Detection and Characterization of UDP-GalNAc: Chondroitin N-Acetylgalactosaminyltransferase in Bovine Serum Using a Simple Assay Method1

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Background: EXTL2 is one of three EXT-like genes homologous to the tumor suppressor EXT gene family members and encodes an N-acetylhexosaminyltransferase. Results: EXTL2 terminated polymerization of glycosaminoglycan (GAG) chains by transferring an N-acetylglucosamine residue to the phosphorylated tetrasaccharide linkage region. Conclusion: EXTL2 controlled GAG biosynthesis in a xylose kinase-dependent manner. Significance: Lack of EXTL2 causes GAG overproduction associated with pathological processes.

Section: Isolation Of Mouse Extl2 Genomic Clones and Analysis Of The mentioning

confidence: 99%

Section: Isolation Of Mouse Extl2 Genomic Clones and Analysis Of The mentioning

confidence: 99%

EXTL2, a Member of the EXT Family of Tumor Suppressors, Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Nadanaka

Zhou

Kagiyama

et al. 2013

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“…Fractions were collected at 1-or 2-min intervals, and radioactivity was quantified by liquid scintillation counting (LS6500, Beckman Coulter). The reaction products of CS isoforms were subjected to gel filtration chromatography using a syringe column packed with Sephadex G-25 (superfine) resin (28). The incorporation of [ 35 S]sulfate into polysaccharides was quantified by measuring the radioactivity in the flow-through fractions by liquid scintillation counting.…”

Section: Materials-mentioning

confidence: 99%

Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

Hashiguchi

Mizumoto

Nishimura

et al. 2011

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Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (␤-TM) and a non-PG form without a CS chain (␣-TM) and hence is a part-time PG. An ␣-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUA␤1-3Gal␤1-3Gal␤1-4xylose, and the nonreducing terminal GlcUA residue is 3-Osulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3-phosphoadenosine 5-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUA␤1-3Gal␤1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of ␣-and ␤-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3-phosphoadenosine 5-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.

“…Briefly, the standard reaction mixture (60 l) contained 10 l of the resuspended beads, 50 mM imidazole-HCl, pH 6.8, 2 mM dithiothreitol, 10 M [ 35 S]PAPS (ϳ1 or 3 ϫ 10 5 dpm), and an acceptor polysaccharide preparation (10 nmol as GlcUA). The reaction mixtures were incubated at 37°C for 1 h and subjected to gel filtration using a syringe column packed with Sephadex G-25 (superfine) (44). [ 35 S]Sulfate incorporation into polysaccharides was quantified by determination of the radioactivity in the flow-through fractions by liquid scintillation counting.…”

Section: Construction Of Expression Vectors Encoding Soluble Forms Ofmentioning

confidence: 99%

Specificities of Three Distinct Human Chondroitin/Dermatan N-Acetylgalactosamine 4-O-Sulfotransferases Demonstrated Using Partially Desulfated Dermatan Sulfate as an Acceptor

Mikami

Mizumoto

Kago

et al. 2003

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111

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-GalNAc-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 35 S-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-GalNAc-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.