Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate

被引:40
作者
Akama, TO [1 ]
Misra, AK [1 ]
Hindsgaul, O [1 ]
Fukuda, MN [1 ]
机构
[1] Burnham Inst, Glycobiol Program, La Jolla, CA 92037 USA
关键词
D O I
10.1074/jbc.M207412200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Among the enzymes of the carbohydrate sulfotransferase family, human corneal GIcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GIcNAc6ST-5/ GST4beta]) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4alpha) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/ GST4beta) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce keratan sulfate (Akama, T.O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, 0., Fukuda, M., and Fukuda, M. N. (2001) J. Biol Chem. 276, 16271-16278). In this study, we analyzed the substrate specificities of these sulfotransferases in vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of keratan sulfate. To clarify further the involvement of hCGn6ST in biosynthe is of keratan sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, beta1,4-galactosyltransferase-1, beta1,3-N-acetylglucosaminyl-transferase-2, hCGn6ST, and keratan sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of keratan sulfate. Based on these results, we propose a biosynthetic pathway for N-linked keratan sulfate on corneal proteoglycans.
引用
收藏
页码:42505 / 42513
页数:9
相关论文
共 54 条
[1]   Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene [J].
Akama, TO ;
Nishida, K ;
Nakayama, J ;
Watanabe, H ;
Ozaki, K ;
Nakamura, T ;
Dota, A ;
Kawasaki, S ;
Inoue, Y ;
Maeda, N ;
Yamamoto, S ;
Fujiwara, T ;
Thonar, EJMA ;
Shimomura, Y ;
Kinoshita, S ;
Tanigami, A ;
Fukuda, MN .
NATURE GENETICS, 2000, 26 (02) :237-241
[2]   Human corneal GlcNAc 6-O-sulfotransferase and mouse intestinal GlcNAc 6-O-sulfotransferase both produce keratan sulfate [J].
Akama, TO ;
Nakayama, J ;
Nishida, K ;
Hiraoka, N ;
Suzuki, M ;
McAuliffe, J ;
Hindsgaul, O ;
Fukuda, M ;
Fukuda, MN .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (19) :16271-16278
[3]   A family of human β4-galactosyltransferases -: Cloning and expression of two novel UDP-galactose:: β-N-acetylglucosamine β1,4-galactosyltransferases, β4Gal-T2 and β4Gal-T3 [J].
Almeida, R ;
Amado, M ;
David, L ;
Levery, SB ;
Holmes, EH ;
Merkx, G ;
van Kessel, AG ;
Rygaard, E ;
Hassan, H ;
Bennett, E ;
Clausen, H .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (51) :31979-31991
[4]   Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide accepters by three distinct α2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III [J].
Angata, K ;
Suzuki, M ;
McAuliffe, J ;
Ding, YL ;
Hindsgaul, O ;
Fukuda, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (24) :18594-18601
[5]   EXPRESSION CLONING OF A CDNA-ENCODING UDP-GLCNAC-GAL-BETA-1-3-GALNAC-R (GLCNAC TO GALNAC) BETA-1-6GLCNAC TRANSFERASE BY GENE-TRANSFER INTO CHO CELLS EXPRESSING POLYOMA LARGE TUMOR-ANTIGEN [J].
BIERHUIZEN, MFA ;
FUKUDA, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (19) :9326-9330
[6]   Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin [J].
Bistrup, A ;
Bhakta, S ;
Lee, JK ;
Belov, YY ;
Gunn, MD ;
Zuo, FR ;
Huang, CC ;
Kannagi, R ;
Rosen, SD ;
Hemmerich, S .
JOURNAL OF CELL BIOLOGY, 1999, 145 (04) :899-910
[7]   Biosynthesis of L-selectin ligands: Sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases [J].
Bowman, KG ;
Cook, BN ;
de Graffenried, CL ;
Bertozzi, CR .
BIOCHEMISTRY, 2001, 40 (18) :5382-5391
[8]  
Chakravarti S, 2000, INVEST OPHTH VIS SCI, V41, P3365
[9]   Lumican regulates collagen fibril assembly: Skin fragility and corneal opacity in the absence of lumican [J].
Chakravarti, S ;
Magnuson, T ;
Lass, JH ;
Jepsen, KJ ;
LaMantia, C ;
Carroll, H .
JOURNAL OF CELL BIOLOGY, 1998, 141 (05) :1277-1286
[10]  
CORNUET PK, 1994, INVEST OPHTH VIS SCI, V35, P870