Glycobiology, Vol 8, 947-953, Copyright © 1998 by Society for Glycobiology
IR Nabi and JW Dennis
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.
ORIGINAL ARTICLES
The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time
Departement de Pathologie et Biologie Cellulaire, Universite de Montreal, Montreal, Quebec, Canada.
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