Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate.
Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-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/GST4) 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, O., 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 biosynthesis 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-I, beta1,3-N-acetylglucosaminyltransferase-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.
Akama
Tomoya O
TO
Glycobiology Program, The Burnham Institute, La Jolla, California 92037, USA. takama@burnham-inst.org
Misra
Anup K
AK
Hindsgaul
Ole
O
Fukuda
Michiko N
MN
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D003315:Cornea / Q000201:enzymology*; D006224:Cricetinae; D004187:Disaccharides / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D007632:Keratan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*; D014162:Transfection; D000097811:Carbohydrate Sulfotransferases
D004187:Disaccharides; D007632:Keratan Sulfate; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013431:Sulfates; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases