Glyceraldehyde-3-Phosphate Dehydrogenase of Babesia microti Is a Plasminogen- and Actin-Binding Protein

Abstract

Babesia microti, an intraerythrocytic protozoa, can cause an emerging tick-borne disease-Human babesiosis. The parasite can successfully invade host red blood cells owing to the assistance of molecules expressed by babesia. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping intracellular glycolytic enzyme, can also be expressed in the external of cells, where contributes to binding to several molecules such as plasminogen and actin. In the present study, we identified B. microti GAPDH (BmGAPDH) and generated the recombinant BmGAPDH (rBmGAPDH) via an E. coli expression system. Furthermore, we confirmed its catalytic dehydration activity in vitro. Moreover, we also demonstrated that rBmGAPDH could bind to human plasminogen and mouse α-actin. In addition, we demonstrated that rBmGAPDH could recognize anti-B. microti mouse serum. In conclusion, BmGAPDH is a multifunctional glycolytic enzyme, which can bind to host plasminogen and α-actin.

Keywords: Babesia microti; binding protein; glyceraldehyde-3-phosphate dehydrogenase; plasminogen; α-actin.

Figure 1

Bioinformatics analysis of BmGAPDH. Multiple sequence alignment of GAPDHs from Babesia microti (XP_012647129), Plasmodium falciparum (AAK30144), Leishmania mexicana (CAA46323), Toxoplasma gondii (AAK20420), Trypanosoma cruzi (CAA37080), Streptococcus cerevisiae (CAD44376), and Homo sapiens (NP_002037) was performed by using the ClustalX program. The alpha helix and beta sheet secondary structures are, respectively, shaded in yellow and turquoise. The NAD⁺ binding domains is boxed with red whereas the catalytic domain is boxed with blue. The conserved NAD⁺ binding and glyceraldehyde-3-phosphate (G3P) interacting residues are, respectively, marked by rectangles and stars. The residues of NAD⁺ binding pocket are marked with underlines. S-loop is indicated with horizontal black line.

Figure 2

Three-dimensional structure of docking complex between BmGAPDH and human plasminogen or mouse α-actin. Molecular docking between BmGAPDH and human plasminogen or mouse α-actin was performed with ClusPro 2.0 server and visualized with PyMOL program. BmGAPDH was marked as green cartoon and human plasminogen (A) or mouse α-actin (B) was marked as gray surface, the putative binding segmentation was enlarged in right of the figure.

Figure 3

Recombinant BmGAPDH (rBmGAPDH) preparation and antigenic analysis. (A) Preparation of rBmGAPDH. Lane M: prestained standard protein marker; Lane 1: total cellular proteins of cell lysates; Lane 2: total cellular proteins of induced cell lysates; Lane 3: supernatant of induced cell lysates; Lane 4: sediment of induced cell lysates; Lane 5: purified rBmGAPDH protein. (B) Western-blot analysis of rBmGAPDH. Lane M: standard protein marker; Lane 1: purified rBmGAPDH proteins were probed with normal mouse serum; Lane 2: purified rBmGAPDH proteins were probed with B. microti-infected mouse serum. (C) immunoprecipitation analysis of the native BmGAPDH. Lane M: standard protein marker; Lane 1: normal mouse erythrocyte lysate; Lane 2: B. microti-infected erythrocyte lysate.

Figure 4

Determination of rBmGAPDH enzymatic activity. (A) The enzymatic activity increased with increasing concentrations of rBmGAPDH. (B) The Lineweaver-Burk plot was used to calculated K_m (0.583 mM) and V_max (0.401 μmol/L/min) for rBmGAPDH. (C) rBmGAPDH enzymatic activity was maximal at 32°C. (D) rBmGAPDH enzymatic activity was maximal at pH 9.5. Relative activity of rBmGAPDH was determined by OD340 absorbance. All data are represented as the mean ± SD from triplicate experiments. Significant differences are denoted by ^*** for p < 0.001.

Figure 5

Binding activity of rBmGAPDH to plasminogen and α-actin. (A) Enzyme-linked immunosorbent assay (ELISA) was performed to characterize the ability of rBmGAPDH binding to human plasminogen, BSA was chosen as a negative control for non-specific binding. rBmGAPDH binding activity was determined by absorbance at OD450. All data shown represent the mean ± SD from triplicate experiments. Significant differences compared to control are denoted by ^***p < 0.001. (B) Pull-down assay was performed to analyze rBmGAPDH binding to mouse α-actin. Lane M: standard protein marker; Lane 1: normal mouse erythrocyte lysate (Input); Lane 2: rBmGAPDH pull-down normal mouse erythrocyte lysate; Lane 3: negative control.