The binding of chondroitin sulfate to pleiotrophin/heparin-binding growth-associated molecule is regulated by chain length and oversulfated structures.
Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTPzeta as a receptor. It has been suggested that the D-type structure (GlcA(2S)beta1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTPzeta and pleiotrophin. Here, we analyzed the interaction of shark cartilage CS-D with pleiotrophin using a surface plasmon resonance biosensor to reveal the importance of D-type structure. CS-D was partially digested with chondroitinase ABC, and fractionated using a Superdex 75pg column. The > or =18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The approximately 46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K(D) = approximately 30 nM), and the binding reactions fitted the bivalent analyte model. However, when the density of the immobilized pleiotrophin was lowered, the strength of affinity remarkably decreased (K(D) = approximately 2.5 microM), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20 approximately 24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K(D) = 3 approximately 20 microM), which seemed to be monovalent. When approximately 22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K(D) = 0.36 approximately >10 microM), and the affinity correlated with the amounts of D- and E- (GlcAbeta1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS approximately 20 mer as a unit and by the amounts of oversulfated structures.
Maeda
Nobuaki
N
Department of Developmental Neuroscience, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. maedan@tmin.ac.jp
Fukazawa
Nobuna
N
Hata
Toshihiro
T
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002356:Cartilage; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016207:Cytokines / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D012754:Sharks; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D016207:Cytokines; D009844:Oligosaccharides; C066845:pleiotrophin; D006493:Heparin; D002809:Chondroitin Sulfates; D019765:Chondroitin ABC Lyase