Position 156 influences the peptide repertoire and tapasin dependency of human leukocyte antigen B*44 allotypes

Abstract

Background: Polymorphic differences between donor and recipient human leukocyte antigen class I molecules can result in graft-versus-host disease due to distinct peptide presentation. As part of the peptide-loading complex, tapasin plays an important role in selecting peptides from the pool of potential ligands. Class I polymorphisms can significantly alter the tapasin-mediated interaction with the peptide-loading complex and although most class I allotypes are highly dependent upon tapasin, some are able to load peptides independently of tapasin. Several human leukocyte antigen B*44 allotypes differ exclusively at position 156 (B*44:02(156Asp), 44:03(156Leu), 44:28(156Arg), 44:35(156Glu)). From these alleles, only the high tapasin-dependency of human leukocyte antigen B*44:02 has been reported.

Design and methods: We investigated the influence of position 156 polymorphisms on both the requirement of tapasin for efficient surface expression of each allotype and their peptide features. Genes encoding human leukocyte antigen B*44 variants bearing all possible substitutions at position 156 were lentivirally transduced into human leukocyte antigen class I-negative LCL 721.221 cells and the tapasin-deficient cell line LCL 721.220.

Results: Exclusively human leukocyte antigen B*44:28(156Arg) was expressed on the surface of tapasin-deficient cells, suggesting that the remaining B*44/156 variants are highly tapasin-dependent. Our computational analysis suggests that the tapasin-independence of human leukocyte antigen B*44:28(156Arg) is a result of stabilization of the peptide binding region and generation of a more peptide receptive state. Sequencing of peptides eluted from human leukocyte antigen B*44 molecules by liquid chromatography-electrospray ionization-mass spectrometry (LTQ-Orbitrap) demonstrated that both B*44:02 and B*44:28 share the same overall peptide motif and a certain percentage of their individual peptide repertoires in the presence and/or absence of tapasin.

Conclusions: Here we report for the first time the influence of position 156 on the human leukocyte antigen/tapasin association. Additionally, the results of peptide sequencing suggest that tapasin chaperoning is needed to acquire peptides of unusual length.

Figure 1.

HLA-B*44 expression on the surface of LCL 721.221 and LCL 721.220 cells. Expression of B*44/156 variants in 721.221 (Tapasin+) and 721.220 (Tapasin−) cells. Flow cytometric analysis for cells stained with anti Bw4-FITC and w6/32-PE labeled monoclonal antibodies. An example for all 20 AA that were exchanged at position 156, here we show the FACS plots for two AA of each group (representative of three separate experiments). (A) and (B) show the FACS plots for the non-polar hydrophobic AA - 156Leu (represented by B*44:03), 156Val (artificial B*44/156 molecule) and polar neutral AA - 156Thr, 156Ser (artificial B*44/156 molecules) in 721.221 and 721.220 cells, respectively. (C) and (D) show the FACS analysis for the acidic AA - 156Asp (represented by B*44:02), 156Glu (represented by B*44:35) and for basic AA 156Arg (represented by B*44:28), 156Lys in 721.221 and 721.220 cells, respectively. All the natural and artificial B*44/156 molecules with the exception of 156Gly were expressed on the surface of LCL 721.221 cells. Only B*44:28^156Arg was exclusively expressed on the surface of LCL 721.220 cells lacking tapasin.

Figure 2.

Tapasin (TPN) silencing results in the lack of surface expression of B*44:02 but not B*44:28 on LCL 221.721 cells. Surface expression of HLA-B*44:02 and B*44:28 on LCL 221.721 cells transduced with shRNA targeting tapasin. Flow cytometric analysis for GFP (reporter gene from the pLVTHm/si plasmid) and w6/32-PE staining shows both B*44:02^156Asp and B*44:28^156Arg to be GFP-positive, but only B*44:28^156Arg to be positive for w6/32-PE staining, thereby indicating that B*44:28^156Arg can be expressed on the cell surface even in the absence of tapasin.

Figure 3.

B*44/156 substitution models. We modeled all amino acids at position 156 with their best rotamer; the results indicate that only arginine and possibly lysine could contact the peptide backbone influencing the stability of the complex. We took the B*44:02 structure (1M6O) and modeled all 20 amino acids at position 156 fitting the best side chain rotamer. Arg156 shows increased hydrogen bonding both to residue Asp114 and to the peptide backbone. This is likely to increase stability of the HLA-peptide complex.

Figure 4.

Western blot analysis of LCL 721.221 cells and sHLA-B*44 expressing cells. (A) LCL 721.221 cells contain all of the minimum essential components of the PLC. The western blot analysis of lymphoblastoid B-cells and 721.221 cells using antibodies against the components of the PLC – ERAP1, TAP, tapasin (TPN), ERp57 and CRT confirmed that 721.221 cells possess all of the minimum components of the PLC. (B) sB*44 molecules are associated with the PLC. Lymphoblastoid B-cells (positive control), LCL 721.221 cells, LCL 721.221/sHLA-B*44:02 and LCL 721.221/sB*44:28 cells were immunopre-cipitated with anti-TAP1 antibody covalently conjugated to protein-A-sepharose beads. Eluted proteins were resolved by SDS gel, transferred to a PVDF membrane and immunoblotted with HRP-conjugated antibodies against V5 tagged recombinant sHLA*B44 protein and the components of the PLC – TPN, ERp57 and CRT.