REGULATION OF CHONDROITIN SULFATE BIOSYNTHESIS BY SPECIFIC SULFATION - ACCEPTOR SPECIFICITY OF SERUM BETA-GALNAC TRANSFERASE REVEALED BY STRUCTURALLY DEFINED OLIGOSACCHARIDES

The relationship between sulfation and polymerization in chondroitin s ulfate (CS) biosynthesis has been poorly understood, In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalN Ac transferase responsible for chain elongation using structurally def ined acceptor substrates, They consisted of tetra- and hexasaecharide- serines that were chemically synthesized and various regular oligosacc harides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme prepara tion was obtained from fetal bovine serum by means of heparin-Sepharos e affinity chromatography, The preparation did not contain the alpha-G alNAc transferase recently demonstrated in fetal bovine serum (Kitagaw a et at, J, Biol., Chem,, 270, 22190-22195, 1995), that utilizes commo n acceptor substrates, The beta-GalNAc transferase used as accepters, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Ga l beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfa te)beta 1-4GlcA beta 1-3Gal(4-sulfate)beta 1- 3Gal beta 1-4Xyl beta 1- O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1- 3GalNAc(4-sulfate)beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl bet a 1-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 res ults indicated that the sulfate group at the Gal-3 C-4 markedly affect ed the transfer of GalNAc to the terminal GlcA, In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasacchari des remarkably enhanced the GalNAc transfer, suggesting that the enzym e recognizes up to the fourth saccharide residue from the nonreducing end, The level of incorporation into a tetra- or hexasaccharide contai ning a terminal 2-O-sulfated GlcA residue was significant, whereas the re was no apparent incorporation into tetra- or hexasaccharides contai ning a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalN Ac residue, These results indicated that sulfation reactions play impo rtant roles in chain elongation and termination.