Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods |
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Authors: | Ghaderi Darius Strauss Holger M Reinke Stefan Cirak Sebahattin Reutter Werner Lucka Lothar Hinderlich Stephan |
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Affiliation: | Charité Universit?tsmedizin Berlin, Campus Benjamin Franklin, Institut für Biochemie und Molekularbiologie, Arnimallee 22, 14195 Berlin-Dahlem, Germany. |
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Abstract: | The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key enzyme for the biosynthesis of sialic acids, the terminal sugars of glycoconjugates associated with a variety of physiological and pathological processes such as cell adhesion, development, inflammation and cancer. In this study, we characterized rat GNE by different biophysical methods, analytical ultracentrifugation, dynamic light-scattering and size-exclusion chromatography, all revealing the native hydrodynamic behavior and molar mass of the protein. We show that GNE is able to reversibly self-associate into different oligomeric states including monomers, dimers and tetramers. Additionally, it forms non-specific aggregates of high molecular mass, which cannot be unequivocally assigned a distinct size. Our results also indicate that ligands of the epimerase domain of the bifunctional enzyme, namely UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid, stabilize the protein against aggregation and are capable of modulating the quaternary structure of the protein. The presence of UDP-N-acetylglucosamine strongly favors the tetrameric state, which therefore likely represents the active state of the enzyme in cells. |
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Keywords: | GNE, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase SEC, size-exclusion chromatography AUC, analytical ultracentrifugation DLS, dynamic light-scattering SV, sedimentation velocity SE, sedimentation equilibrium |
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