3-phosphoglycerate kinase: a glycolytic enzyme protein present in the cell wall of Candida albicans.
We have used a polyclonal antiserum to cell wall proteins of Candida albicans to isolate several clones from a cDNA lambda gt11 expression library. Affinity-purified antibody prepared to the fusion protein of one clone identified a 40 kDa moiety present in cell wall extracts from both morphologies of the organism. Indirect immunofluorescence demonstrated expression of this moiety at the C. albicans cell surface. Sequencing of a pBluescript II genomic clone identified with the cDNA clone revealed an open reading frame for a 417 amino acid protein. The nucleotide sequence showed significant homology with 3-phosphoglycerate kinase (PGK) genes, with 88%, 77% and 76% nucleotide homology with the PGK genes from Candida maltosa, Saccharomyces cerevisiae and Kluyveromyces lactis, respectively. The deduced amino acid sequence was consistent with this identification of the sequence as PGK1 of C. albicans. This finding was confirmed by a positive immunological response of a commercially available purified PGK from S. cerevisiae with the affinity-purified antibody against the fusion protein of the cDNA clone. The presence of PGK in the cell wall was confirmed by two additional methods. Cell wall protein were biotinylated with a derivative that does not permeate the cell membrane to distinguish extracellular from cytosolic proteins. Biotinylated PGK was detected among the biotinylated proteins obtained following streptavidin affinity chromatography. Immunoelectron microscopy revealed that the protein was present at the outer surface of the cell membrane and cell wall as well as expected in the cytoplasm. Northern blot analysis revealed that the gene transcript was present in C. albicans cells growing under different conditions, including different media, temperatures and morphologies. Most of the enzyme activity was found in the cytosol. Low enzymic activity was detected in intact cells but not in culture filtrates. These observations confirmed that PGK is a bona fide cell wall protein of C. albicans.
Alloush
Habib M
HM
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
López-Ribot
José L
JL
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Masten
Barbara J
BJ
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Chaffin
W LaJean
WL
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
D000595:Amino Acid Sequence; D000908:Antibodies, Fungal; D000918:Antibody Specificity; D001483:Base Sequence; D001710:Biotin; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics*; D002451:Cell Compartmentation; D002473:Cell Wall / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005800:Genes, Fungal; D006019:Glycolysis; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000908:Antibodies, Fungal; D018076:DNA, Complementary; D001710:Biotin; D010735:Phosphoglycerate Kinase