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.

SCHEME 1.

Synthesis of 4-deoxy-4-fluoroxylosides. DAST, diethylaminosulfur trifluoride; TMSN₃, tetramethylsilyl azide; rt, room temperature; DMF, N,N-dimethylformamide; DCM, dichloromethane.

FIGURE 1.

Assembly of the PG tetrasaccharide linkage region. XYL T1 and XYL T2, xylosyltransferase-1 and xylosyltransferase-2; GAL T1, galactosyltransferase-1.

FIGURE 2.

Inhibition of PG biosynthesis by xyloside derivatives. ³⁵S-Labeled GAG chains were isolated from control and 4-deoxy-4-fluoroxyloside-treated cells by protease digestion and purification as described under “Experimental Procedures.” Radiolabeled GAG chains were then fractionated on a DEAE HPLC column and analyzed for radioactivity with the aid of an in-line radiodetector. The bound GAG chains were eluted with a linear gradient of 1 m NaCl as described under “Experimental Procedures.” The elution profiles of GAG chains isolated from cells that were untreated (gray trace; control) or treated (black trace) with PG biosynthetic inhibitors (compounds 5a–5i) are shown above.

FIGURE 3.

Dose-dependent decrease in GAG biosynthesis by 4-deoxy-4-fluoroxylosides in CHO cells. Samples of ³H-labeled GAG chains from control and 4-deoxy-4-fluoroxyloside-treated (compounds 5e (A) and 5g (B)) CHO cells were applied to a DEAE HPLC column, eluted with a linear NaCl gradient starting with 0.2 m NaCl as described under “Experimental Procedures,” and analyzed for radioactivity with an in-line radiodetector.

FIGURE 4.

Cytotoxicity of 4-deoxy-4-fluoroxylosides. CHO cells were treated with different concentrations of xylosides (compounds 5a–5i) for 24 h and then incubated with CellTiter-Blue® reagent for 2 h as described under “Experimental Procedures” to estimate the percent of viable cells. CHO cells were grown in the absence of inhibitors (vehicle alone) as a control (Control 1), and the values of treated cells were compared with Control 1. Cells were also grown in the presence of 3% H₂O₂ as an additional control (Control 2).

FIGURE 5.

Comparison of the effects of various 4-deoxy-4-fluoroxylosides on glycolipid biosynthesis in CHO cells. Cells were incubated with vehicle alone (control) or with xylosides (compounds 5a–5i) for 24 h in the presence of [³H]GlcNH₂. Glycolipids were isolated and quantified as described under “Experimental Procedures.” All values are expressed as a percent of the control sample without any xyloside derivative.