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The Serpin-like Loop Insertion of Ovalbumin Increases the Stability and Decreases the OVA 323-339 Epitope Processing Efficiency.

ABSTRACT: Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323-339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323-339 epitope in vitro. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

SUBMITTER: Moss DL

PROVIDER: S-EPMC8253479 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Serpin-like Loop-insertion of Ovalbumin Increases Stability and Reduces OVA 323-339 Epitope Immunogenicity

Project description:Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive-center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution-variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins to study how changes in loop size and protein stability influences CD4+ T-cell priming in vivo. We observed that the OVA R339T loop-insertion increases stability and protease resistance, resulting in reduced CD4+ T-cell priming of the OVA 323-339 epitope in SJL mice. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell-epitope prediction tools.

2021-05-07 | PXD021756 | Pride

Flanking residues are central to DO11.10 T cell hybridoma stimulation by ovalbumin 323-339.

Project description:T cell activation requires formation of a tri-molecular interaction between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323-339 binds the murine class II MHC, I-A(d), in at least three distinct registers. The DO11.10 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using exogenous peptides and peptide insertions into a carrier protein in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weakly binding register is stabilized by the presence of n-terminal flanking residues active in MHC binding. The DO11.10 hybridoma is sensitive to the presence of specific wild-type residues extending to at least the P-3 peptide position. Transfer of the P-4 to P-2 flanking residues to a hen egg lysozyme epitope also presented by I-A(d) increases the activity of that epitope substantially. These results illustrate the inherent complexity in delineating the interaction of multiple registers based on traditional thermodynamic measurements and demonstrate the potential of flanking residue modification for increasing the activity of weakly bound epitopes. The latter technique represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a responding T cell.

| S-EPMC3479146 | biostudies-literature

SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), member 3).

Project description:Review on SERPINB3, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

| S-EPMC4430857 | biostudies-literature

Dynamic local unfolding in the serpin ?-1 antitrypsin provides a mechanism for loop insertion and polymerization.

Project description:The conformational plasticity of serine protease inhibitors (serpins) underlies both their activities as protease inhibitors and their susceptibility to pathogenic misfolding and aggregation. Here, we structurally characterize a sheet-opened state of the serpin ?-1 antitrypsin (??AT) and show how local unfolding allows functionally essential strand insertion. Mutations in ??AT that cause polymerization-induced serpinopathies map to the labile region, suggesting that the evolution of serpin function required sampling of high risk conformations on a dynamic energy landscape.

| S-EPMC3074950 | biostudies-literature

Expression data from ovalbumin (OVA) induced asthma model

Project description:MiRNAs are involved in the pathogenesis of bronchial asthma and are involved in the regulation of airway inflammation, airway remodeling and airway hyperreactivity. In this experiment, we constructed OVA asthma model and identified the differentially expressed miRNAs in asthma and normal models by microarray technology, providing a preliminary basis for future studies on the mechanism of asthma. We used microarrays to detail the global program of gene expression in asthma models and identify miRNAs that are differentially expressed in this process.

2022-02-22 | GSE197090 | GEO

Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop.

Project description:A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1-P1' cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113-120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented further cleavage of the P1-P1' pre-cleaved R339T mutant by subtilisin, which cleaved the second P8-P7 loop site in the P1-P1' pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1-P1' and double P1-P1'/P8-P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8-P7 site in the immediate intermediate after the P1-P1' cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0 x 10(-3)/s in the R339T mutant. The second-order rate constant for the P8-P7 cleavage in the immediate P1-P1' cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.

| S-EPMC1222492 | biostudies-other

Project description:Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys(367)-His(368) is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

| S-EPMC3682532 | biostudies-literature

Ovalbumin and angiotensinogen lack serpin S-R conformational change.

Project description:Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.

| S-EPMC1133235 | biostudies-other

Reactive centre loop dynamics and serpin specificity.

Project description:Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as ?1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from ?1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

| S-EPMC6405850 | biostudies-literature

Loop-sheet mechanism of serpin polymerization tested by reactive center loop mutations.

Project description:The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central β-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.

| S-EPMC2945569 | biostudies-literature

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