MOLECULAR CHARACTERIZATION OF XENOPUS EMBRYO HEPARAN-SULFATE - DIFFERENTIAL STRUCTURAL REQUIREMENTS FOR THE SPECIFIC BINDING TO BASIC FIBROBLAST GROWTH-FACTOR AND FOLLISTATIN

Enzymatic elimination of heparan sulfate (HS) causes abnormal mesoderm al and neural formation in Xenopus embryos, and HS plays an indispensa ble role in establishing the embryogenesis and tissue morphogenesis du ring early Xenopus development (Furuya, S., Sera, M., Tohno-oka, R., S ugahara, K., Shiokawa, K. and Hirabayashi, Y. (1995) Dev. Growth Diffe r. 37, 337-346). In this study, HS was purified from Xenopus embryos t o investigate its disaccharide composition and binding ability to basi c fibroblast growth factor (bFGF) and follistatin (FS), the latter bei ng provided in two isoforms with core sequences of 315 and 288 amino a cids (designated FS-315 and FS-288) originating from alternative mRNA splicing. Disaccharide composition analysis of the purified Xenopus HS showed the preponderance of a disulfated disaccharide unit with uroni c acid 2-O-sulfate and glucosamine S-N-sulfate, which has been implica ted in the interactions with bFGF. Specific binding of the HS to bFGF and FS-288, the COOH-terminal truncated form, was observed in the filt er binding assay, whereas HS did not bind to FS-315, indicating that t he acidic Glu-rich domain of FS-315 precluded the binding. The binding of the HS to bFGF or FS-288 was markedly inhibited by heparin (HP) an d various HS preparations, but not by chondroitin sulfate, supporting the binding specificity of HS. The binding specificity was further inv estigated using FS-288 and bovine intestinal [H-3]HS. Competitive inhi bition assays of the HS binding to FS-288 using size-defined HP oligos accharides revealed that the minimum size required for significant inh ibition was a dodecasaccharide, which is larger than the pentasacchari de required for bFGF binding. The binding affinity of FS to HS increas ed in the presence of activin, a growth/differentiation factor, which could be inactivated by direct binding to FS. These results, taken tog ether, indicate that the structural requirement for binding of HS to b FGF and FS is different. HS may undergo dynamic changes in its structu re during early Xenopus embryogenesis in response to the temporal and spatial expression of various growth/differentiation factors.