A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO(4)) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) because GalNAc(4SO(4)) and DeltaHexA(2SO(4))-GalNAc(6SO(4)) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO(4)) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [S-35]PAPS and GalNAc4S-6ST and the S-35-labeled product was digested with chondroitinase ACII, a S-35-labeled trisaccharide (Oligo III) containing [ 35S] GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.