LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences.
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.
Department of Pathology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Johanna
J
Nilsson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Adnan
A
Halim
Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Erik
E
Larsson
Department of Pathology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Ali-Reza
AR
Moslemi
Department of Pathology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Anders
A
Oldfors
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Göran
G
Larson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden. Electronic address: jonas.nilsson@clinchem.gu.se.
Jonas
J
Nilsson
D002384:Catalysis; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D006023:Glycoproteins; C051887:glycogenin; D005964:Glucosyltransferases; C109150:GYG2 protein, human
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CID; Cell-free expression; DTT; ECD; Glc; Glucosylation; Glycogenin; Glycopeptides; Glycoproteomics; HEK; HEPES; Human Embryonic Kidney; LC; MS; PAS; XIC; collision-induced dissociation; dithiothreitol; electron capture dissociation; extracted ion chromatogram; glucose; liquid chromatography; mass spectrometry; periodic acid-Schiff