Complex assembly, crystallization and preliminary X-ray crystallographic analysis of duck MHC class I complexed with a TUMV viral peptide
Introduction
In recent years, the duck industry has played a pivotal role in China's animal husbandry. With the expansion of scale, the duck farming industry is facing the threat of increasingly serious viral diseases. Since 2010, many provinces in China have successively erupted acute infections caused by duck contact. The main manifestations are that ducks have significantly reduced egg production, viral encephalitis, and frequent neurological symptoms (Su et al., 2011). The study found that the infection caused by a duck Tembusu virus (TMUV, genus Flavivirus, family Flaviviridae) has brought huge economic losses to the duck breeding industry in China. At present, there is no effective therapeutic drug for the disease. However, it has been reported that vaccination immunity can exert certain immunoprotective effects on duck tambourine virus (He et al., 2019), but little is known about the cellular immunity research of the virus.
The major histocompatibility complex I (MHC I) molecule is an important immune molecule that mediates cellular immunity in the body's adaptive immune response, participates in the processing and presentation of antigens, and antigens through forming a ternary complex with β2-microglobulin and antigen peptide. Cytotoxic T lymphocyte (CTL) responses are activated by binding to TCR on CD8+ T cells(Kane and Clark, 1984). MHC I consists of two chains, one being an alpha chain (also known as a heavy chain) and one being a beta chain (also known as a light chain). The heavy chain extracellular domain has three domains, α1, α2 and α3, and the β2-microglobulin as light chain binds to the α3 region of the heavy chain by non-covalent forces. The α1 and α2 of the MHC I molecule together constitute a peptide binding groove (PGB) (Saper et al., 1991). From the N-terminus to the C-terminus of PBG, they are artificially defined as A, B, C, D, E, and F pockets(Saper et al., 1991). These variable pockets are filled by complementary variable amino acid side chains of peptides that called an anchor residue (Rock et al., 2016), and a polypeptide sequence containing these anchor residues is referred to as a polypeptide binding motif. In general, the decisive role is the B pocket and the F pocket that bind to P2 and P9 of the peptide, respectively. Different MHC I molecules are capable of recognizing different polypeptide binding motifs.
Ducks mainly express one MHC class I locus, which is adjacent to TAP2(Mesa et al., 2004). The genome carries 5 MHC class I genes, and the TAP genes are arranged in the following gene order: TAP1, TAP2, UAA, UBA, UCA, UDA and UEA(Moon et al., 2005). Among them, UBA, UCA, UEA are not expressed, UDA is weakly expressed, while UAA is predominantly expressed and plays a major antigen presentation role and generates CTL immune response (Fleming-Canepa et al., 2016;Chan et al., 2016; Wu et al., 2017). Previous studies have reported that MHC I-mediated CTL immune responses in ducks play an important role in anti-IAV(Influenza A virus, IAV) infection, and can provide cross protection for chickens, mice and humans against different IAV strains (Wu et al., 2017). And the structure of duck MHC I (Anpl-UAA*01) and duck β2m with peptides from the H5N1 strains was determined (PDB entry 5GJY). However, the reports of MHC molecules presenting Tembusu virus polypeptides are still poorly understood.
In this study, we describe the expression, refolding and crystallization of the complex assembled by MHC class I molecules Anpl-UAA*SD and duck β2-microglobulin and the polypeptide derived from TMUV, with a preliminary crystallographic analysis. The complex crystal structure provides data support for further research on duck anti-TMUV infection in the future.
Section snippets
Macromolecule production
The gene fragment encoding the Anpl-UAA*SD mature peptide residues 22 to 292 (extracellular domain, GenBank accession MN398410) and the mature protein of duck β2-microglobulin (Anpl-β2m, GenBank accession NP_001297750.1, residues 19 to 119) from Anas platyrhynchos were synthesized by Shanghai Invitrogen Life Technologies. And then ligated into the pET-21a(+) expression vector (Novagen, Merck KGaA, Darmstadt, Germany) and transformed into Escherichia coli strain BL21 (DE3) (Table 1). The
Results and discussion
The complex of LRK-9 and Anpl-UAA*SD and Anpl-β2m was co-refolded in vitro for 48 h and purified by Superdex 200 16/60 HiLoad gel exclusion chromatography. There is a significant elution peak around 85 ml (Fig. 1a; peak 2), and the protein size at the peak is about 43 kDa according to the elution standard of the column. The size of the two bands identified by SDS-PAGE was 31.4 kDa and 11.6 kDa, which were consistent with the molecular weight of Anpl-UAA*SD and Anpl-β2m, and identified as pAnpl
Conclusions
In this article, we first crystallized duck MHC class I complex with a TMUV viral peptide and obtain high quality diffraction data. We also lay a structural foundation for the study of duck anti-TMUV research and interaction with MHC class I molecules and peptide presentation. At the same time, it provides guidance on the design of the duck anti-TMUV peptide vaccine.
The following are the supplementary data related to this article. . Structural based sequence alignment of Anpl-UAA*SD with Anpl
Author contributions
N Zhang conceived and designed the experiments. Z Liu, X Xie and Z Li completed the experimental work. Z Liu completed data collection and structure determination. L Zhang provided the resources. The original draft of the manuscript was written by Z Liu.
Declaration of Competing Interest
The authors declare that they have no conflicts of interest with the contents of this article.
Acknowledgments
This work was supported by the National Key R&D Program of China (Grant No. 2016YFD0500106).
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