Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Abstract

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His 6 ) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono-and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogenrelated enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.

The Transition from Glycogen to Starch Metabolism in Cyanobacteria and Eukaryotes

“…These enzymes are notoriously unable to prime glucan elongation and require the presence of glycogenin. Glycogenin defines an autoglucosylating protein known to glucosylate selective tyrosine residues from UDP-Glc and then extend them to over 10 glucose residues (Albrecht et al 2004). It is therefore believed that the GT3 GS uses this glucosylated primer to further extend glucans.…”

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

“…The yeast Glg2p isoform has two glucosylation sites (Tyr230 and Tyr232), which are very close together, but in this protein both sites contributed equally to the self‐glucosylation [7]. A recent study [23] has shown that in bacterially produced Glg2p, both Tyr residues can be modified simultaneously in the same Glg2p monomer.…”

Rare and unusual glycosylation of peptides and proteins

“…Indeed, glycogenin has the ability to use UDP-Glc as substrate to catalyze the formation of a polymer of about 10 glucose residues and to further act as the substrate for elongation by glycogen synthase. Therefore, a-D-Glc linked to a Tyr has been isolated in this glycoprotein from rabbit-muscle 16 and -retina, 17 rabbit-18 and human-liver 19 and more recently from yeast, like Saccharomyces cerevisiae 20 and Neurospora crassa 21 (Fig. 5A).…”