Glycogenin is a self-glucosylating protein, involved in the de novo biosynthesis of glycogen in eukaryote organisms. The enzyme catalyzes two distinct chemical reactions, the incorporation of glucose to the hydroxyl group of its tyrosine 194 and the subsequent incorporation of glucoses to the first acquired one. This reaction decreases its efficiency as the oligoglucan is ~10 residues long. The sugar donor for both reactions is UDP-glucose (UDPG), and the presence of Mn+2 cations is absolutely required. At the end of the biosynthetic pathway glycogenin is found as a constituent of the newly synthesized proteoglycogen molecule.
In our laboratory, the amphiphatic character of glycogenin was described for the first time1. We continued these studies, using the monolayer technique in order to compare the amphiphatic behavior of glucosylated and non glucosylated glycogenin.
We first performed penetration experiments in preformed lipids monolayers to study the behavior of a recombinant partially glucosylated glycogenin obtained by expression in E. coli wild type (GR-WT). GR-WT was characterized by MALDI-TOF mass spectrometry showing a glucopolymerization degree which varies between 0-10 glucoses. GR-WT preferentially interacts with sphyngolipids and glycoesphyngolipids monolayers rather than with phospholipids ones. The better interaction found was with ceramide, a lipid known to form condensed monolayers.
Glycogenin devoid of covalently attached glucose (GR-P) was prepared by expression in an E. coli strain defective in UDPG production. The absence of glucose was confirmed by mass spectrometry. Due to the great susceptibility of GR-P to proteolysis, we decided to express the protein lacking a portion of 42 aminoacids of its C-terminus (GR31-P) in order to obtain a homogeneous preparation. Incubating GR31-P with UDPG and Mn+2 ions we obtained GR31-Pglc, with a ~12 glucose oligoglucan attached. Both proteins adsorb from the subphase as Gibbs monolayers inducing a rapid increase of the surface pressure and a variation in surface potential. We also study the compressional behavior of the Langmuir monolayers of both proteins. Reproducible isotherms for both were observed, but the collapse pressure of GR31-P was slightly higher and occurred at smaller cross-sectional areas than those of GR31-Pglc. When comparing the penetration of GR-31P and of GR31-Pglc to preformed lipids monolayers no major differences were found, but both species showed greater interaction with all the lipids tested in comparison with GR-WT. We extend our studies to other lipids, and found that GR31-P penetrates better when injected beneath monolayers of lipids that are self?organized in more condensed states rather than in expanded ones.
The amphiphilic behavior of glycogenin whether linked to an oligoglucan and lacking a portion of its C-terminus is discussed.
[1] Carrizo, M.E., Miozzo, M.C., Maggio, B. and Curtino J.A. FEBS Let.,2001, 509, 323-326.
Acknowledgments: This work was supported by FONCYT and CONICET. We special thanks to Universidad de La Laguna,
