Biosynthesis of the Chondroitin Sulfate-Protein Linkage Region: Purification and Properties of a Glucuronosyltransferase From Embryonic Chick Brain

Brandt, Alan E.; Distler, Jack J.; Jourdian, George W.

doi:10.1073/pnas.64.1.374

Cited by 26 publications

(13 citation statements)

References 25 publications

(1 reference statement)

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“…GalL1-3GalL1-4Xyl) but also to disaccharides with analogous structures such as lactose (GalL1-4Glc) and N-acetyllactosamine (GalL1-4GlcNAc) [8,11,12]. In strong contrast, neither lactose nor N-acetyllactosamine was utilized by the recombinant enzyme in the present study (Table 1).…”

Section: Acceptormentioning

confidence: 75%

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Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan‐protein linkage region of proteoglycans

et al. 1999

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We characterized the recombinant glucuronyltransferase I (GlcAT-I) involved in the glycosaminoglycan-protein linkage region biosynthesis. The enzyme showed strict specificity for GalL L1-3GalL L1-4Xyl, exhibiting negligible incorporation into other galactoside substrates including GalL L1-3GalL L1-O-benzyl, GalL L1-4GlcNAc and GalL L1-4Glc. A comparison of the GlcAT-I with another L L1,3-glucuronyltransferase involved in the HNK-1 epitope biosynthesis revealed that the two L L1,3-glucuronyltransferases exhibited distinct and no overlapping acceptor substrate specificities in vitro. Nevertheless, the transfection of the GlcAT-I cDNA into COS-1 cells induced the significant expression of the HNK-1 epitope. These results suggested that the high expression of the GlcAT-I gene rendered the cells capable of synthesizing the HNK-1 epitope.z 1999 Federation of European Biochemical Societies.

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Section: Acceptormentioning

confidence: 75%

“…GlcAT-I activity was ¢rst detected in an embryonic chick cartilage extract [8] and was subsequently partially puri¢ed from embryonic chick brain [11] and mouse mastocytoma cells [12]. However, attempts to purify GlcAT-I to homogeneity have not been successful due to the low concentrations and the di¤culty in solubilizing the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan‐protein linkage region of proteoglycans

et al. 1999

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“…In addition, these microsomal preparations have been shown to contain GlcA transferase activity for adding GlcA to non-reducing terminal Gal residues such as that found in the Gal-Gal-Xyl linkage region (25,26). Mixed substrate experiments have indicated that the transfers of [ 14 C]GlcA to the linkage oligosaccharide and to chondroitin oligosaccharide are each catalyzed by separate enzymes (25), which have been termed GlcA transferase I and GlcA transferase II (2).…”

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confidence: 99%

Biosynthesis of Chondroitin Sulfate

Sugumaran

Katsman

Sunthankar

et al. 1997

Journal of Biological Chemistry

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A photoaffinity analogue, [␤-32 P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDPGlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50 -60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinitylabeled active fractions from the Q-Sepharose, heparinSepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [␤-32 P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.

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“…This intermediate serves as the primer for heparan sulfate and chondroitin sulfate assembly, which arises from the alternating addition of ␤-GlcNAc and ␤-GlcUA or ␤-GalNAc and ␤-GlcUA residues, respectively, to the linkage tetrasaccharide. Three GlcUA-transferases are thought to catalyze the addition of GlcUA: one involved in the formation of the linkage region tetrasaccharide (GlcUAT-I) (11,12) and two that polymerize the different chains (13). The latter activities may be part of bifunctional enzymes in which the same protein catalyzes the alternating addition of a HexNAc residue and GlcUA (14,15).…”

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confidence: 99%

Formation of HNK-1 Determinants and the Glycosaminoglycan Tetrasaccharide Linkage Region by UDP-GlcUA:Galactose β1,3-Glucuronosyltransferases

Ge¹,

Bai

Sarkar³

et al. 1999

Journal of Biological Chemistry

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While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein ␤1,3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO 3 GlcUA␤1, 3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in ␤-linkage to Gal␤1,3/4GlcNAc␤-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Gal␤1,3Gal␤-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Gal␤1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.

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Biosynthesis of the Chondroitin Sulfate-Protein Linkage Region: Purification and Properties of a Glucuronosyltransferase From Embryonic Chick Brain

Cited by 26 publications

References 25 publications

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan‐protein linkage region of proteoglycans

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan‐protein linkage region of proteoglycans

Biosynthesis of Chondroitin Sulfate

Formation of HNK-1 Determinants and the Glycosaminoglycan Tetrasaccharide Linkage Region by UDP-GlcUA:Galactose β1,3-Glucuronosyltransferases

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