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Figure 6
Calculation of dissociation constants
(Kd) for the binding between 293- and CHO-derived glypican-AP fusion proteins against Type I collagen (A), bFGF (B), and laminin-1 (C). For each of the ACE electrophoretograms in Figure 4, retardation coefficients R’s were determined and plotted against ligand concentrations as described in Materials and Methods.  For each plot a Kd value was calculated. The results are shown in Figure 7.

Taken together, these results suggest that the HS moieties on glypican are able to mediate selective binding against extracellular molecules. The fact that glypican produced from different cell lines bind some extracellular molecules with similar binding affinities and other molecules with significantly different affinities suggest that HS-mediated binding may depend on subtle differences in glycanation patterns, consistent with previous findings (Sanderson et al. 1994). An alternative explanation is that binding against bFGF depends primarily on the core protein, which is the same across cell lines; binding against collagen depends primarily on the glycanation patterns, which likely differ across cell lines. This interpretation, however, is not likely to be correct since the biological activity of bFGF is found to be greatly influenced by direct binding to heparin and HS (Ishihara et al. 1994).

 

Using AP-tagging and ACE to measure the binding affinities of glypican-AP fusion proteins derived from two different cell lines against three common extracellular ligands, we have found that differences exist in glycanation patterns for the two mammalian cell lines­293 and CHO (inferred from cell-type dependent differences in binding affinities against collagen)­and these differences affect the binding of glypican against some, but not all, ligands. Similarities across cell types in the binding affinities of glypican against bFGF and laminin-1 suggest that some features in the glycanation pattern of the glypican molecule are preserved across the cell types examined. On the other hand, cell-type dependent differences in the binding affinity of glypican against collagen suggest that glypican has at least some differences in glycanation patterns in different cell lines. These differences may not simply reflect gross discrepancies in chain length or charge. J. D. San Antonio et al. (1993) found that differences in the binding affinities of subpopulations of low-molecular weight heparin against extracellular matrix molecules are likely to be the result of more subtle structural features of heparin, such as specific sugar chain composition and structure rather than chain length or charge. Further studies are necessary to determine the differences in glycanation patterns across cell types that affect PG binding and the significance of these differences in vivo.

Acknowledgements

I would like to thank Dr. Arthur Lander for his guidance and support as well as Rob Chen and Asli Khumbasar for their generosity and helpful advice. This project was supported in part by a grant from the Undergraduate Research Opportunities Program at the University of California, Irvine.

Works Cited

Aviezer D., E. Levy, M. Safran, C. Svahn, E. Buddecke, A. Schmidt, G. David, I. Vlodavsky, and A. Yayon. "Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor." J. Biol. Chem. 269 (1994): 114-21.

Bonneh-Barkay D., M. Shlissel, B. Berman, E. Shaoul, A. Admon, I. Vlodavsky, D. J. Carey, V. K. Asundi, R. Reich-Slotkey, and D. Ron. "Identification of

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