Investigating the Elusive Mechanism of Glycosaminoglycan Biosynthesis

Abstract

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-...

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro

“…Variations in the HS/CS/DS ratio of xyloside-primed GAGs depending on aglycon have been extensively studied over the years (10,16,31,32). The amount of HS has been of particular interest, as xylosides were initially thought to only induce synthesis of CS.…”

Processing by Convertases Is Required for Glypican-3-induced Inhibition of Hedgehog Signaling

“…Alternatively, the conformational change generated by the lack of cleavage of GPC3 could alter its ability to interact with other proteins, and could consequently change the Golgi compartment in which GPC3 becomes glycanated. It has been proposed that different Golgi compartments could harbor different relative amounts of the various enzymes involved in the synthesis of GAG chains (29).…”

Chondroitin Sulfate Accelerates Trans‐Golgi‐to‐Surface Transport of Proteoglycan Amyloid Precursor Protein

“…The range of GAG chain lengths of proteoglycans typically extends from 20 to 60 kDa corresponding to ∼40-120 disaccharides. Xyloside-primed free chains were shown to have molecular weights of 6-34 kDa (65). With an axial rise of ∼1 nm per disaccharide unit (63), CS chains may extend to ∼70-120 nm and thus -even if not fully extended -sterically hinder the entry into coated vesicles, which were shown to have inner diameters of ∼34-42 nm by cryoelectron tomography (66).…”