Inhibition of Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis

Abstract

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...

Ultra-performance ion-pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis

“…They are accepted by the FDA as a host for the production of human therapeutics, and protocols exist for removal of host cell proteins, nucleic acids, and viral contaminants. CHO cells are known to biosynthesize HS but not heparin [49; 50]. Current research in our laboratory is aimed at the metabolic engineering of the HS biosynthetic pathway to produce secreted CHO cell heparin.…”

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine

“…It is possible to stimulate HS and CS biosynthesis by utilizing xylosides to prime GAG chains, however with no specific properties [356]. In another approach, it is possible to inhibit HS/CS biosynthesis by utilizing 4-deoxy-4-fluoro-xylosides [357]. Decreasing overall levels of HS and CS would affect HS/CS-matrix interactions and prevent tumor proliferation, invasion, metastasis, and angiogenesis by reducing for example FGF and VEGF signaling.…”

Sugar glues for broken neurons

“…A number of fluoro-xylosides have been found to inhibit GAG biosynthesis in CHO and endothelial cells. (178, 179) Small molecular inhibitors of GAG sulfotransferases are yet another option that can be utilized to modulate the HS or CS sulfation pattern at the damage site. By selectively targeting one or more sulfotransferase enzymes, highly specific HS or CS motifs can be generated to stimulate neuronal regeneration.…”