Crystal Structure of β1,4-Galactosyltransferase Complex with UDP-Gal Reveals an Oligosaccharide Acceptor Binding Site

Ramakrishnan, B.; Balaji, Petety V.; Qasba, Pradman K.

doi:10.1016/s0022-2836(02)00020-7

Cited by 114 publications

(108 citation statements)

References 34 publications

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“…The central feature of the crystallographic structure of bovine ␤4GalT1 is a large open pocket (24,25). The amino acids DVD, GG, and EDD within motifs ii, iii, and iv are all solvent-exposed and are located near the bottom of the pocket (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Correia

Papayannopoulos

Panin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Fringe proteins are ␤1,3-N-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying Olinked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted Drosophila proteome identifies a set of four sequence motifs shared among Fringe and other putative ␤1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in Drosophila fringe. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among ␤1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine ␤1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity. Although the Notch pathway mediates a wide range of cell fate decisions, the influence of fringe ( fng) genes on Notch signaling has been best studied during the development of the Drosophila wing. In the developing wing imaginal disk, FNG inhibits the ability of Serrate to activate Notch, and potentiates the ability of Delta to activate Notch. These effects of FNG position a stripe of Notch activation along the border between dorsal and ventral cells, which is essential for the subsequent patterning, growth, and compartmentalization of the wing (reviewed in refs. 5 and 6).Drosophila and vertebrate FNG proteins are highly conserved and possess an unusual ␤1,3-N-acetylglucosaminyltransferase activity that elongates O-linked fucose within EGF domains (7,8). Although FNG is not closely related to other glycosyltransferases, it does share a few short sequence motifs with certain other glycosyltransferases (9), and we report here that it is most closely related to members of a ␤1,3-glycosyltransferase (␤3GT) superfamily. These enzymes use a variety of sugar donors and acceptors, but all transfer the sugar from a UDP-sugar donor, creating a ␤ linkage between the 1 carbon of the donor and the 3 carbon of the acceptor. With the exception of site-directed mutations in a DDD motif of FNG (7,8,10,38), the functional requirements for conserved sequence motifs in FNG or other ␤3GTs have not been assessed. Moreover, the only crystallographic structure for a ␤3GT determined to date is that of human glucuronyltransferase I (GlcAT-I), which does not share significant primary sequence similarity with FNG.Here, we employ two complementary approaches to identify functionally important amino acids within FNG: sequence conservation, and genetic and mole...

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Section: Resultsmentioning

confidence: 99%

“…6, which is published as supporting information on the PNAS web site). The structure of ␤4GalT1 has been solved in complex with its cofactor, Mn ϩϩ , and its donor substrate, UDP-galactose (24,25). Amino acids within all three of these motifs make contacts with UDP-galactose.…”

Section: Resultsmentioning

confidence: 99%

Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Correia

Papayannopoulos

Panin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…These enzymes have low sequence homology, and because there was no structural information on them until recently, they have been classified into dozens of different families (1) (http:͞͞afmb.cnrs-mrs.fr͞ϳcazy͞CA ZY͞in-dex.html). In the past few years, 11 different UDP͞TDP-GTase structures from 10 different families have been reported (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), but all belong to only two different structural superfamilies, GT-A and GT-B (16). Eight of the eleven crystal structures obtained to date belong to the GT-A superfamily, which includes most of the GTases found in the Golgi apparatus and the endoplasmic reticulum.…”

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confidence: 99%

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Chen

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-Å crystal structure of Escherichia coli MurG in complex with its donor substrate, UDPGlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

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“…Human ␤4Gal-T1 has been studied by x-ray crystallography, which has led to detailed insights into its kinetic mechanism (25)(26)(27). To determine how deacetylated 1 interacts with ␤4Gal-T1, we co-crystallized it with the open form of the human enzyme, hM340H-Gal-T1, in the presence of Mn 2ϩ and UDP-hexanolamine (Fig.…”

Section: Glcnac␤1-3gal␤-o-nm Binds To ␤1-4-galactosyltransferase 1-wementioning

confidence: 99%

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

Brown

Yang

Sinha

et al. 2009

Journal of Biological Chemistry

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The disaccharide peracetylated GlcNAc␤1-3Gal␤-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Ac␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc; sLe X ) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe X assembly, and (iii) generation of several glycans related to sLe X . In this report, we show that GlcNAc␤1-3Gal␤-O-naphthalenemethanol binds to the acceptor site of human ␤1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAc␤1-2Man␤1-6Man, which is present on N-linked glycans. The 4-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe X formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N 3 , -NH 2 , or -OCH 3 instead of the hydroxyl groups at C-3-and C-4-positions of the terminal N-acetylglucosamine residue also blocked sLe X formation in cells. The reduction of sLe X by the 4-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes. . Although many of these analogs are effective in vitro, they generally do not exhibit inhibitory activity in cells due to poor membrane permeability. The large number of polar hydroxyl groups and the lack of membrane transporters for oligosaccharides in most cells presumably prevent their uptake (6).In contrast to many of the inhibitors described above, peracetylated disaccharides (e.g. acetylated Gal␤1-4GlcNAc␤-Onaphthalenemethanol (NM), acetylated Gal␤1-3GalNAc␣-O-NM, and acetylated GlcNAc␤1-3Gal␤-O-NM) inhibit sLe X biosynthesis in cells (6 -9). These compounds are taken up by cells by passive diffusion and acted on by cytoplasmic or membrane-associated carboxyesterases, which remove the acetyl groups. The compounds gain access to the biosynthetic enzymes located in the Golgi complex, where they serve as substrates, priming oligosaccharide synthesis and generating products related to O-GalNAc-linked mucin oligosaccharides. Priming in this manner diverts the assembly of the O-linked chains from endogenous glycoproteins, resulting in inhibition of expression of terminal Lewis antigens that are recognized by * This work was supported, in whole or in part, by National Institutes of Health Grants CA46462 and CA112278 (to J. D. E.) and by the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research. The crystallographic part of this project has been funded, in whole or in part, with federal funds from NCI, National Institutes of...

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Crystal Structure of β1,4-Galactosyltransferase Complex with UDP-Gal Reveals an Oligosaccharide Acceptor Binding Site

Cited by 114 publications

References 34 publications

Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

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