Ubiquitylation of unbranched glycogen and glycogenin-1 in branching enzyme-deficient cells

Glycogen molecules deficient in α1:6 glycosidic bonds (branch points) must be eliminated rapidly from cells, otherwise they accumulate and precipitate causing human disease, but how unbranched glycogen is marked selectively for destruction is unclear. We report here that unbranched glycogen is polyubiquitylated in glycogen branching enzyme knockout HEK293T cells, but normally branched glycogen in wild-type cells is not. The polyubiquitin liberated from unbranched glycogen by incubation with hydroxylamine or digestion with α-amylase was similar in size and quantity, indicating that polyubiquitin is linked to unbranched glycogen by an ester bond(s). The ubiquitin molecules forming the polyubiquitin were linked predominantly via Met1, Lys11 and Lys48 of ubiquitin, with smaller amounts linked via Thr12 and Ser20 of ubiquitin. We also report that glycogenin-1, the enzyme that initiates glycogen synthesis, is attached to unbranched glycogen covalently and ubiquitylated at Lys342 and Lys344. Our study has established that polysaccharides can be ubiquitylated in cells as well as proteins. We suggest that polyubiquitylation marks unbranched glycogen for uptake into lysosomes and discuss the E3 ligases that may participate in the polyubiquitylation of unbranched glycogen and glycogenin-1.