Mass spectrometric profiling of HLA-B44 peptidomes provides evidence for tapasin-mediated tryptophan editing

Abstract

Activation of CD8 ⁺ T cells against pathogens and cancers involves the recognition of antigenic peptides bound to human leukocyte antigen (HLA) class-I proteins. Peptide binding to HLA class I proteins is coordinated by a multi-protein complex called the peptide loading complex (PLC). Tapasin, a key PLC component, facilitates the binding and optimization of HLA class I peptides. However, different HLA class I allotypes have variable requirements for tapasin for their assembly and surface expression. HLA-B*44:02 and HLA-B*44:05, which differ only at residue 116 of their heavy chain sequences, fall at opposite ends of the tapasin-dependency spectrum. HLA-B*44:02 (D116) is highly tapasin-dependent, whereas HLA-B*44:05 (Y116) is highly tapasinindependent. Mass spectrometric comparisons of HLA-B*4405 and HLA-B*44:02 peptidomes were undertaken to better understand the influences of tapasin upon HLA-B44 peptidome compositions. Analyses of the HLA-B*44:05 peptidomes in the presence and absence of tapasin reveal that peptides with the C-terminal tryptophan residues and those with higher predicted binding affinities are selected in the presence of tapasin. Additionally, when tapasin is present, C-terminal tryptophans are also more highly represented among peptides unique to B*44:02 and those shared between B*44:02 and B*44:05, compared with peptides unique to B*44:05. Overall, our findings demonstrate that tapasin influences the C-terminal composition of HLA class I-bound peptides and favors the binding of higher affinity peptides. For the HLA-B44 family, the presence of tapasin or high tapasin-dependence of an allotype results in better binding of peptides with C-terminal tryptophans, consistent with a role for tapasin in stabilizing an open conformation to accommodate bulky C-terminal residues.

Figure 1:. Expression and purification of HLA-B*44:05 from wild type (WT) and tapasin knockout (TPN-KO) 721.221 cells:

(A) Representative blots showing tapasin (TPN) expression in the lysates of 721.221-B*44:05 WT (lane 1) and tapasin knockout (lane 2) cells. The sgRNA sequence is given in the materials and methods. (B) The graph shows the mean of the number of HLA-B*44:05 molecules expressed per cell on the surface of WT (n=7) and TPN-KO (n=6) 721.221-B*44:05 cells measured by quantitative flow cytometry following staining with W6/32-FITC antibody. The number of HLA-B molecules per cell is quantified from standard curves of the geometric mean fluorescence intensity (MFI) values of W6/32-FITC antibody binding to Quantum Simply Cellular beads (Bangs Laboratory, Inc.) that contain a known number of Fc receptors and are stained concurrently with the cells. Each data point represents an independent experiment. Statistical significance was calculated using an unpaired t-test assuming equal variance. (C) Representative plot shows changes in the expression (normalized relative to the 0h condition) of B*44:05 on the surface of WT and TPN-KO 721.221-B*44:05 cells, assessed using the W6/32 antibody, at different time points following brefeldin A treatment. (D) The graph shows calculated half-lives of B*44:05 on the surface of B*44:05-WT and B*44:05-TPN-KO 721.221 cells based on the surface stability measurements shown in C (n=4). Statistical significance is calculated using a two-tailed unpaired t-test. Graphs are plotted in GraphPad Prism. (E and F) Representative blots (of six independent experiments) show the purification of HLA-B*44:05 from B*44:05-WT (E) and B*44:05-TPN-KO 721.221 cells (F). “Beads” indicates protein A beads used for pre-clearing of lysates and “W6/32 beads” indicates protein A beads crosslinked to W6/32 antibody. HLA-B heavy chain and β2m are probed using HC10 and anti-β2m antibodies, respectively. (G) Representative Coomassie brilliant blue stained gels (of six independent experiments) showing elution of B*44:05 from W6/32 beads following affinity purification from B*44:05-WT or B*44:05-TPN-KO 721.221 cells. The red outlined box marks the HLA-B heavy chain bands in the eluates. E, Elution; FT, Flow through; IB, Immunoblot

Figure 2:. Charateristics of HLA-B*44:05 peptides purified from wild type or tapasin-KO cells.

(A) Venn diagram showing total number of peptides identified from six MS/MS runs grouped into the unique to B*44:05-TPN-KO, unique to B*44:05-WT, or shared categories. Only peptides observed in 2 or more runs are included. (B) Length distributions of peptides identified for the B*44:05-WT and B*44:05-TPN-KO categories, indicated as a fraction of total number of peptides. Significant differences in distribution were determined using the Chi-squared test of independence. (C) Shannon Entropy (SE) plots for indicated positions within the 9-mer, 10-mer, and 11-mer peptides from (A). The P_C, P_C-1 and P_C-2 represent the C-terminal, and the −1 and −2 positions relative to the C-terminus, respectively. Significant differences in SE at each position were determined using a bootstrap hypothesis test with Bonferroni corrected p-values. (D) The fractional distribution of all amino acids at the P_C position of 9-mer, 10-mer, and 11-mer peptides, grouped according to the side chain properties of individual amino acids. Significant differences in the amino acid distributions were determined using the Chi-squared test of independence, with standardized residual post-hoc tests using Benjamini-Hochberg corrections. *: p < 0.05; **: p < .01; ***: p < .0001

Figure 3:. Peptides unique to B*44:05 from 721.221-WT cells show increased preference for tryptophan at the C-terminus:

(A to C) Seq2Logo motifs for 9-mer, 10-mer, and 11-mer peptides from the peptide set highlighted in Figure 2A, grouped into the unique to B*44:05-WT (A), unique to B*44:05-TPN-KO (B) and peptides shared across the two conditions (C). (D and E) The Seq2Logo motifs for the total peptides (including the unique and shared peptides) for the B*44:05-WT (D) and B*44:05-TPN-KO (E) conditions are also shown.

Figure 4.. Tapasin knockout results in reduced predicted binding affinities of peptides bound to B*44:05:

The predicted binding affinities (nM) of B*44:05 peptides were calculated for the indicated peptide groups and plotted separately for the 9-mer, 10-mer, and 11-mer peptides. Peptides with predicted affinities >10,000 nM were excluded.

Figure 5:. Expression and purification of HLA-B*44:02 from 721.221 cells:

(A) The graph shows the mean number of HLA-B molecules expressed per cell on the surface of B*44:02 (n=9 independent experiments) and B*44:05 (n=6 independent experiments) 721.221 cells measured by quantitative flow cytometry following staining with W6/32-FITC antibody. The number of HLA-B molecules per cell is quantified from standard curves of the geometric mean fluorescence intensity (MFI) values of W6/32-FITC antibody binding to Quantum Simply Cellular beads (Bangs Laboratory, Inc.) that contain a known number of Fc receptors and are stained concurrently with the cells. Each data point represents an independent experiment. Statistical significance was calculated using an unpaired t-test assuming equal variance. (B) Representative plot shows changes in the expression (normalized relative to the 0h condition) of HLA-B on the surface of 721.221-B*44:02 and 721.221-B*44:05 cells assessed using the W6/32 antibody, at different time points following brefeldin A treatment of cells. (C) The graph shows calculated half-lives of B*44:02 and B*44:05 on the surface of 721.221 cells based on the surface stability measurements shown in B (n=4). Statistical significance is calculated using a two-tailed unpaired t-test. Graphs are plotted in GraphPad Prism. (D) Representative blots (of six independent experiments) show the purification of HLA-B*44:02 from 721.221 cells. “Beads” indicates protein A beads used for pre-clearing of lysates and “W6/32 beads” indicates protein A beads crosslinked to W6/32 antibody. HLA-B heavy chain and β2m are probed using HC10 and anti-β2m antibodies, respectively. (E) Representative Coomassie brilliant blue stained gel (of six independent experiments) showing elution of HLA-B*44:02 from W6/32 beads following affinity purification from 721.221-B*44:02 cells. The red outlined box marks the HLA-B heavy chain bands in the eluates. E, Elution; FT, Flow through; IB, Immunoblot

Figure 6:. Characteristics of peptides purified from B*44:02 compared with those from B*44:05.

(A) Venn diagram showing the total number of peptides identified from six MS/MS runs grouped into those unique to B*44:02, unique to B*44:05, or shared between the two categories. Only peptides observed in 2 or more runs are included for further analyses. (B) Length distributions of identified peptides, indicated as a fraction of total number of peptides. Significant differences in distribution were determined using the Chi-squared test of independence. (C) Shannon Entropy (SE) plots for indicated positions within 9-mer, 10-mer, and 11-mer peptides from (A). The P_C, P_C-1 and P_C-2 represent the C-terminal, and the −1 and −2 positions relative to the C-terminus, respectively. Significant differences in SE at each position were determined using a bootstrap hypothesis test with Bonferroni corrected p-values. (D, E) The fractional distribution of all amino acids at the P_C (D) and P_C-2 (E) positions of 9-mer, 10-mer, and 11-mer peptides. Peptides are grouped by side chain properties. Significant differences in distributions were determined using the Chi-squared test of independence, with standardized residual post-hoc tests using Benjamini-Hochberg corrections. *: p < 0.05; **: p < .01; ***: p < .0001

Figure 7:. Tryptophan is under-represented among peptides unique to B*44:05 relative to those shared with B*44:02:

(A to C) Seq2Logo motifs for 9-mer, 10-mer, and 11-mer peptides among the peptides unique to B*44:05 (A), unique to B*44:02 (B) and shared between B*44:02 and B*44:05 (C). (D and E) The Seq2Logo motifs for the total set of peptides (including the unique and shared peptides) for B*44:05 (D) and B*44:02 (E) are also shown.

Figure 8.. Reduced predicted binding affinities of peptides unique to B*44:05 compared to those shared with B*44:02:

(A) The predicted affinities (nM) of B*44:05 peptides were calculated for the indicated groups, and plotted separately for the 9-mer, 10-mer, and 11-mer peptides. Peptides with predicted affinities >10,000 nM are excluded. (B) The predicted affinities (nM) of B*44:02 peptides were calculated for indicated groups, and plotted separately for the 9-mer, 10-mer, and 11-mer peptides. Peptides with predicted affinities >10,000 nM are excluded.