Abstract
The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 microM) reduced the quantity (by 64.1-76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K(av) shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7-73.0%, and the size showed a K(av) shift from 0.30 to 0.50. Treatment of ASM cells with 45 microM and 179 microM 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-beta-D-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K(av) of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.
Highlights
The synthesis and physical properties of proteoglycans are altered under some pathological conditions such as cancer [1], spinal cord injury [2], atherosclerosis [3], and asthma [4]
airway smooth muscle (ASM) Cells—To understand the effects of 4-F-GlcNAc on gycosaminoglycan biosynthesis, we characterized the types of glycosaminoglycans synthesized by cultures of murine ASM cells that were radiolabeled with [3H]glucosamine and [35S]sulfate
heparan sulfate (HS) synthesis is blocked by 4-F-GlcNAc addition to the nonreducing terminal GlcUA residue, thereby preventing further addition of GlcUA via a fluorine residue at the requisite 4-position for HS chain elongation
Summary
F, fluorine; HS, heparan sulfate; CS, chondroitin sulfate; HA, hyaluronan; ASM, airway smooth muscle; GlcUA, glucuronate; DS, dermatan sulfate; DMEM, Dulbecco’s modified Eagle’s medium; C’ase ABC, chondroitinase ABC. Airway smooth muscle (ASM) cells produce HS- and CS/DScontaining proteoglycans, including perlecan, versican, and decorin [12]. Using these cells, we observed that 4F-GlcNAc inhibits CS/DS synthesis nearly as effectively as it inhibits HS synthesis. To explore this putative mechanism, we analyzed the inhibitory effects of 4-F-GlcNAc on intrinsic and xyloside-stimulated CS synthesis in ASM cells [13]
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