A NOVEL METHOD TO CO-LOCALIZE GLYCOSAMINOGLYCAN-CORE OLIGOSACCHARIDE GLYCOSYLTRANSFERASES IN RAT-LIVER GOLGI - COLOCALIZATION OF GALACTOSYLTRANSFERASE-I WITH A SIALYLTRANSFERASE
Jr. Etchison et al., A NOVEL METHOD TO CO-LOCALIZE GLYCOSAMINOGLYCAN-CORE OLIGOSACCHARIDE GLYCOSYLTRANSFERASES IN RAT-LIVER GOLGI - COLOCALIZATION OF GALACTOSYLTRANSFERASE-I WITH A SIALYLTRANSFERASE, The Journal of biological chemistry, 270(2), 1995, pp. 756-764
4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminogly
can synthesis by first serving as an acceptor for the addition of 2 ga
lactoses and 1 glucuronic acid residue to make the typical core struct
ure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigat
e the relative localization of these biosynthetic enzymes, intact and
properly oriented rat liver Golgi preparations were incubated with Xyl
beta MU and 1 mu M UDP-[H-3]Gal and then chased with 5 mu M of unlabe
led UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under
these conditions, no intervesicular transport occurs and acceptor labe
ling depends entirely upon transporter-mediated delivery of the labele
d sugar nucleotides into the lumen of a vesicle and co-localization of
the appropriate glycosyltransferases. The labeled products were isola
ted from the incubation medium and from within the Golgi and their str
uctures analyzed by C18, anion-exchange, and amine adsorption high per
formance liquid chromatography in combination with glycosidase digesti
ons, Surprisingly, the major products within the Golgi mere two sialyl
ated xylosides (Sia alpha 2,3Gal beta 1,4Xyl beta MU and Sia alpha 2,8
Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group o
f partially completed GAG core structures, Less than 10% of the produc
ts within the Golgi are the expected core structures containing a seco
nd Gal residue or, in addition, GlcUA. The amount of the sialylated pr
oducts is only partially decreased if the chase is omitted or if the c
hase is done in the absence of added CMP-Sia, suggesting a pool of pre
viously transported CMP-Sia drives synthesis of the major products, Co
nversely, when detergent permeabilized vesicles are provided with high
concentration of the same sugar nucleotides, the ratio of sialylated
products is reduced and replaced by an increase in GAG-like products.
These results argue that GAG core-specific Gal transferase I and II ar
e not extensively co-localized within the same Golgi compartment. By c
ontrast, glycosaminoglycan core Gal transferase I is substantially co-
localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialylt
ransferase. Incubating intact Golgi vesicles with exogenous diffusible
accepters offers a novel method to assess the functional co-localizat
ion of glycosyltransferases of multiple pathways within the Golgi comp
artments.