Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods.
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.
Ghaderi
Darius
D
Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Biochemie und Molekularbiologie, Arnimallee 22, 14195 Berlin-Dahlem, Germany.
Strauss
Holger M
HM
Reinke
Stefan
S
Cirak
Sebahattin
S
Reutter
Werner
W
Lucka
Lothar
L
Hinderlich
Stephan
S
D000818:Animals; D001703:Biophysics / Q000379:methods; D002238:Carbohydrate Epimerases / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008027:Light; D008968:Molecular Conformation; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D014461:Ultracentrifugation / Q000379:methods
D008024:Ligands; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; D019158:N-Acetylneuraminic Acid