Depression of T-cell Epitope Generation by Stabilizing Hen Lysozyme

Abstract

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular crosslink. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen. An antigen-specific CD4ϩ T-cell recognizes an antigen-derived peptide that is mounted on a major histocompatibility complex class II molecule on a cell surface of antigen-presenting cells, via its antigen receptor (1, 2). Therefore, the conformation of protein antigens is unlikely to have any role in the step of T-cell recognition. Prior to T-cell activation, however, antigen processing is necessary for a protein antigen to stimulate the specific T-cells; this processing consists of multiple steps of cellular events, i.e. internalization of proteins by antigen-presenting cells, reduction of the disulfide bond and unfolding of proteins, enzymatic digestion, and assembly of the generated peptides with major histocompatibility complex class II molecules (3, 4). Proteases preferentially digest proteins in an unfolded state rather than those in a folded state (5-7); thus, the unfolding may be a crucial step for intracellular antigen processing. In this context, we can expect that depression of protein unfolding by increasing protein stability would reduce the antigenicity of proteins for T-cells.Several reports have demonstrated a relationship between increased antigenicity and the decreased stability of proteins (8 -10). However, the influence of protein stability on the antigenicity remains unknown. To address this issue, we prepared three derivatives of hen egg white lysozyme (HEL) 1 possessing different conformational stabilities (see Table I). A three-disulfide derivative of HEL, S-carboxymethylated HEL at Cys ...

A Protein’s Conformational Stability Is an Immunologically Dominant Factor: Evidence That Free-Energy Barriers for Protein Unfolding Limit the Immunogenicity of Foreign Proteins

“…1-15 CL-HEL . 35-108 HEL (18). Thus, the IgG production results were consistent with the T cell-stimulating capacities of the HEL derivatives.…”

“…Because protein unfolding may be a crucial step in intracellular Ag processing, the conformational stability of the protein Ag is important in determining the immune response. In a previous study using modified hen egg white lysozyme (HEL), we found that the T cell-triggering response and, therefore, the immune response, were governed by the degree of conformational stability of the protein Ags (18,19). This finding was supported by a study using derivatives of snake toxin with different stabilities, which found that Ag presentation was controlled by the stability of the toxin (20).…”

“…There have been few reports on the correlations between conformational stability and immune response (18)(19)(20). In this study, we demonstrated that there were inverse and linear correlations between free-energy changes for the protein unfolding and the amount of IgG production against Ags in BALB/c mice with both HEL derivatives and ML mutants.…”

“…Proteolytic-degradation analysis in vitro previously showed that restriction of denaturation by increasing the stability of lysozyme prevented the generation of antigenic peptides, resulting in the suppression of T cell activation (18). Generally, protein Ags are degraded in the cell by proteases, such as cathepsins B and D, prior to Ag-specific T cell activation (32,33).…”

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non‐specific immunostimulatory effects

“…It has been reported that some enzymes and toxins are inactivated by mechanisms involving either oxidation of specific amino acids and formation of disulfide bonds after periodate and formaldehyde treatments [40][41][42]. In addition, some authors have described that increasing protein conformational stability might either increase [17] or diminish the immunogenicity of certain antigens [43][44][45]. The question that arises is why Ox-CCH presents higher immunogenicity than CCH.…”