Conformational dependence and conservation of an immunodominant epitope within the babesia equi erythrocyte-stage surface protein equi merozoite antigen 1

Abstract

Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.

FIG. 1.

Alignment of deduced amino acid sequences of EMA-1 from B. equi isolates recovered worldwide. The sequences were obtained from GenBank and aligned by use of the AlignX program from the Vector NTI Suite. The line indicates the region where the epitope defined by MAb 36/133.97 is located. Black boxes designate nonhomologous residues, and gray boxes designate conserved substitutions. Gaps are represented by dots.

FIG. 2.

Physical map of the recombinant EMA-1 proteins relative to the hydropathicity scale of the protein. The method of Kyle and Doolittle (16) was used to calculate hydropathicity over a window of 31 amino acids. Regions with values below zero are hydrophilic in character. The x axis represents the amino acid position in EMA-1. The full-length protein (EMA-1) and the truncated recombinant clones (EMA-1.2, EMA-1.3, and EMA-1.5) are plotted against the same x axis.

FIG. 3.

MAb 36/133.97 and horse serum reactivities to full-length (EMA-1) and truncated (EMA-1.2, EMA-1.3, and EMA-1.5) recombinant proteins by Western blotting. Recombinant E. coli lysates were used as antigens. (A) Reaction with MAb 36/133.97; (B) reaction with serum from a horse persistently infected with B. equi. Lanes 1, recombinant EMA-1; lanes 2, recombinant EMA-1.2; lanes 3, recombinant EMA-1.3; lanes 4, recombinant EMA-1.5; lanes 5, E. coli lysate. The positions of the full-length and truncated proteins are indicated on the right, and standard molecular masses (in kilodaltons) are indicated on the left.