Fig 1.
K562 and C1R cells expressing DLA-88*50101.
DLA and HLA class I expression of transfected and non-transfected K562 (A) and C1R cells (B) was determined by flow cytometry using BBM.1 antibody (anti-β2-microglobulin). Staining with W6/32 antibody (anti-HLA-A, -B, -C) and BB7.2 (anti HLA-A2,) characterizes the HLA expression of C1R (C) and JY cells (D). (A) non-transfected K562 cells unstained (grey line); non-transfected K562 cells stained with BBM.1 antibody (black dotted line), and transfected K562 cells stained with BBM.1 antibody (black line). (B) non-transfected C1R cells unstained (grey line), non-transfected C1R cells stained with BBM.1 antibody (black dotted line), and transfected C1R cells (black line) stained with BBM.1 antibody. (C) non-transfected C1R cells unstained (grey line), C1R cells stained with W6/32 antibody (black stippled line), or stained with BB7.2 antibody (black line). (D) JY cells unstained (grey line), stained with W6/32 antibody (black dotted line), or stained with BB7.2 antibody (black line). (E) MHC class I expression on JY cells, DLA-88*50101 transfected and non-transfected C1R cells. QIFIKIT (Dako) and BBM.1 antibody were used in flow cytometry for absolute quantification of MHC class I molecules per cell.
Table 1.
Summary of MHC class I immunoaffinity purification assay of C1R and K562 cells transfected with DLA-88*50101.
Fig 2.
The MHC binding motif of peptides deriving from C1R-DLA-88*50101 and K562-DLA-88*50101 cells.
(A) The average length distribution of MHC peptide ligands deriving from C1R and K562 cells transfected with DLA-88*50101 shows that the majority of peptides are 9 residues in length (n = 2436; 65.5%). (B) Summary binding motif of DLA-88*50101. Nonamers deriving from C1R-DLA-88*50101 cells, (Exp. 1–3) as well as peptides eluted from K562-DLA-88*50101 cells (Exp. 4), were characterized and revealed dominant anchor residues at position 2 and 9. (C) Binding motif of HLA-A*02:01. Derivation of data is described in the “Materials and Methods” section.
Table 2.
Quantitative binding motif for DLA-88*50101.
Fig 3.
Alignment of MHC class I α1 and α2 domain amino acid sequences of HLA-A*02:01 and DLA-88*50101.
(A) Alignment of MHC class I α1 and α2 domain amino acid sequences of human (HLA-A*02:01; AAA76608.2a) and dog (DLA-88*50101; AF100577, AF101496, [19]b). Identity of amino acid sequences to human HLA-A*02:01 is shown by dashes, disposability of data is illustrated by a dot. Position numbers for DLA-88*50101 are indicated in black above the sequences. Deviating position numbers for HLA-A*02:01 resulting from the amino acid deletion are given in grey bold type on top of the corresponding DLA-88*50101 position. Residues allocated to hypervariable regions I-III are indicated above the alignment ([19]b). (B) Comparison of selected residues of HLA-A*02:01 (AAA76608.2a) and DLA-88*50101 (AF100577, AF101496, [19]b) with reference to pockets A-F. The indicated positions refer to HLA-A*02:01 ([39–41]c). For DLA-88*50101, corresponding positions are shifted by one amino acid starting with the C-terminal of position 154, due to an amino acid insertion [44].
Fig 4.
Alignment of MHC class I α1 and α2 domain amino acid sequences of selected species.
(A) Alignment of MHC class I α1 and α2 domain amino acid sequences of human (Homo sapiens; AAA76608.2), tree shrew (Tapaia belangeri; AFM54604.1), giant panda (Ailuropoda melanoleuca; ABY27206.1), donkey (Equus asinus; ADE61511.1), horse (Equus caballus; BAI45218.1), wild pig (Sus scrofa; ACA33856.1), black rhinoceros (Diceros bicornis minor; AAD02690.1), porpoise (Neophocaena asiaeorientalis; ALB25881.1), cow (Bos taurus; AFS51741.1), dog (Canis lupus familiaris; NP_001014767.1), sheep (Ovis aries; AGN98174.1) and cat (Felis catus; ACK99133.1). Identity of residues to human HLA-A*02:01 (Homo sapiens; AAA76608.2) is shown by dashes, disposability of data by dots. Residues allocated to hypervariable regions I-III are indicated above the alignment ([19]a). (B) Same as (A) but focusing on the MHC class I residues with reference to pocket A-F. The indicated positions refer to HLA-A*02:01 [39–41]a).
Table 3.
Bioinformatic MHC binding scores for peptides used for experimental binding analysis into HLA and DLA.
Fig 5.
Peptide binding on HLA-A*02:01 versus DLA-88*50101.
The cells were incubated with peptides pol HIV-1 reverse transcriptase 476–484, ILKEPVHGV; S-adenosylmethionine synthase 220–227 DALKEKVI; Spatacsin 1296–1304 SVAEKLSKL and mediator of RNA polymerase II transcription subunit28 110–118 VIKEDVSEL for 1 h 30 min at 37°C in a 5% CO2 atmosphere. DMSO was used as negative control to determine the intrinsic fluorescence. Samples were analyzed by flow cytometry and data derived from two biological experiments and three technical replicates was analyzed as described in the “Materials and Methods”. The standard deviations of the analyzed data are shown in bars. For HLA-A*02:01 loading, the fluorescence intensity of peptide binding regarding ILKEPVHGV on HLA-A*02:01 was set to 100%, whereas for DLA-88*50101 loading, SVAEKLSKL binding on DLA-88*50101 was set to 100%.