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 cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1-6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2-6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks 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 treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. 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 molecular 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. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.
Nabi
I R
IR
Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada.
Dennis
J W
JW
D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002460:Cell Line; D004285:Dogs; D006031:Glycosylation / Q000187:drug effects; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013696:Temperature
D000606:Amino Sugars; D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D011134:Polysaccharides; C066929:polylactosamine; D016695:Glycosyltransferases; D015739:Nocodazole