Regulation of chondroitin sulfate biosynthesis by specific sulfation: acceptor specificity of serum β-GalNAc transferase revealed by structurally defined oligosaccharides

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS β-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the α-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al, J. Biol Chem., 270, 22190–22195, 1995), that utilizes common acceptor substrates. The β-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and GlcAβ1-3GalNAc(4-sulfate)β1-4GlcAβ1-3Gal(4-sulfate)β1-3Galβ1-4Xylβ1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcAβ1-3GalNAc(4-sulfate)β1-4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-o-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.