Journal
GLYCOCONJUGATE JOURNAL
Volume 37, Issue 6, Pages 667-679Publisher
SPRINGER
DOI: 10.1007/s10719-020-09955-7
Keywords
Fluorogenic branched dextrin; Glycogen debranching enzyme; Glycogen storage disease type III; Phosphorylase limit dextrin; Pyridylamination
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Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (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 alpha-1,6-coupling of maltotetraose with Glc(7)-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4GlcPA (B3/31) was prepared from commercially available Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4Glc. Using alpha-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-alpha-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-alpha-1,6-glucosidase.
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