Characterization of a novel Candida albicans 29 kDa IgE-binding protein--purification, cDNA isolation and heterologous expression of Cand a 3.
BACKGROUND
Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)-reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29 kDa IgE-binding protein from C. albicans.


METHODS
The 29 kDa protein was partially purified and its tryptic digests subjected to mass spectrometric analysis. The cDNA encoding this protein was isolated and heterologously expressed in Escherichia coli. Monoclonal antibodies (MoAbs) were raised against the 29 kDa protein purified from C. abicans extracts.


RESULTS
We isolated a 29 kDa IgE-reacting component from C. albicans. The protein was digested on-gel with trypsin and the masses of the resulting fragments were determined in a MALDI-TOF mass spectrometer. The data were searched against protein sequences deduced from the C. albicans genome. An open reading frame that possibly encodes the 29 kDa IgE-reacting component was identified. The cDNA corresponding to the open reading frame was isolated. It encodes a 236 residues protein that has 62% sequence identity to that of a hypothetical protein (YDR533c) from Saccharomyces cerevisiae. Conserved domain search suggests that the encoded protein belongs to the ThiJ/PfpI family. The cDNA isolated was inserted into a pQE-30 vector for protein expression in Escherichia coli. The recombinant protein can react with IgE antibodies in sera from asthmatic patients and two MoAbs that were generated against the purified native 29 kDa protein from C. albicans.


CONCLUSIONS
We identified and cloned a novel 29 kDa IgE-reacting component (Cand a 3) from C. albicans. The recombinant proteins produced from this clone and the MoAbs prepared may be useful in the standardization of diagnostic extracts. They are also instrumental in elucidating the role of C. albicans in clinical allergy.
Chou
H
H
Department of Medical Research, Veterans General Hospital-Taipei, Taiwan.
Tam
M F
MF
Chang
C-Y
CY
Lai
H-Y
HY
Huang
M-H
MH
Chou
C-T
CT
Lee
S-S
SS
Shen
H-D
HD
D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001249:Asthma / Q000276:immunology; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D008875:Middle Aged; D008970:Molecular Weight; D016366:Open Reading Frames
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D007073:Immunoglobulin E