Structure of pleiotrophin- and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches.
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.
Li
Fuchuan
F
Faculty of Advanced Life Science, Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan.
Nandini
Chilkunda D
CD
Hattori
Tomohide
T
Bao
Xingfeng
X
Murayama
Daisuke
D
Nakamura
Toshikazu
T
Fukushima
Nobuhiro
N
Sugahara
Kazuyuki
K
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*
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