N-Acetylgalactosamine (GalNAc) Transfer to the Common Carbohydrate-Protein Linkage Region of Sulfated Glycosaminoglycans

doi:10.1074/jbc.270.38.22190

N-Acetylgalactosamine (GalNAc) Transfer to the Common Carbohydrate-Protein Linkage Region of Sulfated Glycosaminoglycans

IDENTIFICATION OF UDP-GalNAc:CHONDRO-OLIGOSACCHARIDE α-N-ACETYLGALACTOSAMINYLTRANSFERASE IN FETAL BOVINE SERUM (*)

From the (1) 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
(2) Graduate School for Agriculture and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan, and the
(3) Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, S-751 23 Uppsala, Sweden

^§ To whom correspondence should be addressed. Tel.: 81-78-441-7570; Fax: 81-78-441-7571.

Abstract

During the course of a study to elucidate the role of modification of the common polysaccharide-protein linkage structure, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, in biosynthetic sorting mechanisms of the different sulfated glycosaminoglycan chains, a novel N-acetylgalactosamine (GalNAc) transferase was discovered in fetal bovine serum. The enzyme catalyzed the transfer of [H]GalNAc from UDP-[H]GalNAc to linkage tetrasaccharide and hexasaccharide serines synthesized chemically and to various regular oligosaccharides containing terminal D-glucuronic acid (GlcA), which were prepared from chondroitin and chondroitin sulfate using testicular hyaluronidase digestion. The labeled products obtained with the linkage tetra- and hexasaccharide serines and with the tetrasaccharide (GlcAβ1-3GalNAc) were resistant to digestion with chondroitinase AC-II and β-N-acetylhexosaminidase but sensitive to α-N-acetylgalactosaminidase digestion, indicating that the enzyme is an α-N-acetylgalactosaminyltransferase. This finding is in contrast to that of Rohrmann et al. (Rohrmann, K., Niemann, R., and Buddecke, E.(1985) Eur. J. Biochem., 148, 463-469), who reported that a corresponding product was susceptible to digestion with β-N-acetylhexosaminidase. The presence of a sulfate group at C4 of the penultimate GalNAc or Gal units markedly inhibited the transfer of GalNAc to the terminal GlcA, while a sulfate group at C6 of the GalNAc had little effect on the transfer. Moreover, a slight but significant transfer of GalNAc was observed to an oligosaccharide containing terminal 2-O-sulfated GlcA as acceptor, whereas no incorporation was detected into oligosaccharides containing terminal unsaturated or 3-O-sulfated GlcA units. These results suggest that this novel serum enzyme is a UDP-GalNAc:chondro-oligosaccharide α1-3- or 1-4-N-acetylgalactosaminyltransferase. The possibility of involvement of this enzyme in glycosaminoglycan biosynthesis is discussed.

Footnotes

↵* This work was supported in part by the Science Research Promotion Fund from Japan Private School Promotion Foundation, Grants-in-aid for Encouragement of Young Scientists 07857169, for Scientific Research 06808061, and for Scientific Research on Priority Areas 05274107 from the Ministry of Education and Culture of Japan, the Swedish Medical Research Council Grants 2309, 10155, and 10440, and the Mizutani Foundation for Glycoscience. 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:

GalNAc

N-acetylgalactosamine

GlcA

D-glucuronic acid

MES

2-(N-morpholino)ethanesulfonic acid

HPLC

high performance liquid chromatography.
↵²K. Sugahara, Y. Tanaka, K. Masayama, H. Kitagawa, N. Seno, and S. Yamada, manuscript in preparation.
³K. Lidholt, M. Fjelstad, U. Lindahl, F. Goto, T. Ogawa, Y. Tanaka, K. Tsuchida, H. Kitagawa, and K. Sugahara, manuscript in preparation.
- Received May 23, 1995.
- Revision received July 13, 1995.