Abstract
Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4-8 glucose units on Tyr 195 of glycogenin-1, but only 0-4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2-4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.
| Original language | English |
|---|---|
| Journal | B B A - Reviews on Cancer |
| Volume | 1844 |
| Issue number | 2 |
| Pages (from-to) | 398-405 |
| Number of pages | 8 |
| ISSN | 0006-3002 |
| DOIs | |
| Publication status | Published - Feb 2014 |
| Externally published | Yes |
Keywords
- Catalysis
- Catalytic Domain
- Chromatography, Liquid
- Enzyme Activation
- Gene Expression Regulation, Enzymologic
- Glucosyltransferases
- Glycoproteins
- Glycosylation
- HEK293 Cells
- Humans
- Protein Processing, Post-Translational
- Substrate Specificity
- Tandem Mass Spectrometry