THE ROLE OF THE CARBOHYDRATE CHAINS OF GAL-BETA-1,4-GLCNAC-ALPHA-2,6-SIALYLTRANSFERASE FOR ENZYME-ACTIVITY

Galbeta-1,4-GlcNAcalpha2,6-sialyltransferase (CMP-N-acetylneuraminate: beta-galactosidealpha2,6 sialyltransferase, EC 2.4.99.1) is a glyco-pr otein containing carbohydrate chains of the complex type (Jamieson, J. C. (1989) Life Sci. 43, 691-697). The carbohydrate chains may be impor tant for controlling the expression of sialyltransferase catalytic act ivity during transit of the enzyme from the rough endoplasmic reticulu m to the Golgi complex where it is active as a membrane bound enzyme a nchored to the luminal face. To study the role of the carbohydrate cha ins of sialyltransferase for enzyme activity, conditions were establis hed in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains f rom native sialyltransferase, but methanol or ethanol had to be presen t for rapid and complete deglycosylation. Presence of methanol or etha nol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sia lyltransferase with increased deglycosylation. After deglycosylation w ith Glycanase for 18 h catalytic activity was largely eliminated and t here was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylat ed with Glycanase. At shorter times of incubation partially deglycosyl ated forms of the enzyme were detected. Complete deglycosylation of na tive or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of cataly tic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the nativ e and deglycosylated enzyme and a third form corresponding to a partia lly deglycosylated enzyme. Sialyltransferase was also subjected to seq uential treatment with exoglycosidases. Removal of NeuAc and Gal had l ittle effect on catalytic activity, but subsequent removal of GlcNAc r esulted in a significant loss in catalytic activity suggesting that th e presence of the trimannose core with GlcNAc attached is important fo r the expression of catalytic activity. The presence of organic solven ts during deglycosylation with Glycanase may be a useful method that c an be applied to other glycoproteins.