Polymorphism in position 156 influences the peptide repertoire
of HLA-B*4402 and B-4403 molecules
Hernando Escobar, Eduardo Reyes-Vargas, David Crockett, Peter Jensen, and Julio C. Delgado
ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah
Strong CTL-mediated allogeneic reactions have been reported between HLA-B*4402 and
–B*4403 alleles. These alleles differ only by a single residue located in position 156 of the
α chain, with HLA- B*4402 expressing Asp and HLA-B*4403 expressing Leu. Position 156
influences the conformation of pocket D and E of the peptide binding groove of HLA class I
molecules but has little influence on the specificity of peptide anchor residues P2 and P9.
This study was performed to investigate the influence of position 156 in the repertoire
peptides presented by these two HLA-B44 subtypes. HLA-B44-peptide complexes were
separated by immuno-affinity columns from whole cell lysates of K562 cells expressing
HLA-B*4402 or HLA-B*4403. Peptide pools were obtained by acid elution followed by
HPLC fractionation and sequencing by CID and TOF MS/MS. We found that over 2/3 of
peptides eluted from HLA- B*4402 differ from those eluted from HLA-B*4403. Close
analysis of the peptide binding motif of non-over-lapping peptide sequences of these 2
molecules revealed that in the non-anchor position P7, HLA-B*4402 prefers polar,
uncharged and basic residues such as Gly, Gln or His, whereas HLA*B4403 shows
preference for non-polar residues Leu, Ile or Val. No differences were observed in the
specificity of residues located in anchor positions P2 and P9. Position 156 has direct effect
on the specificity of the residue located in position P7 of the peptide binding motif of HLAB*4402 and HLA-B*4403 alleles. Given that P7 position is important for T-cell receptor
interaction and subsequent T-cell activation, we suggest that the polymorphism in
position156 between HLA-B*4402 and HLA-B*4403 alleles might play an important role in
the alloreactivity between these 2 alleles.
Eluted peptides from K562 cells expressing HLA-B*4402 or B*4403 were processed as described in Escobar, H.
a.
et al., J. Immunol. 2008; 181,4874-4882. In brief, MHC molecules were immunopurified from whole cell lysates
and peptides were acid-eluted and fractionated by HPLC. Peptides fractions were analyzed by LC/MS/MS using
an Agilent 6510 Q-TOF (Agilent Technologies, Santa Clara, CA) equipped with nanospray ChipCube source and
Agilent 1200 HPLC system. Peptide separation was performed on a C-18SB-ZX Chip (40nL trap, 150mm x
43mm, 5mm particles). Data acquisition was performed with MassHunter QTOF Acquisition software B.02 with
an acquisition rate of 3 scans/s followed by MS/MS scans of 3 most intense ions. Exclusion was set for 12s after
two consecutive MS/MS scans of the precursor ion. TOF mass analyzer was calibrated and tuned prior to each
set of samples with mass accuracy < 1 ppm. The acquired MS/MS spectra were searched using Spectrum Mill
MS Proteomics Workbench Rev A.03.03.075 (Agilent) against SwissProt human or mouse database. Spectra
were validated automatically and filtered with the following settings: minimum score 7.0, minimum 60 percent
scored peak intensity SPI, minimum unmatched ions 15 of 30 total ions, maximum precursor error 20ppm.
Sequences were then manually validated after the initial database search as reported in Delgado, J.C. et al
Immunogenetics, 61: 241–246.
b.
Using large-scale sequencing of eluted endogenous peptides we identified 52 peptides from HLA- B*4402 and
MHC class I molecules are highly polymorphic structures that bind peptide antigens for
62 HLA-B*4403. As expected, both peptide repertoires originate from intracellular compartments and have
presentation to T cells and generate an immune response against pathogens. Several
similar function (Fig 2). Approximately 50% of B*4402-derived peptides are shared with B*4403, whereas only
MHC class I molecules can be classified in 10-12 major HLA class I supertypes which are
37% of B*4403-derived peptides are shared with B*4402. Analysis of peptide sequences confirmed previous
characterized by specific anchor binding motifs. The HLA-B44 supertype is present in
reports showing a dominant anchor Glu residue in P2 position and the presence of aromatic or hydrophobic
most human populations where HLA-B*4402 is common in Caucasian and HLA-B*4403 is
residues as Phe, Tyr and Leu in the last position (Fig 3). Analysis of non-common peptide sequences revealed
common in African and Asian populations. Between this two B44 alleles there is only a
that in position P7, HLA-B*4402 prefers polar, uncharged and basic residues such as Gly, Gln or His, whereas
single residue difference located in position 156 (Asp in HLA- B*4402 and Leu in HLA-
HLA*B4403 shows preference for non-polar residues Leu, Ile or Val.
B*4403). Although position 156 is buried in the antigen-binding cleft of MHC class I
molecule, its effect on T cell recognition is crucial for allograft rejection in clinical
transplantation (Macdonald WA, et al. J. Exp. Med, 2003; Ely LK, et al. J. Immunol, 2005).
As seen in Fig 1, position 156 is located in the floor of the binding cleft and affects pockets
Figure 3. Logos displaying peptide-binding motifs of non-common peptides found
in: (a) HLA-B*4402 and (b) HLA-B*4403. Residues acidic (red), basic (blue),
hydrophobic (black), neutral (green) and aromatic (purple) are illustrated.
Dominant P2 position occupied by Glu has been removed to show in better detail
the frequencies of the remaining positions.
D and E of MHC class I molecules and has a strong effect on T cell recognition (Herman
JV et al. Tissue Antigens, 1999)
a.
1. Our results demonstrate that position 156 in has direct effect on the specificity of
the residue located in position P7 of the peptide binding HLA-B*4402 and HLAB*4403 molecules.
b.
2. Previous studies have shown that P7 position has a potential T-cell receptor
interaction and subsequent T-cell activation (Archbold JK, et al. JEM, 2009 ).
Thus, the polymorphism in position 156 might play an important role in the
observed alloreactivity between HLA-B*4402 and HLA-B*4403 alleles.
Figure 1. Top side view of peptide binding cleft of (a) HLA-B*4402 and (b) HLA–B*4403
showing Asp and Leu residues in position 156, respectively, and its influence in pockets D
and E.
Figure 2. Gene Ontology classification of identified peptides from (A) HLA-B*4402 and (B) HLA-B*4403.
3. In-vitro binding assays are currently being performed using sets of peptides with
variation of residues in position P7 to demonstrate the effect of this position in
the binding to HLA-B*4402 and HLA-B*4403 molecules