Unusual aggregation property of recombinantly expressed cancer-testis antigens in mammalian cells

Abstract

Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions, and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein–protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell.

Hydrophobicity and molecular mass‐based separation method for autoantibody discovery from mammalian total cellular proteins

“…This inclination of autoantibodies towards linear epitopes is believed to be influenced by autoantigen conformation. Notably, the majority of cancer‐testis antigens belong to a class of intrinsically disordered proteins (Ahmadi et al, 2021; Rajagopalan et al, 2011). Although most intrinsically disordered proteins adopt biologically active structures through interactions with binding partners, proteins aberrantly expressed in cancer cells lack such associations, resulting in their aggregation or degradation (Ahmadi et al, 2021).…”

“…Notably, the majority of cancer‐testis antigens belong to a class of intrinsically disordered proteins (Ahmadi et al, 2021; Rajagopalan et al, 2011). Although most intrinsically disordered proteins adopt biologically active structures through interactions with binding partners, proteins aberrantly expressed in cancer cells lack such associations, resulting in their aggregation or degradation (Ahmadi et al, 2021). This biophysical attribute potentially contributes to the antigenicity of the humoral immune responses.…”

Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading

“…Purified antibodies reliably validated control in the MUSCAT assay system. Most CTAs have unstable and aggregation-favored properties ( 30 ); therefore, S- cationization is a powerful technique to prepare highly purified and water-soluble antigens. We are currently preparing comprehensive sets of CTAs/TAAs and each specific antibody for use as a validation tool using these procedures.…”

“…Full-length cancer antigen preparation is favored for monitoring antigen spreading based on these requirements. However, many recombinant CTAs appear to show an aggregation-favored unstable property ( 30 ). This property is consistent with the bioinformatics prediction of the structure of CTAs, which shows that the majority of CTAs are intrinsically disordered proteins (IDPs) ( 31 ).…”