Comparative Analysis of the Endogenous Peptidomes Displayed by

Publication Date (Web): January 26, 2016 ... In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV r...
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Comparative Analysis of the Endogenous Peptidomes Displayed by HLA-B*27 and Mamu-B*08: Two MHC Class I Alleles Associated with Elite Control of HIV/SIV Infection Miguel Marcilla,*,† Iñaki Alvarez,‡ Antonio Ramos-Fernández,§ Manuel Lombardía,† Alberto Paradela,† and Juan Pablo Albar†,∇ †

Proteomics Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049 Madrid, Spain Immunology Unit, Department of Cell Biology, Physiology and Immunology and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain § Proteobotics SL, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Indian rhesus macaques are arguably the most reliable animal models in AIDS research. In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV replication. A similar scenario is observed in humans where the expression of HLA-B*27 or HLA-B*57 has been linked to slow or no progression to AIDS after HIV infection. Despite having large differences in their primary structure, it has been reported that HLA-B*27 and Mamu-B*08 display peptides with sequence similarity. To fine-map the Mamu-B*08 binding motif and assess its similarities with that of HLA-B*27, we affinity purified the peptidomes bound to these MHC class I molecules and analyzed them by LC-MS, identifying several thousands of endogenous ligands. Sequence analysis of both sets of peptides revealed a degree of similarity in their binding motifs, especially at peptide position 2 (P2), where arginine was present in the vast majority of ligands of both allotypes. In addition, several differences emerged from this analysis: (i) ligands displayed by Mamu-B*08 tended to be shorter and to have lower molecular weight, (ii) Mamu-B*08 showed a higher preference for glutamine at P2 as a suboptimal binding motif, and (iii) the second major anchor position, found at PΩ, was much more restrictive in Mamu-B*08. In this regard, HLA-B*27 bound efficiently peptides with aliphatic, aromatic (including tyrosine), and basic C-terminal residues while Mamu-B*08 preferred peptides with leucine and phenylalanine in this position. Finally, in silico estimations of binding efficiency and competitive binding assays to Mamu-B*08 of several selected peptides revealed a good correlation between the characterized anchor motif and binding affinity. These results deepen our understanding of the molecular basis of the presentation of peptides by Mamu-B*08 and can contribute to the detection of novel SIV epitopes restricted by this allotype. KEYWORDS: peptidomics, peptides, mass spectrometry, MHC, HLA-B*27, Mamu-B*08, binding motif, HIV, SIV

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control viremia even without antiretroviral treatment.3 A comparable protective role has been described for the rhesus macaque (Macaca mulatta) allotype Mamu-B*008:01 (MamuB*08) in the context of Simian Immunodeficiency Virus (SIV) infection.4 This allele was expressed by about 40% of elite controllers and only 3% of progressors in a cohort of infected macaques, and its presence correlated with reduced viral loads in plasma during the chronic phase of the disease.4 HLA-B*27:05 and Mamu-B*08 differ in 37 residues in their amino acid sequence, 25 of them located at the α1 or α2 domains, which form the peptide binding cleft (Supporting Information S1). Employing a positional scanning combinato-

INTRODUCTION

MHC class I antigens are cell membrane glycoproteins that facilitate immunological surveillance by binding and presenting peptides to cytotoxic T lymphocytes (CTLs). The peptidome displayed by these molecules derives from the degradation of endogenous polypeptides, including tumor-specific or viral proteins, in the nucleus or cytosol. The crucial role of class I antigens in immunity against intracellular parasites is highlighted by the association of some MHC class I allotypes with control of particular viral infections. Such is the case of Human Leukocyte Antigen (HLA)-B*27 that is well recognized as a protective factor against HIV.1−3 HLA-B*27 is significantly overrepresented among individuals that progress slowly to AIDS after HIV infection. Remarkably, a fraction of these, the so-called elite controllers or long-term nonprogressors, can © XXXX American Chemical Society

Received: December 21, 2015

A

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cells were lysed in an isotonic buffer (20 mM TRIS, 150 mM NaCl, pH 7.5) containing 1% Igepal CA-630 (Sigma-Aldrich) and a mixture of protease inhibitors (Complete, Roche). The lysate was then centrifuged for 10 min at 2000g, 30 min at 10 000g and 1 h at 100 000g. Afterward, the supernatant was subjected to affinity chromatography using the W6/32 mAb coupled to CNBr-activated sepharose beads (GE Healthcare). MHC class I-bound peptides were eluted with 0.1% TFA and filtered through Centricon 3 devices (Amicon). The peptide fraction was concentrated in a speedvac, desalted with an OMIX C18 tip (Varian), dried to completeness and redissolved in 0.1% formic acid prior to LC-MS analysis.

rial peptide library, Loffredo et al. reported a similar peptide binding motif for both molecules and showed that HLA-B*27 and Mamu-B*08 share an almost absolute preference for peptides with arginine at peptide position 2 (P2) and a partially similar structural motif at the C-terminus (PΩ).5 Emphasizing the importance of the elucidation of binding motifs for the understanding of MHC class I function, this information allowed the characterization of 6 novel SIV-specific epitopes restricted by Mamu-B*08. In view of this similar binding motif and the well reported role of CD8+ T-cells in the control of immunodeficiency virus replication both in humans6−8 and macaques,9−12 the existence of HLA-B*27 and Mamu-B*08 positive elite controllers suggests that these allotypes could present conserved immunodominant epitopes to CTLs promoting disease resistance. Recent advances in mass spectrometry and bioinformatics have laid the foundations for the development of peptidomics -the large scale characterization of physiologically relevant peptides- which, in turn, has brought a deeper understanding of the structure and function of MHC antigens and their role in the immune response.13 In contrast to other approaches such as the above-mentioned combinatorial peptide libraries, highthroughput peptide sequencing provides information about the ligands that are actually displayed on the cell surface for recognition by T-cells. In addition, it can reveal subtle variations in residue usage between different MHC-I molecules or identify suboptimal binding motifs that may escape detection by other methods. In this work, we have used high-resolution tandem mass spectrometry together with a multiengine search strategy to identify several thousands of Mamu-B*08 and HLA-B*27 endogenous ligands. Sequence analysis of these sets of peptides allowed the characterization of a refined binding motif for both allotypes and revealed differences between the two molecules affecting mainly P2 and PΩ. Finally, the NetMHC algorithm14,15 and a competitive binding assay with both naturally presented and mutant ligands were employed to confirm the peptide binding preferences of Mamu-B*08.

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LC-MS Analysis

MHC class I-derived peptidomes were analyzed in a nano-LC Ultra HPLC (Eksigent) coupled online with a 5600 triple TOF mass spectrometer (AB Sciex) through a nanospray III source (AB Sciex). The HPLC was equipped with a C18 chromXP trapping column (350 μm x 0.5 mm, 3 μm particle diameter and 120 Å pore size, Eksigent) and a C18 chromXP column (75 μm × 150 mm, 3 μm particle diameter and 120 Å pore size, Eksigent). Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. The flow-rate of the nanopump was 300 nL/min. The nanospray interface was equipped with a fused silica PicoTip emitter (10 μm x 12 cm, New Objective). The ion source was operated in positive ionization mode at 150 °C with a potential difference of 2800 V. The peptide pool obtained from 3 × 109 C1R cells was analyzed in a single LC-MS run under gradient elution conditions consisting of a linear increase from 2 to 30% B in 109 min. Each acquisition cycle comprised a survey scan (350− 1250 m/z) of 250 ms and up to 50 MS/MS scans (100−1500 m/z) of 50 ms. Two biological replicates of the peptide pools isolated from C1R-B*27 and C1R-Mamu-B*08 cells were analyzed injecting 3 × 109 and 5 × 109 cell equivalents per LC run, respectively. In a set of experiments the samples were analyzed exactly as described for the peptidome of C1R cells. The second replicates were fractionated with a linear gradient from 2 to 30% in 182 min. The acquisition cycle consisted of a survey scan (350−1250 m/z) of 250 ms and a maximum of 25 MS/MS scans (100−1500 m/z) of 100 ms. Two technical replicates for each sample were analyzed with this latter setup.

EXPERIMENTAL PROCEDURES

Cell Lines and Monoclonal Antibodies

HMy2.C1R (C1R) is a human lymphoid cell line with no expression of its endogenous HLA class I molecules, except HLA-C*04:01 and HLA-B*35:03 that are expressed at low levels.16 The stable transfectant expressing HLA-B*27:05 (C1R-B*27) has been described elsewhere.17 The transfectant expressing Mamu-B*08 (C1R-Mamu-B*08) was generated by electroporation of 107 C1R cells at 250 mV and 950 μF with 10 μg of a full length cDNA of Mamu-B*008:01 cloned in pcDNA3.1- (a kind gift of Dr. David Watkins, University of Miami, FL). Stable transfectants were selected by growing the cells in the presence of 1 mg/mL Geneticin (Gibco) and surface expression of Mamu-B*08 was confirmed by flow cytometry. Cells were cultured in DMEM medium supplemented with 7.5% FCS (both from Sigma). The mAb W6/32 (IgG2a specific for a monomorphic HLA class I determinant) has been described elsewhere.18

MS/MS Ion Search and Peptide Identification

MS/MS data were converted to mgf files with PeakView (ABSciex, version 1.1) and searched against a composite target/ decoy database built from the 67 911 sequences in the Homo sapiens reference proteome at Uniprot Knowledgebase (as of February 2015), together with commonly occurring contaminants. Three independent search engines were used: MASCOT (Matrix Science, version 2.4.0), X!Tandem2 (The Global Proteome Machine Organization, version 2013.02.01.1), and Myrimatch (version 2.2.140). Search parameters were set as follows: no enzyme, MS tolerance: 15 ppm, MS/MS tolerance: 0.02 Da and oxidation of methionine, protein N-terminal acetylation and pyroglutamic acid formation from either Nterminal glutamine or glutamic acid as variable modifications. The individual outputs of the search engines were combined by converting each engine-specific scoring scheme to a common probability-based scale as previously described.20 Score distribution models were used to compute peptide-spectrum match p-values,20 and identifications at a FDR ≤ 0.02 at the peptide level were selected for analysis.

Isolation of the HLA Class I-Bound Peptide Pool

The immunoprecipitation of MHC class I molecules and the isolation of their associated peptidomes was carried out as previously described19 starting with pellets of about 1010 (C1RB*27 and C1R-Mamu-B*08) or 3 × 109 cells (C1R). In brief, B

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Table 1. Characterization of the Peptidomes Bound to MHC Class I Molecules in C1R, C1R-B*27, and C1R-Mamu-B*08 Cells

a

Cell Line

Unique Peptides

FDRa

8−13mers

Background Contaminants

HLA-C*04:01

HLA-B*35:03

HLA-B*27:05

C1R C1R-B*27 C1R-Mamu-B*08

246 2569 1479

2% 2% 2%

236 (96%) 2341 (91%) 1359 (92%)

36 10 14

156 121 368

44 42 97

2168

Mamu-B*008:01

880

The FDR at the peptide level is indicated.

In Silico Prediction of MHC Binding Affinities

biological model, a lysate of untransfected C1R cells was subjected to immunoprecipitation with the mAb W6/32 and acid extraction of the bound peptide pool before LC-MS analysis. A total of 246 sequences were identified at 2% FDR of which 236 (96%) corresponded to peptides of 8 to 13 residues, the typical length of MHC class I ligands (Table 1 and Supporting Information S2). In this subset, 44 and 156 sequences matched the B*35:0323 and the C*04:01 binding motif,24,25 respectively. Additionally, 36 peptides could not be assigned to any of these allotypes and were thus classified as background contaminants. To fine-map and compare the peptide binding motifs of HLA-B*27 and Mamu-B*08, the ligandomes associated with these molecules were isolated and analyzed as described above. This resulted in 2569 and 1479 peptide identifications at 2% FDR, respectively (Table 1 and Supporting Information S3 and S4). More than 90% of these sequences were 8 to 13 residues long. To define a precise list of HLA-B*27 and Mamu-B*08 ligands, peptides showing the C*04:01 or B*35:03 binding motifs were set apart. Additionally, those sequences that had been classified as contaminants of the C1R peptidome were rejected. After this, a total of 2168 (HLA-B*27) and 880 ligands (Mamu-B*08) were compiled and considered for subsequent analysis.

Theoretical binding affinities for HLA-B*27:05 and MamuB*008:01 were calculated independently for ligands of 8 to 13 residues long using the NetMHC 3.4 server14,15 available at http://www.cbs.dtu.dk/services/NetMHC. Statistical Analysis

Differences in the molecular weight distributions of the MamuB*08 and the HLA-B*27-associated ligands were evaluated with a two-tailed Mann−Whitney test. Preferences in residue usage were determined by comparing the observed frequency of each amino acid at each peptide position (fobs) with the frequency of the same amino acid in the database ( fexp) using a binomial test with Bonferroni correction. A detailed description of this procedure can be found elsewhere.21 P-values 5, red letters) or weak ( fobs/fexp < 5, blue letters). The number of peptides (n) in each set is indicated.

Table 2. Sequences and IC50 Values of the Peptides Assayed for Binding to Mamu-B*08 Sequence

Description

IC50 (μM)

SRIPVQALL SQIPVQALL GRIEILSGF LRFPEILQKa GQIEILSGF GRLTSQLLR ARFVNVLGY GEFSRFYSL

Common to HLA-B*27 and Mamu-B*08 P2 mutant Common to HLA-B*27 and Mamu-B*08 Common to HLA-B*27 and Mamu-B*08 P2 mutant HLA-B*27 specific HLA-B*27 specific Negative control

0.14 0.29 0.42 0.47 1.89 2.02 4.05 21.80

a

This sequence was identified at a FDR > 2% in the Mamu-B*08 peptidome and confirmed by fragmentation of the corresponding synthetic peptide (Supporting Information S6).

this limitation, we applied our peptidomic workflow to untransfected C1R cells. As expected, this analysis revealed the presence of a relatively large set of peptides that matched the C*04:0124,25 and the B*35:0323 binding motifs. Moreover, we detected a number of sequences that were classified as contaminants based on the lack of binding motifs for these molecules. This information was used to filter out the peptide sequences of the C1R-B*27 and C1R-MamuB*08 cell lines in order to generate a curated list of ligands of both allotypes. In agreement with a previous report,5 the analysis of the identified sequences revealed large similarities between both peptidomes, with the almost absolute preference for Arg2 being the most obvious. In addition, several differences were also detected: (i) HLA-B*27 bound longer peptides of higher molecular weight, (ii) Mamu-B*08 showed greater tolerance for peptides with Gln at P2, and (iii) residues selected at PΩ in Mamu-B*08 were a subset of those found in HLA-B*27.

Figure 5. Theoretical binding affinity of the peptides associated with HLA-B*27 and Mamu-B*08 estimated with the NetMHC 3.4 server. Median and interquartile range are shown. (A) Binding affinity for Mamu-B*08 of HLA-B*27-specific ligands (HLA-B*27), MamuB*08-specific ligands (Mamu-B*08), or ligands shared by both allotypes (Shared). (B) Binding affinity for HLA-B*27 of the same sets of ligands.

spectrometers, some authors have expressed their concerns about the likely negative effect of the MHC class I background of this cell line on the study of exogenous alleles.25 To bypass G

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Figure 6. Peptide binding assay to Mamu-B*08. Cells were acid stripped and washed before the addition of β2m, the reference peptide, and different concentrations of the indicated test peptides. Afterward, cells were incubated overnight at 4 °C before FACS analysis. Experimental values are represented by dots while the best-fitting sigmoid curve is depicted as a continuous line. Peptide concentration is plotted against percentage of maximum fluorescence. Three independent experiments are shown: (A) Inhibition curves for HLA-B*27-specific (red lines) or shared ligands (blue lines). The negative control is represented in black. (B−C) Inhibition curves for two endogenous ligands of Mamu-B*08 (blue lines) and their corresponding mutant peptides with Gln2 (green lines).

Table 3. Comparison of the B and F Pockets of HLA-B*27:05 and Mamu-B*008:01a B Pocket HLA-B*27:05 Mamu-B*008:01

HLA-B*27:05 Mamu-B*008:01 a

7 Y 74 D -

9 H S 77 D -

34 V -

24 T S 80 T -

81 L -

45 E 84 Y -

63 E F Pocket 95 L -

97 N T

66 I R 114 H -

67 C A 116 D Y

70 K H 123 Y -

99 Y 133 W -

143 T -

146 K -

147 W -

Conserved residues are represented by dashes. Polymorphic positions are shown in bold type.

The peptide C-terminus showed some divergence in residue usage as well. Confirming previous reports,30−33 HLA-B*27associated ligands showed some variability at PΩ, and peptides with aliphatic, aromatic (except Trp), and basic residues at this position were frequently displayed. Mamu-B*08 also bound effectively ligands with Leu and Phe at the C-terminus. These similarities are explained by the conservation of several residues that contribute to the hydrophobicity of the F pocket, including Leu81, Tyr123, and Thr143.26,28 Regarding basic residues, ArgΩ was shown to be incompatible with an efficient binding to Mamu-B*08 while LysΩ was a suboptimal anchor motif. The preference of HLA-B*27 for peptides with basic C-terminal has been attributed to the acidic residues Asp74, Asp77, and, fundamentally, Asp116.26,27 It is likely that the lack of the latter in Mamu-B*08 hinders the stabilization of the side chain of Arg residues in the F pocket. In addition, the low frequency of LysΩ could be due to a compensatory effect of Asp74 and Asp77. Finally, peptides with the TyrΩ motif were completely absent from the Mamu-B*08 peptide repertoire, most likely reflecting the absence of Asp116 in this molecule. In this regard, the correlation between the presence of Asp116 and the

By far, Arg was the most overrepresented residue at P2 in both peptidomes. This fact can be explained by the conservation of the key residues Glu63 and, specially, Glu45 that interact via a salt bridge with Arg at P2, determining the specifity of the B pocket (Table 3). Additionally, Gln was also tolerated at this position. The presence of ligands with Gln2 has been already described in HLA-B*27:0542 and, to our knowledge, is reported here for the first time in the context of Mamu-B*08. The frequency of this suboptimal motif is significantly higher in this latter allomorph, probably due to the absence of Cys67 in the B pocket (Table 3). In this regard, it is known that the mutation C67S favors the binding of peptides with Gln2 in HLA-B*27.42 In agreement with this observation, the substitution of Arg2 by Gln2 in an endogenous ligand of Mamu-B*08 did not prevent the binding of the mutant peptide. In a second case, however, the same substitution caused a significant decrease in affinity. This fact suggests that Gln2 contributes to the stability of the peptide-MHC complex only in an appropriate sequence context, explaining, in turn, the low frequency of this motif at P2. H

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and Dr. Francisco Gavilanes (Universidad Complutense, Madrid) for his expert help in peptide quantification. The Proteomics Unit of the Spanish National Biotechnology Centre belongs to ProteoRed (PRB2-ISCIII) and is supported by FIS grant PT13/0001. M.M. was partially funded by the JAE-Doc 2009 CSIC program.

presentation of ligands with TyrΩ is well established for HLAB*27 subtypes.32 Consistent with these results, binding assays confirmed that Mamu-B*08-specific ligands containing ArgΩ or TyrΩ showed much lower affinities, probably incompatible with efficient binding, than those with LeuΩ, PheΩ, and LysΩ, which were shared by both molecules.

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DEDICATION This paper is dedicated to the memory of our mentor and colleague Juan Pablo Albar, who recently passed away.

CONCLUSIONS We have employed high-throughput mass spectrometry in combination with a multiengine search approach to confidently identify thousands of peptides associated with HLA-B*27 and Mamu-B*08. A comprehensive comparison of the two peptidomes together with peptide binding assays prompted the following conclusions: (i) relative to Mamu-B*08, HLAB*27 binds longer peptides of higher molecular weight, (ii) both allotypes use Arg2 as a major anchor motif though peptides with Gln2 can also be presented, (iii) the suboptimal motif Gln2 is comparatively favored by Mamu-B*08, (iv) Mamu-B*08 binds preferentially peptides with Phe and aliphatic residues at the peptide C-terminus and, unlike HLAB*27, excludes ligands with ArgΩ or TyrΩ, and (v) LysΩ is a major binding motif for HLA-B*27 but a suboptimal one for Mamu-B*08. This study may contribute to the identification of novel SIV- or HIV-specific epitopes with suboptimal binding motifs that may have been previously overlooked.

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ABBREVIATIONS CTL, cytotoxic T lymphocyte; MHC, Major Histocompatibility Complex; FDR, False Discovery Rate; HIV, Human Immunodeficiency Virus; HLA, Human Leukocyte Antigen; mAb, Monoclonal Antibody; SIV, Simian Immunodeficiency Virus; TFA, Trifluoroacetic Acid

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b01146. Sequence alignment of HLA-B*27:05 and MamuB*008:01. (PDF) Peptides identified from C1R cells after MHC class I immunoprecipitation. (XLSX) Peptides identified from C1R-B*27 cells after MHC class I immunoprecipitation. (XLSX) Peptides identified from C1R-Mamu-B*08 cells after MHC class I immunoprecipitation. (XLSX) Residue usage at each peptide position of 9-mers, 10mers, and 11-mers associated with HLA-B*27:05 and Mamu-B*008:01. (PDF) MS2 spectrum of the synthetic peptide LRFPEILQK and the corresponding spectra acquired during the analysis of the HLA-B*27 and the Mamu-B*08-bound peptidomes. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel: 34 91 5854540, Fax: 34 91 5854506, E-mail address: [email protected]. Present Address

(M.M., M.L., and A.P.) Centro Nacional de Biotecnologia,́ Darwin 3, 28049 Madrid, Spain. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. David Watkins (University of Miami, FL) for providing the cDNA clone of Mamu-B*008:01 I

DOI: 10.1021/acs.jproteome.5b01146 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research (42) Alvarez, I.; Marti, M.; Vazquez, J.; Camafeita, E.; Ogueta, S.; Lopez de Castro, J. A. The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation. J. Biol. Chem. 2001, 276 (52), 48740−7.

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DOI: 10.1021/acs.jproteome.5b01146 J. Proteome Res. XXXX, XXX, XXX−XXX