Ir. Nabi et Jw. Dennis, THE EXTENT OF POLYLACTOSAMINE GLYCOSYLATION OF MDCK LAMP-2 IS DETERMINED BY ITS GOLGI RESIDENCE TIME, Glycobiology, 8(9), 1998, pp. 947-953
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.