THE EXTENT OF POLYLACTOSAMINE GLYCOSYLATION OF MDCK LAMP-2 IS DETERMINED BY ITS GOLGI RESIDENCE TIME

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cu ltured for 1 day relative to cells cultured for 3 days has been correl ated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mel. Biol, Cell., 4, 627-635), To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosylt ransferase expression levels, the activities of beta 1-6GlcNAc-TV, bet a 1-3GlcNAc-T(i), beta 1-2GlcNAc-TI, beta 1,4Gal-T, alpha 2-6sialyl-T, and alpha 2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts mere dete cted. During MDCK epithelial polarization, the Golgi apparatus undergo es morphological changes and apiconuclear Golgi networks were more evi dent in 3 day cells. Treatment with nocodazole disrupted Golgi network s and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by trea tment with endo-beta-galactosidase, indicating that gross Golgi morpho logy did not influence the extent of LAMP-2 polylactosamine glycosylat ion, Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecula r size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells, Extendi ng the time of incubation at 20 degrees C did not influence the size o f LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi p assage at the lower temperature in both 1 and 3 day cells. An identica l effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine g lycosylation, even in isolated Golgi clusters.