Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms.Proteoglycans play a major role in various cellular/physiological processes, including blood clotting, growth factor signaling, embryogenesis, axon growth and guidance, angiogenesis, and others (1-4). Proteoglycans consists of a core protein and glycosaminoglycan (GAG) 2 chains. GAG chains account for Ͼ50% of the total molecular weight and are primarily responsible for physiological activity of the proteoglycans (5, 6). GAG chains are composed of repeating disaccharide units of a hexosamine residue and a hexuronic acid residue. The three major types of GAG chains found in the proteoglycans are heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). These GAG chains are differentiated by the type of hexosamine (glucosamine/galactosamine), the percentage of uronic acid epimers (glucuronic/iduronic acid), the extent of sulfation, and the nature of glycosidic linkage (␣-/-). One of the key steps in the proteoglycan biosynthesis is the xylosylation of certain specific serine residues of the core protein (7-10), which occurs in the late endoplasmic reticulum and/or cis-Golgi compartments (11-13). This key event is an essential prelude for the construction of the proteoglycan linkage region (14) that is followed by sequence of events resulting in the assembly of mature GAG chains by alternative addition of hexosamine and glucuronic acid residues. The maturation of GAG chains occurs in the medial and trans-Golgi compartments and involves the following events: N-sulfation of glucosamine units by N-deacetylase-...
Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4-fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.Proteoglycans are composed of a core protein and one or more glycosaminoglycan (GAG) 4 side chains such as chondroitin sulfate (CS) and heparan sulfate (HS). A common linkage tetrasaccharide, GlcA(1-3)Gal(1-3)Gal(1-4)Xyl(1-O-Ser), is found between serine residues in core proteins and the GAG polysaccharide side chains (1, 2). Various biological activities of proteoglycans depend critically on interactions of the sulfated GAG side chains with a wide array of proteins, including proteases and protease inhibitors, growth factors and receptors, morphogens, cytokines, and extracellular matrix structural proteins (3, 4). These proteins differentially recognize and bind to specific sulfate groups of GAG chains. GAG chain formation involves the following events: chain initiation by the transfer of xylose residues to certain serine amino acids in the core proteins, assembly of the tetrasaccharide linkage region, elongation by alternate addition of D-glucuronic acid and N-acetyl-D-hexosamine (GlcNAc for HS and GalNAc for CS) units to the linker tetrasaccharide, and finally highly coordinated multiple sulfation/epimerization steps along the GAG chains (5-8).The linkage tetrasaccharide is synthesized by sequential transfer of xylose, galactose, galactose, and glucuronic acid residues from their corresponding sugar nucleotides. The following glycosyltransferases have been shown to be involved in assembly of the linkage region: xylosyltransferase-1/2, galactosyltransferase-1, galactosyltransferase-2, and glucuronyltransferase-1 (see Fig. 1) (9 -14). Furthermore, various chemical modifications such as phosphorylation of xylose at C-2 or sulfation of galactose residues at C-4 and C-6 in the linkage region are predicted t...
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