LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences

TY - JOUR

T1 - LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences

AU - Nilsson, Johanna

AU - Halim, Adnan

AU - Larsson, Erik

AU - Moslemi, Ali-Reza

AU - Oldfors, Anders

AU - Larson, Göran

AU - Nilsson, Jonas

N1 - Copyright © 2013 Elsevier B.V. All rights reserved.

PY - 2014/2

Y1 - 2014/2

N2 - 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.

AB - 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.

KW - Catalysis

KW - Catalytic Domain

KW - Chromatography, Liquid

KW - Enzyme Activation

KW - Gene Expression Regulation, Enzymologic

KW - Glucosyltransferases

KW - Glycoproteins

KW - Glycosylation

KW - HEK293 Cells

KW - Humans

KW - Protein Processing, Post-Translational

KW - Substrate Specificity

KW - Tandem Mass Spectrometry

U2 - 10.1016/j.bbapap.2013.11.002

DO - 10.1016/j.bbapap.2013.11.002

M3 - Journal article

C2 - 24239874

SN - 0304-419X

VL - 1844

SP - 398

EP - 405

JO - Biochimica et Biophysica Acta - Reviews on Cancer

JF - Biochimica et Biophysica Acta - Reviews on Cancer

IS - 2

ER -