Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

Hans Bakker,Takuji Oka,Angel Ashikov,Ajit Yadav,Monika Berger,Nadia A Rana,Xiaomei Bai,Yoshifumi Jigami,Robert S Haltiwanger,Jeffrey D Esko,Rita Gerardy-Schahn

doi:10.1074/jbc.m804394200

Abstract

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A approximately 200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.

Highlights

Rasaccharide, GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-O-Ser, attached to proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans (GAGs)2 [1]
Two variants of O-xylosyltransferases (XylT1 and XylT2) are responsible for the initiation of glycosaminoglycan biosynthesis, which differ in terms of acceptor specificity and tissue distribution [4,5,6,7], and two different enzymatic activities have been identified that catalyze xylosylation of O-glucose residues added to epidermal growth factor (EGF) repeats [8,9,10]
Several different Chinese hamster ovary (CHO) cell lines with defects in GAG biosynthesis have been isolated by screening for reduced incorporation of sulfate [18] and reduced binding of fibroblast growth factor 2 (FGF-2) [19, 20] and by direct selection with FGF-2 conjugated to the plant cytotoxin saporin [21]

Summary

Introduction

Rasaccharide, GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-O-Ser, attached to proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans (GAGs)2 [1]. We provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. A ϳ200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant.

Results

Conclusion