A Novel Method to Co-localize Glycosaminoglycan-Core Oligosaccharide Glycosyltransferases in Rat Liver Golgi

doi:10.1074/jbc.270.2.756

A Novel Method to Co-localize Glycosaminoglycan-Core Oligosaccharide Glycosyltransferases in Rat Liver Golgi

CO-LOCALIZATION OF GALACTOSYLTRANSFERASE I WITH A SIALYLTRANSFERASE (*)

From the La Jolla Cancer Research Foundation, Glycobiology/Carbohydrate Chemistry Program, La Jolla, California 92037

^§ To whom correspondence should be addressed:
La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., San Diego, CA 92037.
Tel.: 619-455-6480; Fax: 619-450-2101.

Abstract

4-Methylumbelliferyl-β-xyloside (XylβMU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUAβ1, 3Galβ1,3Galβ1,4XylβMU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with XylβMU and 1 μM UDP-[³H]Gal and then chased with 5 μM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Siaα2,3Galβ1,4XylβMU and Siaα2,8Siaα2,3Galβ1,4XylβMU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, 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 are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an α-2,3-sialyltransferase and an α-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.

Footnotes

↵* This work was supported in part by a grant from the Mizutani Foundation for Glycoscience and by United States Public Health Service Grant R01-CA38701. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GAG

glycosaminoglycan

4MU

4-methylumbelliferyl

CHO

Chinese hamster ovary cells

HPLC

high performance liquid chromatography

NDV

Newcastle disease virus

AUS

Arthrobacter ureafaciens sialidase

XylβMU

4-methylumbelliferyl-β-D-xylose

CS

crude cell smooth membrane

SE

supernatant extract

SP

supernatant pellet

XT

extract supernatant

XP

extract pellet.
↵²P. V. Salimath and H. H. Freeze, unpublished results.
- Received October 18, 1994.
- Revision received November 15, 1994.