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, PTPζ as a receptor. It has been suggested that the D-type structure (GlcA(2S)β1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTPζ 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 ≥18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The ∼46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (KD = ∼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 (KD = ∼2.5 μm), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20∼24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (KD = 3∼20 μm), which seemed to be monovalent. When ∼22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (KD = 0.36 ∼ >10 μm), and the affinity correlated with the amounts of D- and E- (GlcAβ1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS ∼20 mer as a unit and by the amounts of oversulfated structures.