UDP-N-ACETYL-ALPHA-D-GALACTOSAMINE-POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE - IDENTIFICATION AND SEPARATION OF 2 DISTINCT TRANSFERASE ACTIVITIES

Using a defined acceptor substrate peptide as an affinity chromatograp hy ligand we have developed a purification scheme for a unique human p olypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase ( GalNAc-transferase) (White, T., Bennett, E. P., Takio, K., Sorensen, T ., Bonding, N., and Clausen, H. (1995) J, Biol. Chem, 270, 24156-24165 ). Here we report detailed studies of the acceptor substrate specifici ty of GalNAc-transferase purified by this scheme as well as the GalNAc -transferase activity, which, upon repeated affinity chromatography, e vaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separa ted transferase preparations was identified. Analysis of GalNAc-transf erase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the exist ence of multiple GalNAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of Gal NAc-transferases existing is unknown, as is the specificity of the unt il now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme prote ins. The identification of acceptor peptides that can be used to discr iminate GalNAc-transferase activities is an important step toward unde rstanding the molecular basis of GalNAc O-linked glycosylation in cell s and organs and in pathological conditions.