N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3-phosphoadenosine 5-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO 4 )) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3-phosphoadenosine 5-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO 4 ) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO 4 ) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.

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 (CS-A). From the nonreducing terminal of CS-A, a GlcAcontaining 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 ⌬HexA(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 )-GlcAGalNAc(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 [35 S]PAPS and GalNAc4S-6ST and the 35 S-labeled product was digested with chondroitinase ACII, a 35 Slabeled trisaccharide (Oligo III) containing [ 35 S]GalNAc(4,6-SO 4 ) 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.