title: Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches.
abstract: Endogenous pleiotrophin and hepatocyte growth factor (HGF) mediate the neurite outgrowth-promoting activity of chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains isolated from embryonic pig brain. CS/DS hybrid chains isolated from shark skin have a different disaccharide composition, but also display these activities. In this study, pleiotrophin- and HGF-binding domains in shark skin CS/DS were investigated. A high affinity CS/DS fraction was isolated using a pleiotrophin-immobilized column. It showed marked neurite outgrowth-promoting activity and strong inhibitory activity against the binding of pleiotrophin to immobilized CS/DS chains from embryonic pig brain. The inhibitory activity was abolished by chondroitinase ABC or B, and partially reduced by chondroitinase AC-I. A pentasulfated hexasaccharide with a novel structure was isolated from the chondroitinase AC-I digest using pleiotrophin affinity and anion exchange chromatographies. It displayed a potent inhibitory effect on the binding of HGF to immobilized shark skin CS/DS chains, suggesting that the pleiotrophin- and HGF-binding domains at least partially overlap in the CS/DS chains involved in the neuritogenic activity. Computational chemistry using molecular modeling and calculations of the electrostatic potential of the hexasaccharide and two pleiotrophin-binding octasaccharides previously isolated from CS/DS hybrid chains of embryonic pig brain identified an electronegative zone potentially involved in the molecular recognition of the oligosaccharides by pleiotrophin. Homology modeling of pleiotrophin based on a related midkine protein structure predicted the binding pocket of pleiotrophin for the oligosaccharides and provided new insights into the molecular mechanism of the interactions between the oligosaccharides and pleiotrophin.
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan.
authors.forename: Fuchuan
authors.initials: F
authors.lastname: Li
authors.forename: Chilkunda D
authors.initials: CD
authors.lastname: Nandini
authors.forename: Tomohide
authors.initials: T
authors.lastname: Hattori
authors.forename: Xingfeng
authors.initials: X
authors.lastname: Bao
authors.forename: Daisuke
authors.initials: D
authors.lastname: Murayama
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Nakamura
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Fukushima
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D003198:Computer Simulation; D016207:Cytokines / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D055672:Static Electricity; D013464:Sulfuric Acids / Q000737:chemistry*
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D009844:Oligosaccharides; D013464:Sulfuric Acids; C066845:pleiotrophin; D003871:Dermatan Sulfate; D017228:Hepatocyte Growth Factor; D002809:Chondroitin Sulfates; D002810:Chondroitinases and Chondroitin Lyases; C033158:sulfuric acid