Biosynthesis of the Linkage Region of Glycosaminoglycans

Bai, Xiaomei; Zhou, Dapeng; Brown, Jillian R.; Crawford, Brett E.; Hennet, Thierry; Esko, Jeffrey D.

doi:10.1074/jbc.m107339200

Cited by 158 publications

(53 citation statements)

References 46 publications

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“…(C) GlcUA͞ GalNAc, presumed by similarity to mammalian chondroitin synthase (33). (D) Gal͞Gal, presumed by similarity to mammalian galactosyltransferase II (34). (E) Unknown donor͞acceptor.…”

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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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%

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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“…GalTII has been recently cloned and shown to also overlap with ␣-mannosidase II as wells as CALNUC, a marker of the cis Golgi (35). The physical interaction of GalTII with GlcATI or GalTI has not yet been explored.…”

Section: Discussionmentioning

confidence: 99%

Enzyme interactions in heparan sulfate biosynthesis: Uronosyl 5-epimerase and 2- O -sulfotransferase interact in vivo

Pinhal

Smith²,

Olson³

et al. 2001

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

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142

106

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The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase͞N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker ␣-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-Osulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.glycosaminoglycans ͉ sulfation ͉ epimerization ͉ enzyme localization ͉ Golgi complex T he biosynthesis of heparan sulfate initiates by the translation of a proteoglycan core protein and the assembly of the so-called linkage region tetrasaccharide on specific serine residues (-GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-O-Ser). The chain then polymerizes by the addition of alternating N-acetylglucosamine (GlcNAc␣1,4) and glucuronic acid (GlcA␤1,4) residues. A series of modification reactions takes place simultaneously that involves at least six enzymatic activities: (i) N-deacetylation of a portion of GlcNAc residues, (ii) N-sulfation of the resulting unsubstituted amino groups to form GlcNS units, (iii) 5-epimerization of adjacent D-GlcA residues to form L-iduronic acid (IdoA), (iv) 2-O-sulfation of L-IdoA and more rarely D-GlcA residues, (v) 6-O-sulfation of glucosamine units, and (vi) occasional 3-O-sulfation of glucosamine residues (Fig. 1A). A major question concerns how these enzymes orchestrate the formation of specific oligosaccharide sequences with unique binding properties for ligands (1, 2). Multiple isozymes exist for several of the transferases that differ in substrate specificity and temporal͞spatial expression during developmen...

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“…Although examples of the other core-synthesizing enzymes, i.e. galactosyltransferase I (␤4GalTVII) (5,36,37), galactosyltransferase II (␤3GalTVI), (38) and glucuronyltransferase I (5, 39), have been characterized from mammalian and nematode sources, the relevant fly genes have not yet been described. For example, it is not known which of the three ␤1,4-galactosyltransferase homologues from fly acts as galactosyltransferase I and which may have roles in elongation of Fringe-modified O-fucosyl glycans, for example (40).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Drosophila melanogaster Peptide O-Xylosyltransferase, the Key Enzyme for Proteoglycan Chain Initiation and Member of the Core 2/I N-Acetylglucosaminyltransferase Family

Wilson

2002

Journal of Biological Chemistry

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Chondroitin and heparan sulfates are essential players in animal development and are synthesized by a series of glycosyltransferases, the first of which is UDP-␣-D-xylose:proteoglycan core protein ␤-D-xylosyltransferase (EC 2.4.2.26). In the present study, a Drosophila melanogaster gene (CG17771), previously designated as a homologue of core 2 and I ␤1,6-N-acetylglucosaminyltransferases, was shown to encode an active peptide O-xylosyltransferase. A novel coupled assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated transfer of xylose to the peptide DDDSIEGSGGR. Analysis of sequences of various peptide O-xylosyltransferase and ␤1,6-N-acetylglucosaminyltransferase sequences indicates that they are members of a large multifunctional protein family with a range of roles in ␤-glycosylation of either peptide or glycan substrates. Because in contrast to mammals, there is only one fly peptide O-xylosyltransferase gene, it is anticipated that, given the key roles of proteoglycans, the hereby designated oxt gene is essential for viability.

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Biosynthesis of the Linkage Region of Glycosaminoglycans

Cited by 158 publications

References 46 publications

Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila

Enzyme interactions in heparan sulfate biosynthesis: Uronosyl 5-epimerase and 2- O -sulfotransferase interact in vivo

Functional Characterization of Drosophila melanogaster Peptide O-Xylosyltransferase, the Key Enzyme for Proteoglycan Chain Initiation and Member of the Core 2/I N-Acetylglucosaminyltransferase Family

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