Abstract
Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1–4Glcα1–4Glcα1–4Glcα1–4(Glcα1–4Glcα1–4Glcα1–4Glcα1–6)Glcα1–4Glcα1–4Glcα1–4GlcPA (B4/84, where Glc = d-glucose and GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-d-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1–4Glcα1–4Glcα1–4Glcα1–4(Glcα1–4Glcα1–4Glcα1–4Glcα1–6)Glcα1–4Glcα1–4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1–6Glcα1–4Glcα1–4GlcPA (B3/31) was prepared from commercially available Glcα1–6Glcα1–4Glcα1–4Glc. Using α-cyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1–4Glcα1–4Glcα1–4Glcα1–4(Glcα1–6)Glcα1–4Glcα1–4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase.
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The data that supported the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We are grateful to Prof. Takaaki Miyaji and Ms. Asako Kawakami from the Department of Genomics & Proteomics, Okayama University for performing the MALDI-TOF MS analysis of the fluorogenic branched dextrins. We would also like to thank Amano Enzyme (Nagoya, Aichi, Japan) for providing CGTase (trade name, Contizyme).
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Sakaguchi, M., Makino, Y. & Matsubara, H. New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme. Glycoconj J 37, 667–679 (2020). https://doi.org/10.1007/s10719-020-09955-7
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DOI: https://doi.org/10.1007/s10719-020-09955-7