Molecular Cloning and Expression of Glucuronyltransferase I Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

doi:10.1074/jbc.273.12.6615

Molecular Cloning and Expression of Glucuronyltransferase I Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans*

From the Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan, ^‡RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-01, Japan, the ^§Graduate School for Agriculture and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan, and the ^¶Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

Abstract

We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galβ1–3Galβ1–4Xylβ1-O-Ser. In contrast, asialoorosomucoid, which contains the Galβ1–4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to β-glucuronidase digestion and co-chromatographed with authentic GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a β1,3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, of proteoglycans.

Footnotes

↵* This work was supported in part by the Science Research Promotion Fund of the Japan Private School Promotion Foundation (to K. S.), the Mizutani Foundation for Glycoscience (to K. S.), the Kanae Medical Research Promotion Fund (to H. K.), Grants-in-aid for Encouragement of Young Scientists 09772013 (to H. K.) and for Scientific Research (B) 09470509 (to K. S.) from the Ministry of Education, Science, Culture, and Sports of Japan.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AB009598.
↵‖ To whom correspondence should be addressed: Dept. of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7571; E-mail:k-sugar{at}kobepharma-u.ac.jp.
↵1 The abbreviations used are: GAG, glycosaminoglycan; bp, base pair(s); DABS-Cl, 4-dimethylaminoazobenzene-4′-sulfonyl chloride; GlcA,d-glucuronic acid; GlcAT-I, glucuronyltransferase I (EC2.4.1.135); GlcAT-P, glycoprotein-specific glucuronyltransferase (10); HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction.
↵2 K. Sugahara and H. Tsuda, unpublished results.
↵3 Y. Mitsumoto, S. Oka, and T. Kawasaki, unpublished results.
↵4 Y. Tone, H. Kitagawa, and K. Sugahara, unpublished results.
- Received December 22, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.