Characterization of the Heparan Sulfate and Chondroitin Sulfate Assembly Sites in CD44

doi:10.1074/jbc.274.4.2511

Characterization of the Heparan Sulfate and Chondroitin Sulfate Assembly Sites in CD44*

From the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543 and the ^§Department of Dermatology, University of Washington, Seattle, Washington 98105

Abstract

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.

Footnotes

↵* This work was supported in part by Public Health Service Grant AR 21557 from the National Institutes of Health, Department of Health and Human Services.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.
↵‡ These authors contributed equally to this work.
↵¶ To whom correspondence should be addressed: BMS-PRI, P. O. Box 4000, Princeton, NJ 08543. Tel.: 609-252-6719; Fax: 609-252-6058; E-mail: bennettk{at}bms.com.
Abbreviations:

HS

heparan sulfate

GAG(s)

glycosaminoglycan(s)

CS

chondroitin sulfate

PCR

polymerase chain reaction

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

HPLC

high performance liquid chromatography

b-FGF

basic fibroblast growth factor

Rg

human IgG1 immunoglobulin region

aa

amino acid(s)

wt

wild type

SG

serine/glycine.
- Received March 16, 1998.
- Revision received August 21, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.