Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis

Renato M. De Paula, Wayne A. Wilson, Peter Roach, Héctor F. Terenzi, Maria Célia Bertolini

Research output: Contribution to journalArticle

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Abstract

Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K m (4.41 μM) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN.

Original languageEnglish
Pages (from-to)2208-2214
Number of pages7
JournalFEBS Letters
Volume579
Issue number10
DOIs
StatePublished - Apr 11 2005

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Neurospora crassa
Glycogen
Glycogen Synthase
Biosynthesis
Uridine Diphosphate Glucose
glycogenin
Mutagenesis
Two-Hybrid System Techniques
Arches
Site-Directed Mutagenesis
Oligosaccharides
Yeast
Escherichia coli
Saccharomyces cerevisiae
Assays
Mass Spectrometry
Catalytic Domain
Complementary DNA
Spectroscopy
Peptides

Keywords

  • Glycogen
  • Glycogenin
  • Mass spectrometry
  • Neurospora crassa
  • Site-directed mutagenesis
  • Yeast complementation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis. / De Paula, Renato M.; Wilson, Wayne A.; Roach, Peter; Terenzi, Héctor F.; Bertolini, Maria Célia.

In: FEBS Letters, Vol. 579, No. 10, 11.04.2005, p. 2208-2214.

Research output: Contribution to journalArticle

De Paula, Renato M. ; Wilson, Wayne A. ; Roach, Peter ; Terenzi, Héctor F. ; Bertolini, Maria Célia. / Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis. In: FEBS Letters. 2005 ; Vol. 579, No. 10. pp. 2208-2214.
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