Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions |
| |
| Authors: | Nobuo Sugiura Tatsumasa Shioiri Mie Chiba Takashi Sato Hisashi Narimatsu Koji Kimata Hideto Watanabe |
| |
| Institution: | From the ‡Institute for Molecular Science of Medicine and ;¶Research Complex for the Medicine Frontiers, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195 and ;§Research Center for Medical Glycoscience, Advanced Industrial Science and Technology, Tsukuba 305-8568, Japan |
| |
| Abstract: | Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions. |
| |
| Keywords: | Chondroitin Sulfate Glycosaminoglycan Polysaccharide Sulfotransferase Surface Plasmon Resonance (SPR) ELISA |
|
|
