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, PTP zeta as a receptor. It has been suggested that the D-type structure (GlcA(2S)beta 1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTP zeta 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 similar to 46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K-D = similar to 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 = similar to 2.5 mu M), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20 similar to 24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K-D = 3 similar to 20 mu M), which seemed to be monovalent. When similar to 22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K-D = 0.36 similar to > 10 mu M), and the affinity correlated with the amounts of D- and E- (GlcA beta 1-3GalNAc(4S, 6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS similar to 20 mer as a unit and by the amounts of oversulfated structures.