Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

Abstract

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.Asparagine deamidation is the most common spontaneous post-translational protein modification and is increasingly recognized as having an important impact on protein function. For example, a recent study demonstrated that the DNA-damaging antineoplastic drug cisplatin induced the deamidation of two asparagine residues in a flexible loop of the antiapoptotic protein Bcl-xL, preventing its interaction with other proapoptotic Bcl family members and thereby inducing apoptosis (1). Other studies document the deleterious effects of asparagine deamidation on the CD4 binding activity of human immunodeficiency virus gp120 (2), the co-mitogenic and receptor binding activity of murine IL1-␤ 1 (3), and the angiogenic activity of angiogenin (4). Immune recognition of proteins and peptides can also be affected by deamidation/isomerization of asparagine and glutamine residues. For example, T cells have been described that specifically recognize peptides containing asparagine residues that have either cyclized (5) or become deamidated to aspartic acid (6, 7). As has been pointed out (8), the appearance of such modifications in "self"-proteins may disturb the delicate balance that the immune system establishes between tolerance to self-proteins on the one hand but an ability to respond to foreign entities on the other. For example, mice showed no response to unmodified forms of a self-cytochrome c peptide (90 -104) or to a small nuclear ribonucleoprotein D peptide (65-79) but developed strong T and B cell responses to forms of these peptides in which a single Asp residue had been modified to iso-Asp (9). Importantly, the B cell response to the iso-Asp containing peptide cross-reacted with the native protein, suggesting a plausible chain of events initiated by post-translational protein modification that may lead to autoreactivity. In the case of ␣-gliadin, a single glutamine residue, likely deamidated as a result of tissue transglutaminase action, is found in two overlapping T cell epitopes recognized by T cells from human leukocyte antigen-DQ2-positive celiac disease patien...

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…7,23,42 Deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-displayed peptides to T cells and thus contributing to exacerbation of auto immune diseases. 43,44 It has also been suggested that in vivo deamidation of proteins could serve as a molecular timer of many important biological events, such as aging. 5,[45][46][47] Few studies have attempted to investigate the consequences of deamidation by directly investigating the altered properties or by mutating the deamidated site.…”

Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

“…However, the deamidation that occurs in vitro during the isolation of proteins and subsequent storage can adversely affect the biological properties of the protein. Effects on epitope structure, antigen processing, and antigen presentation represent negative outcomes that are particularly relevant to vaccine development (15,16). The rate of deamidation of any given Asn or Gln residue depends on a number of parameters, including primary structure (neighboring amino acids), pH, temperature, and ionic strength.…”

Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

“…Alternatively, changes in the amino acid sequence of rPA due to deamidation might affect antigen processing by altering critical target sites for the proteases involved in antigen processing. Such a phenomenon has been described for tetanus toxin fragment C (12). Those authors showed that deamidation of Asn residues interfered with processing of the protein, which affected generation of a T-cell response as assessed using an in vitro assay.…”

“…Deamidation of Asn occurs in vivo on a time scale ranging from a few seconds to centuries, depending on the local environment (10). In theory, deamidation might adversely affect the interaction of rPA with the aluminum adjuvant of the vaccine, might alter the epitope structure of the antigen, and/or might affect the ability of the protein to elicit T-cell help for antibody production which, in turn, might affect the vaccine immunogenicity (11)(12)(13)(14)(15).Earlier studies have demonstrated that certain Asn residues of rPA deamidate during the purification process and upon storage (8,16,17). Previously, we generated a mutant form of rPA in which the six Asn residues that are the most prone to deamidation were changed to Asp residues (six-Asp mutant rPA).…”

“…Deamidation of Asn occurs in vivo on a time scale ranging from a few seconds to centuries, depending on the local environment (10). In theory, deamidation might adversely affect the interaction of rPA with the aluminum adjuvant of the vaccine, might alter the epitope structure of the antigen, and/or might affect the ability of the protein to elicit T-cell help for antibody production which, in turn, might affect the vaccine immunogenicity (11)(12)(13)(14)(15).…”