological changes.
Highlights
Akaogi et al [1] have purified a cell adhesion glycoprotein of 30 kDa, tentatively designated as tumor-derived adhesion factor (TAF),1 from conditioned medium of the human bladder
We demonstrated that the binding of AGM to the ECV-304 cell surface and the cord-like structure formation of these cells were completely inhibited by exogenously added heparin (Hep), a chemical analog of heparan sulfate (HS) [4]
ECV-304 cells produce AGM, which promotes the adhesion of these cells to type IV collagen and morphological changes to form cord-like structures when they are stimulated using exogenous type I collagen [4]
Summary
Materials—Hep preparations from bovine lung and porcine intestine were purchased from Sigma and Nacalai Tesque (Kyoto, Japan), respectively. Analysis of 3H-labeled GAGs—ECV-304 cell [3H]GAGs were isolated from each trypsinate fraction by actinase E digestion and subsequent trichloroacetic acid treatment as described previously [19] and purified by chromatography on a DEAE-cellulose column through stepwise elution with 0.5, 1.0, and 2.0 M LiCl. Because 2.0 M LiCl-eluted fractions contained little radioactivity, 0.5 and 1.0 M LiCl-eluted fractions were used for the composition analysis of GAGs. Because 2.0 M LiCl-eluted fractions contained little radioactivity, 0.5 and 1.0 M LiCl-eluted fractions were used for the composition analysis of GAGs These fractions were desalted using a Sephadex G-50 column, and the GAGs were identified by enzymatic digestion using Streptomyces hyaluronidase, chondroitinase ABC, or a mixture of heparinase and heparitinases I and II. The cell morphology was examined under a phase-contrast microscope
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