Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.
We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.
Sundstrom
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Department of Microbiology and Immunology, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
Aliaga
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics*; D002177:Candidiasis / Q000175:diagnosis; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005455:Fluorescent Antibody Technique; D006019:Glycolysis; D016056:Immunodominant Epitopes; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D004274:DNA, Recombinant; D016056:Immunodominant Epitopes; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins; D010751:Phosphopyruvate Hydratase