Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

Abstract

Background: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Methods: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64-100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64-100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits.

Results and conclusions: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Figure 1

The molecular model of Pf-DHFR. The Pf-DHFR model (green) with the aa64-100 loop highlighted (red). The N-terminal part of the Pf-DHFR (white) was not included in the Pf-DHFR model and it is here shown together with the Leishmania major-TS structure (white and cyan, respectively) to illustrative that the aa64-100 loop does not interfere with these parts of the DHFR-TS complex. The Pf-DHFR substrate and cofactor are just visible at the lower right part of Pf-DHFR (orange). The surfaces are solvent-accessible surfaces generated with SYBYL.

Figure 2

Dot blot analysis of mouse polyclonal and monoclonal antibodies raised against the aa64-100 peptide when tested against the loop peptide and crude P. falciparum 3D7 protein extract in the presence of various reagents interfering with protein structure. The reactivity to two-fold dilutions of crude 3D7 protein extract of polyclonal and monoclonal antibodies are shown. As a positive control the reactivity to the aa64-100 peptide solution (10 μg/ml, two-fold diluted) is shown. Crude 3D7 protein extract was pre-treated before dotting as indicated: 1: untreated, 2: boiled, 3: β-ME, 4: β-ME + boiled, 5: β-ME + IAA, 6: β-ME + IAA + boiled, 7: 0.1 % SDS, 8: 0.1 % SDS + boiled, 9: 0.1 % SDS + β-ME, 10: 0.1 % SDS + β-ME +boiled, 11: 0.1 % SDS + β-ME + IAA, 12: 0.1 % SDS + β-ME + IAA + boiled, B: Buffer control.

Figure 3

Western blots of crude P. falciparum 3D7 protein extract reacted with aa64-100-monoclonal antibody and rabbit polyclonal antibody. (M) Pre-stained protein marker, (1) aa64-100 (10 μg/ml) of the purified antibody; (2) 1:50 dilution of rabbit-antibody. In each lane approximately 6,25 μg total protein was added.