Elucidating the structure of the 12 kDa ES protein from T. gondii is necessary to understand the functionality of the antigen. It is interesting to find that BLAST against nr protein database and PDB database, showed the 12 kDa ES protein has high sequence identity with thioredoxin proteins. It is the most abundant cellular-reducing dithiol catalyst which functions include redox regulation, protein folding, intracellular signaling and oxidative stress responses. The thioredoxin family is a large family of proteins consisting of domains that function biochemically by forming disulfide bonds with target molecules, resulting in conformational changes or rearrangement of disulfide bonds in the target. In cancer studies Trxs have been proposed as drug targets. Furthermore, components of the redox cycle have been considered targets in malaria parasites and trypanosomatids. The ES protein has also been identified to be mainly localized at the outer compartment of T.gondii apicoplast and it is discovered that an Apicoplast Thioredoxin-like protein 1 (ATrx1) was the first protein found to reside in apicoplast intermembrane spaces. Several enzymes found in the apicoplast that are potentially regulated by thioredoxin including 1-deoxy-d-xylulose 5-reductoisomerase, Clp protease and the protein translation factors EF-G and EF-Tu .
In secondary structure prediction study, all servers showed similar results except for Prof server. Prof server predicted a β-sheet (residue 63–70) instead of an α-helix, which contradicted with the other three servers. This contradiction could be mainly caused by the small size of the α-helix which consists of only 8 residues (residue 63–70). It was the shortest α-helix among all the α-helices in the built ES protein model. This secondary structure prediction can help in the verification for the tertiary structure of the built protein.
The built ES protein resembled thioredoxin proteins, consisting of four α-helices (α1- α4) flanking five mixed β-sheets (β1- β5) in the center of the protein (Figure 2a). Each α-helix and β-strand was connected through loops and turns. From the ES protein structure, all the loops and turns were located on the surface of the protein where they could be accessed easily and have a higher tendency to be the ES protein epitope. All four helices were also located on the surface flanking the β-sheets, causing the sheets to be buried in the center of the ES protein and thus less likely to be accessible. The β-sheets and α-helices can be subdivided into N- and C-terminal motifs which are connected by loops. The above mentioned orientation of the sheets, helices, loops and turns are similar to the thioredoxin protein family. Thus, this makes the 12 kDa ES protein a variant of thioredoxin .
Thioredoxin is a single-domain monomeric glutaredoxins-like protein with an extra β-sheet and α-helix at the N-terminal . Thioredoxin exist either in a reduced form, with a dithiol, or in an oxidized form when its half-cystine residues form an intramolecular disulfide bridge . The built model of the 12 kDa ES protein model is most likely in the oxidized form because of the presence of disulfide bridge linking two cystine residues (residue 31 and 34) (Figure 2a). Thioredoxin is also involved in reduction/oxidization (redox) reaction through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reaction . By having the backbone structure of a thioredoxin protein, the ES protein is expected to share similar functions with thioredoxins. The 12 kDa ES protein is made of 75% β-sheets and α-helices, which is similar in characteristic to thioredoxin protein, and making it exceptionally stable and highly resistant to heat . Apart from that, the ES protein structure also has the Trp-Cys-Gly-Pro-Cys conserved region (Figure 2b). This region is possessed by thioredoxins and appears to be the location of the disulfide bridge linking the two cystine residues (residue 31 and 34) within the conserved region between the β2 and α2. This conserved region is found in residue 30 to 34 of the 12 kDa ES protein which is known to be the active site for redox activity .
Thioredoxin protein is ubiquitously expressed in all living cells, which has a variety of biological functions related to cell proliferation and apoptosis . Thioredoxins are found to have anti-apoptotic effects and are identified as an interacting partner of ASK1. Study showed that residue Gly-32, Pro-33, Ile-74, Pro-76, Val-91, Gly-92 and Ala-93 of thioredoxin are involved in the binding of thioredoxin to other protein molecules . From the results of the docking simulation performed between ES protein and ASK1, all of the above mentioned residues (ES protein has the similar residue number) are located in the binding site of ES protein and play a role in the binding and interaction of ES protein with ASK1. The expression of thioredoxin interacts with the N-terminal of ASK1 and inhibits ASK1 kinase activity and subsequent ASK1-dependent apoptosis. This interaction is highly dependent on the redox status of thioredoxin, indicating that the redox ability of thioredoxin is very important for apoptosis inhibition . The molecular docking results also suggest that the ES protein is able to bind to ASK1, thus it can possibly inhibiting ASK1 kinase activity which could stop the mechanism of ASK1-dependent apoptosis.
Another study also suggested that mitochondrial thioredoxin is essential for cell viability and regulation of the mitochondrial apoptosis signaling pathway . A novel thioredoxin reductase inhibitor study on human leukemia cell lines showed inhibition activity of cell growth and induction of apoptosis . The inhibition of ES protein could also help in the inhibition of T. gondii proliferation which occurs in the tachyzoites stage. This is because cell proliferation of T. gondii and apoptosis occur in the host cells during T. gondii infection. Thus, inhibiting the ES protein could prevent cell apoptosis. This lead to the hypothesis that the ES protein may play an important role in the pathogenesis of T. gondii infection.
ES proteins are secreted by T. gondii to perform certain tasks such as invading its host cells or excreted as waste products into the blood circulation. Thus it could be useful for diagnostic purposes since it is expected to be present in blood of all actively infected patients . In addition, previous studies showed that the diagnostic sensitivity and specificity of T. gondii ES antigens are higher than the crude parasite antigen by 5% and 6% respectively . ES antigens are also known to improve the sensitivity of diagnostic tools compared to somatic antigens [11–13]. Thus, we conclude that ES antigens are potential biomarker candidates in the development of diagnostics for toxoplasmosis.
Epitope prediction is important to identify the binding site of an antigen which will interact with the antibody. Epitopes are usually located on the surface of an antigen to make it accessible to antibodies. In addition, epitopes also tend to be located on the loops and turns of an antigen . Epitopes are predicted using the calculation of physiochemical properties of the residues and determining the residue’s antigenicity . The results of epitope predictions in this study are consistent with the basic criteria of epitopes whereby most of the predicted epitopes are located in the loops and turns of the 12 kDa ES protein. The conserved region of thioredoxin seen in the ES protein, is one of the predicted epitopes. This region is actually conserved with Trp-Cys-X-X-Cys; where the X represents any amino acid. If the amino acids represented by the Xs vary in thioredoxins of different organism, the ES protein can be a very valuable biomarker as the conserved region will provide a very specific binding site for an antibody. Besides that, epitope can also be termed as “protein binding site”. Protein binding site of the ES antigen was predicted by ProBis [35–37], a protein binding site detection server which detected three of the predicted epitopes as the antigen’s binding sites, corroborating with the predicted epitopes.
An antibody does not necessarily binds to just one of the epitopes. It might bind to more than one epitope which are closely located in a folded protein but not in the linear sequence of an antigen. The epitopes of residue 27–35, 71–76 and 91–98 in ES protein may function as one “single” epitope to be bound with an antibody. Predicted epitopes can be linear and/or conformational depending on the binding mode with an antibody. All coloured region in Figure 2(a) are considered linear epitopes. However, if an antibody is bound to more than one coloured region, it can be considered as conformational epitopes. These predicted epitopes can thus be used in future design of a binder or an antibody (e.g. scFv) which is specific to the 12 kDa ES protein.