Glycobiology Advance Access originally published online on September 23, 2007
Glycobiology 2007 17(12):1365-1376; doi:10.1093/glycob/cwm103
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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
2 Department of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan
3 Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
1 To whom correspondence should be addressed: Fax: +81-566-26-2649; e-mail: ohabuchi{at}auecc.aichi-edu.ac.jp
Received on June 9, 2007; revised on August 23, 2007; accepted on September 20, 2007
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.
Key words: chondroitin sulfate D / chondroitin sulfate E / GalNAc4S-6ST / squid / sulfotransferase