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.