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1.
Mol Immunol ; 112: 151-162, 2019 08.
Article in English | MEDLINE | ID: mdl-31108423

ABSTRACT

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.


Subject(s)
Fungal Proteins/immunology , Nucleotidyltransferases/immunology , Paracoccidioides/immunology , Paracoccidioidomycosis/immunology , Adenosine Triphosphate/immunology , Adolescent , Adult , Aged , Amino Acid Sequence , Cytidine Triphosphate/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Escherichia coli/immunology , Female , Humans , Male , Middle Aged , Recombinant Proteins/immunology , Young Adult
2.
Anal Biochem ; 154(1): 276-81, 1986 Apr.
Article in English | MEDLINE | ID: mdl-3010772

ABSTRACT

A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.


Subject(s)
Deoxycytosine Nucleotides/analysis , Radioimmunoassay/methods , Animals , Chromatography, Affinity , Cross Reactions , Cytidine Triphosphate/immunology , Cytidine Triphosphate/isolation & purification , Deoxycytosine Nucleotides/immunology , Fibroblasts/analysis , Humans , Leukemia L5178/metabolism , Mice , Microchemistry , Periodic Acid
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