Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage.
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed.
Ito
Y
Y
Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542, Japan.
Habuchi
O
O
D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D049832:Decapodiformes; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011509:Proteoglycans / Q000302:isolation & purification; D015238:Sulfotransferases / Q000302:isolation & purification* / Q000378:metabolism; D013464:Sulfuric Acids / Q000378:metabolism
D006025:Glycosaminoglycans; D011509:Proteoglycans; D013464:Sulfuric Acids; C416670:N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; D015238:Sulfotransferases