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Abstract
We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.
Highlights
Glycosylation inhibitors provide powerful tools for understanding the biosynthesis and biological functions of glycoconjugates in animal cells
To identify novel compounds that modulate proteoglycan biosynthesis, we developed a rapid, high throughput assay to screen large collections of natural and synthetic compounds
Pilot experiments comparing a mutant CHO line defective in glycosaminoglycan biosynthesis to the wild-type showed that over 90% of the bound radioactive material consisted of proteoglycans or glycosaminoglycans
Summary
One of the compounds contained a galloyl group at C-3 of the xylose moiety These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells. Other types of glycoside primers include ␣-N-acetylgalactosaminides which target the O-linked pathways of glycoprotein formation [39, 40], -glucosides [41], -galactosides [42], -N-acetylglucosaminides [43], and even disaccharides (44 – 46) These simple compounds resemble natural biosynthetic intermediates and trick cells into assembling oligosaccharide chains on the exogenous primer instead of on endogenous substrates. An alternative discovery strategy consists of screening random chemical libraries The success of this approach depends on rapid, high throughput screening assays to detect potential inhibitors in crude mixtures from microbial or plant sources or from large combinatorial libraries of synthetic compounds. The discovery of primers in plants suggests possible roles for these compounds in chemical defense and ethnobotany, and that large scale screenings might yield other kinds of primers that affect glycosylation
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