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The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose
1Department of Mycology, Nippon Roche Research Center, Kamakura, Kanagawa, Japan.
Abstract:
We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.
Insights
Researchers identified two fungal genes, CaVIG9 and CgVIG9, encoding GDP-mannose pyrophosphorylase, crucial for protein glycosylation. These genes complement yeast mutations, enabling GDP mannose synthesis and high-throughput screening.
Area of Science:
- Molecular Biology
- Mycology
- Biochemistry
Background:
- Protein glycosylation is essential for fungal cell wall integrity and virulence.
- GDP-mannose pyrophosphorylase is a key enzyme in the mannose biosynthesis pathway, critical for protein glycosylation.
Purpose of the Study:
- To identify and characterize GDP-mannose pyrophosphorylase genes from pathogenic fungi Candida albicans and Candida glabrata.
- To assess the functional complementation of these genes in Saccharomyces cerevisiae.
- To develop a high-throughput screening method for enzyme activity.
Main Methods:
- Genomic DNA fragment identification and sequencing.
- Functional complementation assays in a vig9 mutant of Saccharomyces cerevisiae.
- Expression and purification of glutathione S-transferase fusion proteins.
- Enzyme activity assays using a non-radioactive substrate in a heterologous Escherichia coli expression system.
Main Results:
- Identified CaVIG9 and CgVIG9 genes encoding GDP-mannose pyrophosphorylase from Candida albicans and Candida glabrata.
- Demonstrated that CaVIG9 and CgVIG9 functionally complement the vig9 mutation in Saccharomyces cerevisiae.
- Confirmed homology of the predicted gene products to S. cerevisiae GDP-mannose pyrophosphorylase.
- Established enzyme activity of the fungal VIG9 proteins in an E. coli expression system.
- Developed a non-radioactive assay for detecting GDP-mannose pyrophosphorylase activity suitable for high-throughput screening.
Conclusions:
- The identified VIG9 genes from C. albicans and C. glabrata encode functional GDP-mannose pyrophosphorylases essential for protein glycosylation.
- These findings provide insights into the conservation of the mannose biosynthesis pathway in pathogenic fungi.
- The developed assay facilitates efficient screening for enzyme activity, potentially aiding in drug discovery targeting fungal glycosylation.