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Inhibition of lipid-linked mannose and mannoprotein synthesis in yeast by diumycin in vitro
Abstract:
Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP-Man); 50% inhibition was observed at approximately 10 microgram/ml. To a lesser extent also mannosyl transfer from DolP-Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein-serine/threonine acceptor sites as well as into positions within the asparagine-linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP-Man formation is blocked. DolP-Man synthesis as well as mannosyl transfer from DolP-Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates. Glycosyltransfer reactions from GDP-mannose either to protein-serine/threonine-linked mannose (formation of short manno-oligosaccharides) or to dolichyl-diphosphate-linked chitobiose (formation of lipid-linked trisaccharide) are not inhibited by diumycin under conditions where DolP-Man synthesis is blocked by the antibiotic. The inhibitory action of diumycin on DolP-Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.
Insights
Diumycin, a phosphoglycolipid antibiotic, inhibits yeast mannosyl transfer, specifically dolichyl phosphate mannose (DolP-Man) synthesis. This disruption impacts protein glycosylation, affecting mannoprotein formation.
Area of Science:
- Biochemistry
- Microbiology
- Molecular Biology
Background:
- Yeast mannoproteins are crucial for cell wall structure and function.
- Glycosylation, particularly N-linked and O-linked mannosylation, is essential for yeast cell integrity.
- Dolichyl phosphate mannose (DolP-Man) is a key intermediate in the synthesis of mannosylated glycans.
Purpose of the Study:
- To investigate the inhibitory mechanism of the phosphoglycolipid antibiotic diumycin on mannosyl transfer reactions in yeast.
- To determine the specific steps in the mannosylation pathway affected by diumycin.
- To elucidate the non-competitive nature of diumycin's inhibition regarding dolichyl phosphate.
Main Methods:
- Utilized yeast membrane preparations and solubilized enzymes.
- Assayed mannosyl transfer reactions, including DolP-Man formation and transfer to protein acceptors.
- Investigated the effect of varying substrate concentrations (dolichyl phosphate) to determine inhibition type.
Main Results:
- Diumycin significantly inhibited dolichyl phosphate mannose (DolP-Man) formation in yeast membranes (50% inhibition at ~10 µg/ml).
- Mannosyl transfer from DolP-Man to protein acceptors (serine/threonine and asparagine-linked) was also inhibited, particularly when DolP-Man synthesis was blocked.
- Diumycin did not inhibit glycosyltransfer reactions from GDP-mannose, and its inhibition of DolP-Man formation was not overcome by increased dolichyl phosphate concentrations, suggesting non-competitive inhibition.
Conclusions:
- Diumycin effectively inhibits yeast mannosylation by primarily blocking DolP-Man synthesis.
- The antibiotic disrupts both the synthesis of the mannosyl donor (DolP-Man) and its subsequent transfer to proteins.
- Diumycin's non-competitive inhibition with respect to dolichyl phosphate provides insight into its specific molecular target in the mannosylation pathway.