An Animal Cell Mutant Defective in Heparan Sulfate Hexuronic Acid 2-O-Sulfation

doi:10.1074/jbc.271.30.17711

An Animal Cell Mutant Defective in Heparan Sulfate Hexuronic Acid 2-O-Sulfation*

Xiaomei Bai and
Jeffrey D. Esko^‡

From the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

^‡ To whom correspondence and requests for reprints should be addressed. Tel.: 205-934-6034; Fax: 205-975-2547; E-mail: jesko{at}bmg.bhs.uab.edu

Abstract

The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with ¹²⁵I-labeled basic fibroblast growth factor (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with ³⁵SO₄ to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind ¹²⁵I-bFGF, but incorporated ³⁵SO₄ normally (mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO₃)-GlcNSO₃ and IdUA(2OSO₃)-GlcNSO₃(6OSO₃), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO₃(6OSO₃) and IdUA-GlcNSO₃(6OSO₃). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO₃ groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GlcUA

glucuronic acid

IdUA

iduronic acid

HexUA

hexuronic acid

GAG

glycosaminoglycan

CHO

Chinese hamster ovary

HPLC

high performance liquid chromatography

bFGF

basic fibroblast growth factor

aMan_R

anhydromannitol

PAPS

adenosine 3′-phosphate,5′-phosphosulfate.
↵² X. Bai and J. D. Esko, unpublished results.
↵³ Recent work suggests that unsubstituted GlcN residues accumulate in some tissues, but the origin of the material has not been resolved (48, 49).
- Received January 26, 1996.
- Revision received April 25, 1996.