The impact of micropolymorphism in Anpl-UAA on structural stability and peptide presentation.

Micropolymorphism significantly shapes the peptide-binding characteristics of major histocompatibility complex class I (MHC-I) molecules, affecting the host's resistance to pathogens, which is particularly pronounced in avian species displaying the "minimal essential MHC" expression pattern. In this study, we compared two duck MHC-I alleles, Anpl-UAA*77 and Anpl-UAA*78, that exhibit markedly different peptide binding properties despite their high sequence homology. Through mutagenesis experiments and crystallographic analysis of complexes with the influenza virus-derived peptide AEAIIVAMV (AEV9), we identified a critical role for the residue at position 62 in regulating hydrogen-bonding interactions between the peptide backbone and the peptide-binding groove. This modulation affects the characteristics of the B pocket and the stability of the loop region between the 310 helix and the α1 helix, leading to significant changes in the structure and stability of the peptide-MHC-I complex (pMHC-I). Moreover, the proportion of different residues at position 62 among Anpl-UAAs may reflect the correlation between pAnpl-UAA stability and duck body temperature. This research not only advances our understanding of the Anpl-UAA structure but also deepens our insight into the impact of MHC-I micropolymorphism on peptide binding.

Efficient Identification of Tembusu Virus CTL Epitopes in Inbred HBW/B4 Ducks Using a Novel MHC Class I-Restricted Epitope Screening Scheme.

The identification of MHC class I-restricted CTL epitopes in certain species, particularly nonmammals, remains a challenge. In this study, we developed a four-step identification scheme and confirmed its efficiency by identifying the Anpl-UAA*76-restricted CTL epitopes of Tembusu virus (TMUV) in inbred haplotype ducks HBW/B4. First, the peptide binding motif of Anpl-UAA*76 was determined by random peptide library in de novo liquid chromatography-tandem mass spectrometry, a novel nonbiased, data-independent acquisition method that we previously established. Second, a total of 38 TMUV peptides matching the motif were screened from the viral proteome, among which 11 peptides were conserved across the different TMUV strains. Third, the conserved TMUV peptides were refolded in vitro with Anpl-UAA*76 and Anpl-ß2-microglobulin to verify the results from the previous two steps. To clarify the structural basis of the obtained motif, we resolved the crystal structure of Anpl-UAA*76 with the TMUV NS3 peptide LRKRQLTVL and found that Asp34 is critical for the preferential binding of the B pocket to bind the second residue to arginine as an anchor residue. Fourth, the immunogenicity of the conserved TMUV peptides was tested in vivo using specific pathogen-free HBW/B4 ducks immunized with the attenuated TMUV vaccine. All 11 conserved TMUV epitopes could bind stably to Anpl-UAA*76 in vitro and stimulate the secretion of IFN-γ and lymphocyte proliferation, and three conserved and one nonconserved peptides were selected to evaluate the CTL responses in vivo by flow cytometry and their tetramers. We believe that this new scheme could improve the identification of MHC class I-restricted CTL epitopes, and our data provide a foundation for further study on duck anti-TMUV CTL immunity.