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Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates

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Abstract

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO4) (E unit) and CS containing GlcA(2SO4)-GalNAc(6SO4) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15–17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

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Acknowledgments

This work was supported by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a special research fund from Seikagaku Corporation and Yamasa Corporation.

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Authors and Affiliations

  1. Department of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi, 448-8542, Japan

    Mario Numakura, Noriko Kusakabe, Shiori Ohtake-Niimi & Osami Habuchi

  2. Bioproducts Laboratory, Biochemical Division, YAMASA CORPORATION, 10-1, Araoi-cho 2-chome, Choshi, Chiba, 288-0056, Japan

    Mario Numakura & Kazuya Ishige

  3. Aichi Medical University Research Complex for the Medicine Frontiers, Aichi Medical University, Nagakute, Aichi, 480-1195, Japan

    Hiroko Habuchi

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  1. Mario Numakura
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  2. Noriko Kusakabe
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Correspondence to Osami Habuchi.

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Numakura, M., Kusakabe, N., Ishige, K. et al. Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates. Glycoconj J 27, 479–489 (2010). https://doi.org/10.1007/s10719-010-9293-2

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  • DOI: https://doi.org/10.1007/s10719-010-9293-2

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