Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis.
4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-Gal-NAc-IdoUA-than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the 35S-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA- but also in -GlcUA-Gal-NAc-IdoUA- and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.
Mikami
Tadahisa
T
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Mizumoto
Shuji
S
Kago
Naohide
N
Kitagawa
Hiroshi
H
Sugahara
Kazuyuki
K
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D019765:Chondroitin ABC Lyase / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003871:Dermatan Sulfate / Q000737:chemistry; D004187:Disaccharides / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D007067:Iduronic Acid / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014907:Whales
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004187:Disaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D003871:Dermatan Sulfate; D007067:Iduronic Acid; C411659:CHST11 protein, human; C411660:CHST12 protein, human; D015238:Sulfotransferases; C438013:dermatan-4-sulfotransferase-1; C019893:chondroitin 4-sulfotransferase; D019765:Chondroitin ABC Lyase