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(4SO(4)) 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(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), 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(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) 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.
Yamaguchi
Teruyoshi
T
Department of Chemistry, Aichi University of Education, Kariya, Aichi, Japan.
Ohtake
Shiori
S
Kimata
Koji
K
Habuchi
Osami
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D049832:Decapodiformes; D004798:Enzymes / Q000737:chemistry*; D006025:Glycosaminoglycans / Q000737:chemistry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D004798:Enzymes; D006025:Glycosaminoglycans; D011134:Polysaccharides; D011994:Recombinant Proteins; D002809:Chondroitin Sulfates; C416670:N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; D015238:Sulfotransferases