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Papers In Press, published online ahead of print August 23, 2005
Department of Biochemistry, Kobe Pharmaceutical University, Kobe, Hyogo 658-8558
Corresponding Author: k-sugar{at}kobepharma-u.ac.jp
Mammalian brains contain significant amounts of chondroitin sulfate (CS), dermatan sulfate (DS) and CS/DS hybrid chains. CS/DS chains isolated from embryonic pig brains (E-CS/DS) promote the outgrowth of neurites in embryonic mouse hippocampal neurons in culture by interacting with pleiotrophin (PTN), a heparin-binding growth factor. Here, we analyzed oligosaccharides isolated from E-CS/DS, which showed that octasaccharides were the minimal size capable of interacting with PTN at a physiological salt concentration. Five and eight sequences were purified from fluorescently labeled PTN-bound and -unbound octasaccharide fractions, respectively, by enzymatic digestion followed by PTN-affinity chromatography. Their sequences were determined by enzymatic digestion in conjunction with high performance liquid chromatography, revealing a critical role for oversulfated D and/or iD disaccharides in the low yet significant affinity for PTN, which is required for neuritogenesis. The critical D and iD units are GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate) and IdoUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate), respectively, where IdoUA represents L-iduronic acid. In contrast, high-affinity interactions with PTN required decasaccharides with E units [GlcUAbeta1-3GalNAc(4, 6-O-disulfate)], B units [GlcUA(2-O-sulfate)beta1-3GalNAc(4-O-sulfate)] and/or their IdoUA-containing counterparts (iE and iB) in addition to D/iD units, although the biological significance of such strong interactions remains to be investigated. Thus, chain size and composition are crucial to the interaction with PTN, and PTN binds to multiple sequences in E-CS/DS chains with distinct affinity. Notably, not only heparan sulfate but also CS/DS hybrid chain structures of mammalian brains contain a high degree of microheterogeneity with a cluster of oversulfated disaccharides and appear to play roles in regulating the functions of PTN.
J. Biol. Chem, 10.1074/jbc.M507304200
Submitted on July 6, 2005
Revised on August 23, 2005
Accepted on August 23, 2005
Demonstration of the pleiotrophin-binding oligosaccharide sequences isolated from chondroitin sulfate/dermatan sulfate hybrid chains of embryonic pig brains
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