Glycosyltransferase activity can be modulated by small conformational changes of acceptor substrates.
A range of N-acetyllactosamine derivatives (compounds 4-7) that have restricted mobilities around their glycosidic linkages have been employed to determine how small changes in conformational properties of an oligosaccharide acceptor affect catalytic efficiencies of glycosylations by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI. Restriction of conformational mobility was achieved by introducing tethers of different length and chemical composition between the C-6 and C-2' hydroxyl of LacNAc. Compound 4 is a 2',6-anhydro derivative which is highly constrained and can adopt only two unusual conformations at the LacNAc glycosidic linkage. Compound 5 is modified by a methylene acetal tether and can exist in a larger range of conformations; however, the Phi dihedral angle is restricted to values smaller than 30 degrees, which are not entirely similar to minimum energy conformations of LacNAc. The ethylene-tethered 6 can attain conformations in the relatively large energy plateau of LacNAc that include syn conformations A and B, whereas compound 7, which is modified by a methylamide tether, can only reside in the B-conformer. 2',6-Dimethoxy derivative 2 was employed to determine the effect of alkylation of the C-6 and C-2' hydroxyls of 5 and 6 whereas 3 was used to reveal the effects of the C-6 amide and C-2' alkylation of 7. The apparent kinetic parameters of transfer to the conformationally constrained 4-7 and reference compounds 1-3 catalyzed by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI were determined, and the results correlated with their conformational properties. The data for 4-6 showed that each enzyme recognizes N-acetyllactosamine in a low minimum energy conformation. A small change in conformational properties such as in compound 5 resulted in a significant loss of catalytic activity. Larger conformational changes such as in compound 4 abolished all activity of the sialyltransferases whereas the fucosyltransferases showed some activity, albeit very low. The kinetic data for compounds 4 and 5 demonstrate clearly that different glycosyltransferases respond differently to conformational changes, and the fucosyltransferases lost less activity than the sialyltransferases. Correlating apparent kinetic parameters of conformationally constrained 6 and 7 and their reference compounds 2 and 3 further supports the fact that different enzymes respond differently and indicates that sialyltransferases and fucosyltransferases recognize N-acetyllactosamine in a different conformation. Collectively, the data presented here indicate that small conformational changes of an oligosaccharide acceptor induced by, for example, the protein structure can be employed to modulate the patterns of protein glycosylation.
Galan
M Carmen
MC
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602, USA.
Venot
Andre P
AP
Boons
Geert-Jan
GJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D004187:Disaccharides; D007527:Isoenzymes; D011994:Recombinant Proteins; D005647:Fucosyltransferases; D012799:Sialyltransferases