The Binding of Chondroitin Sulfate to Pleiotrophin/Heparin-binding Growth-associated Molecule Is Regulated by Chain Length and Oversulfated Structures

Abstract

Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTP as a receptor. It has been suggested that the D-type structure (GlcA(2S)␤1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTP 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 ϳ46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K D ‫؍‬ ϳ30 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 ‫؍‬ ϳ2.5 M), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20ϳ24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K D ‫؍‬ 3ϳ20 M), which seemed to be monovalent. When ϳ22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K D ‫؍‬ 0.36 ϳ >10 M), and the affinity correlated with the amounts of D-and E-(GlcA␤1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS ϳ20 mer as a unit and by the amounts of oversulfated structures.Pleiotrophin, also known as heparin-binding growth-associated molecule (HB-GAM) 2 or heparin affin regulatory peptide (HARP), is an 18-kDa growth factor that shows 45% amino acid sequence identity with midkine (1, 2). Pleiotrophin and midkine share many biological activities such as the promotion of neurite outgrowth and migration of embryonic neurons and osteoblasts (1, 2). Furthermore, it has been revealed that both use a common signal-transducing receptor, receptortype protein-tyrosine phosphatase (PTP) (3-7). PTP is synthesized as a membrane-bound chondroitin sulfate (CS) proteoglycan, and its extracellular variant, which is generated by alternative splicing, is secreted as a major soluble CS proteoglycan in the brain, phosphacan (8, 9).The binding of phosphacan to pleiotrophin and midkine depends on the CS portion of this proteoglycan, and the removal of CS resulted in a remarkable decrease in the binding affinity (9, 10). It was revealed that pleiotrophin inactivated the tyrosine phosphatase activity of this receptor leading to an increase in the tyrosine phosphorylation levels of specific substrates such as cat-1 and ␤-catenin (4, 11). Several researchers suggested that pleiotrophin induces the dimerization of PTP, which results in the inactivation of its enzymatic activity (4, 11).On the other hand, midkine and pleiotrophin easily formed noncovalently bound multimers, and it has be...

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

“…In SPR analysis, native CSD and CSE showed high affinity to MK and PT, consistent with previous reports (33)(34)(35), and a comparison between the native and synthetic CS reveals distinct features. MK bound to sCS containing diSD units, but with a lower affinity than to one containing diSE units.…”

Receptor Type Protein Tyrosine Phosphatase ζ-Pleiotrophin Signaling Controls Endocytic Trafficking of DNER That Regulates Neuritogenesis

“…The binding of PTN with phosphacan depends on both the chondroitin sulfate and protein portions of this proteoglycan, and removal of chondroitin sulfate resulted in marked decrease of the binding affinity (22,25,26). Phosphacan and PTN are deposited between the Purkinje cell surface and the processes of Bergmann glia, and chondroitinase ABC digestion of cerebellar slices remarkably reduced the PTN content in the tissue (40).…”

Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

“…CS/DS not only play structural roles in connective tissues, such as cartilage and bone, but also participate in various important physiological and pathological processes, such as development (1), neuronal growth (2-5), inflammation (6), tumor progression (7,8), and infection (9 -12), by interacting with a large number of growth factors, growth factor receptors, and other target proteins (13,14). Various functions of CS/DS have been attributed to their variety of structures.…”