Molecular Cloning of Squid N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase and Synthesis of a Unique Chondroitin Sulfate Containing E-D Hybrid Tetrasaccharide Structure by the Recombinant Enzyme

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO₄) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO₄) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO₄) could be sulfated efficiently when the GalNAc(4SO₄) residue was included in the unique nonreducing terminal structure, GalNAc(4SO₄)-GlcA(2SO₄)-GalNAc(6SO₄), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO₄) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO₄)-GlcA(2SO₄)-GalNAc(6SO₄), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO₄)-GlcA(2SO₄)-GalNAc(6SO₄) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E-D hybrid tetrasaccharide structure.