Background: Chondroitin sulfate (CS) is a linear polysaccharide, composed of repeating disaccharide units and modified with sulfate groups at various positions.Results: A CS library was constructed with defined structures using CS polymerase and sulfotransferases. Conclusion: The CS library provided details of interactions with CS-binding molecules. Significance: Chemo-enzymatic synthesis provides a useful tool for studying the biological functions of CS.Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ϳ90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ϳ29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.
Chondroitin sulfate (CS)2 is a glycosaminoglycan that is a linear acidic polysaccharide composed of repeating disaccharide units (-4 D-glucuronic acid (GlcUA)  1-3 N-acetyl-dgalactosamine (GalNAc)1-) n and modified with sulfate groups at various positions on the sugar residues (1). The average molecular weight (M r ) of CS is 10,000ϳ100,000 consisting of 40ϳ400 saccharide residues as a mature molecule (Table 1).The major disaccharide structures (see Fig. 1A) of CS are as follows: a non-sulfated unit (0S, GlcUA-GalNAc), a monosulfated unit at the C-4 position of the GalNAc residue (4S, GlcUA-GalNAc(4S)), a monosulfated unit at ...