Glycosaminoglycan structures required for strong binding to midkine, a heparin-binding growth factor

Abstract

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.

Heterogeneity of the Chondroitin Sulfate Portion of Phosphacan/6B4 Proteoglycan Regulates Its Binding Affinity for Pleiotrophin/Heparin Binding Growth-associated Molecule

“…However, squid CS-E contains a substantial amount of unusual 3-O-sulfated glucuronic acid. Accordingly, Zou et al (14) prepared artificial CS-E without 3-O-sulfation of glucuronic acid using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase and demonstrated that this preparation also bound strongly with midkine. These findings suggested that oversulfated portions in chondroitin sulfate contribute to the strong binding with heparin binding growth factors.…”

Identification and Functions of Chondroitin Sulfate in the Milieu of Neural Stem Cells

“…Recently, neural precursor cells have been shown to synthesize and secrete CSPGs, including some lectican family members (14). In addition, it has been reported that cultured tissue segments of E13 mouse brains synthesize CS rich in a disaccharide unit containing 4,6-disulfated N-acetylgalactosamine (E-unit) and that the CS preparation has an affinity for midkine, a heparin-binding growth factor (15). A commercial preparation including chondroitin sulfate E (CS-E) from the squid cartilage binds with a high affinity to various heparin-binding growth factors such as FGFs, pleiotrophin, and midkine (16).…”

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

“…Some researchers reported that CS-E but not CS-D strongly bound with midkine and suggested that the E-type structure was the critical determinant of the binding affinity for midkine (17)(18)(19). Other studies, however, indicated that CS-E strongly bound with pleiotrophin, but CS-D also bound to * This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, Mizutani Foundation for Glycoscience, and from the Uehara Memorial Foundation.…”

“…Some researchers reported that CS-E but not CS-D strongly bound with midkine and suggested that the E-type structure was the critical determinant of the binding affinity for midkine (17)(18)(19). Other studies, however, indicated that CS-E strongly bound with pleiotrophin, but CS-D also bound to this growth factor with high affinity (16,20,21).…”