Investigating the Elusive Mechanism of Glycosaminoglycan Biosynthesis

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 >50% 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 (α-/β-). 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-N-sulfotransferases (for HS only), epimerization of glucuronic acids to iduronic acids by C-5 epimerase, and sulfation of the repeating disaccharide units by a variety of sulfotransferases and their isoforms.The position, extent, and pattern of sulfation attribute enormous diversity to GAG chains, which confer specificity in binding to a vast array of proteins. These diverse structural features are very tightly regulated in a spatio-temporal manner during and beyond the development of an organism, and these features dictate differential interactions with various growth factors and receptors, and numerous protein targets leading to an array of physiological functions (15, 16).The presence of free GAG chains has been known to disrupt the interaction of endogenous GAG components of proteoglycans with protein ligands thereby altering the physiological activities. Consequently, they have been used as molecular tools in the elucidation of the role of GAG chains in the activation of cellular events (17–19). Free GAG chains can be synthesized in vitro in cell culture by providing exogenous xylosides containing various hydrophobic aglycone moieties. Thus, the xylosides can act as false acceptors for initiation of linkage region and the subsequent elongation of GAG chains. Xylosides have been used for over three decades both in vitro (20–28) and in vivo (25, 29–31) to probe the functional significance of GAG chains in various dynamic systems under different conditions. The quantity and type of GAG chains synthesized depends on the system where it was tested and on the structure of the aglycone moiety of the xylosides (32–34). Most of these studies have utilized a few O-xylosides that are inherently less stable. Furthermore, synthesis of O-xylosides requires very stringent reaction conditions, toxic Lewis acids, and at times leads to inseparable α and β mixtures with unpredictable yields. As a result, it is tedious to generate diverse xylosides in a rapid fashion and utilize them in biological systems. We envisioned that synthesis of metabolically stable xylosides will advance our knowledge of glycosaminoglycan biosynthesis and how they regulate various pathophysiological processes.In our earlier communication, we outlined a simple strategy, utilizing click chemical methodology that addresses the above limitations of O-xylosides, to generate a library of xylosides in a robust manner (35). Several studies have shown that the concentration of the primers and the aglycone moieties influence the composition of GAG chains produced (32). In the current study, we show that the aglycone moieties of click-xylosides may not only influence the composition and quantity of GAG chains but also the extent of sulfation, sulfation pattern, disaccharide composition, and chain length using pgsA-745 Chinese hamster ovary (CHO) cell line as a model cellular system. Our findings provide new insights in to the mechanism of GAG biosynthesis and offer new avenues to decipher the biological actions of GAG chains in model organisms.