Identification and Characterization of Three Drosophila melanogaster Glucuronyltransferases Responsible for the Synthesis of the Conserved Glycosaminoglycan-Protein Linkage Region of Proteoglycans

Abstract

The Drosophila melanogaster genome contains three putative glucuronyltransferases homologous to human GlcAT-I and GlcAT-P. These enzymes are predicted to be ␤1,3-glucuronyltransferases involved in the synthesis of the glycosaminoglycan (GAG)-protein linkage region of proteoglycans and the HNK-1 carbohydrate epitope of glycoproteins, respectively. The genes encode active enzymes, which we have designated DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII (where BS stands for "broad specificity"). Protein A-tagged truncated soluble forms of all three enzymes efficiently transfer GlcUA from UDP-GlcUA to the linkage region trisaccharide Gal␤1-3Gal␤1-4Xyl. Strikingly, DmGlcAT-I has specificity for Gal␤1-3Gal␤1-4Xyl, whereas DmGlcAT-BSI and DmGlcAT-BSII act on a wide array of substrates with non-reducing terminal ␤1,3-and ␤1,4-linked Gal residues. Their highest activities are obtained with asialoorosomucoid with a terminal Gal␤1-4GlcNAc sequence, indicating their possible involvement in the synthesis of the HNK-1 epitope in addition to the GAG-protein linkage region. Gal␤1-3GlcNAc and Gal␤1-3GalNAc, disaccharide structures widely found in N-and O-glycans of glycoproteins and glycolipids, also serve as acceptors for DmGlcAT-BSI and -BSII. Transcripts of all three enzymes are ubiquitously expressed throughout the developmental stages and in adult tissues of Drosophila. Thus, all three glucuronyltransferases are likely involved in the synthesis of the GAG-protein linkage region in Drosophila, and DmGlcAT-BSI and -BSII appear to be involved in various GlcUA transfer reactions for the synthesis of proteoglycans, glycoproteins, and glycolipids. This activity distinguishes these glucuronyltransferases from their mammalian homologs GlcAT-P and GlcAT-D (or -S). Sequence alignment of the Drosophila glucuronyltransferases with homologs in human, rat, and Caenorhabditis elegans demonstrates the conservation of a majority of the critical amino acid residues in the active sites of the three Drosophila enzymes. Proteoglycans (PGs)1 play an essential role in a variety of biological processes such as cell-cell adhesion, cell proliferation, and tissue morphogenesis (1, 2). PGs consist of a core protein and sulfated glycosaminoglycan (GAG) side chains. PGs can be classified into three groups based on the nature of their GAGs: heparan sulfate (HS)-type PGs, chondroitin sulfate (CS)-type PGs, and keratan sulfate PGs. There is increasing evidence that uniquely sulfated domain structures of GAG side chains are critically involved in various functions of these PGs (3-5), and defective synthesis of GAGs causes aberrant morphology and even embryonic lethality during development (6).In biosynthesis, HS or CS linear chains are differentially assembled on the common linkage region tetrasaccharide Glc-UA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-, which takes place on a specific serine residue in a given core protein (7). Transfer of either ␣1,4-GlcNAc or ␤1,4-GalNAc to the tetrasaccharide linkage region terminus mediated by the GAG-specific hexosaminyltransferases determin...

The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

“…3 Instead, it is tempting to speculate that GlcA serves as the sialic acid equivalent in Drosophila. The occurrence of terminal GlcA on Drosophila glycosphingolipids and of terminal sialic acid on the vertebrate ganglioside family of glycosphingolipids is also consistent with functional equivalence between these two acidic monosaccharides (74,75,77). However, distinct differences indicate that this hypothesis is probably an oversimplification.…”

“…Some of these activities have been described in other species or are evident in the biosynthesis of Drosophila glycosphingolipids and glycosaminoglycans (77,95). However, in the context of O-linked core 1 or core 2 or O-Fuc, these modifications are novel, and identification of the relevant biosynthetic enzymes will provide new opportunities for dissecting the functions of O-linked glycans.…”

TheSf1-related nuclear hormone receptorHr39regulatesDrosophilafemale reproductive tract development and function

“…Two genes, GlcAT-P and PAPS appear to be particularly strong candidates. Expression of GlcAT-P, encoding a putative N-acetyllactosamine ␤-1,3-glucuronosyltransferase (Kim et al, 2003) was reduced to one thirtieth of its original levels. This gene has been implicated in glycoprotein, glycosphingolipid and proteoglycan biosynthesis.…”

Incomplete Elongation of the Chondroitin Sulfate Linkage Region on Aggrecan and Response to Interleukin-1β

“…Many enzymes involved in linkage structure formation, sulfation and/or phosphorylation, and chain polymerization of these structures have been carefully characterized, but we have yet to understand even the most rudimentary regulatory mechanism involved in this process [11,23,48,49]. Empirical evidence has shown that CS chain length, as well as the location and degree of modifications, can vary along the core protein of aggrecan [50,51].…”