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A Common Single Nucleotide Polymorphism
in Endoplasmic Reticulum Aminopeptidase 2
Induces a Specificity Switch That Leads to
Altered Antigen Processing
Irini Evnouchidou, James Birtley, Sergey Seregin,
Athanasios Papakyriakou, Efthalia Zervoudi, Martina
Samiotaki, George Panayotou, Petros Giastas, Olivia
Petrakis, Dimitris Georgiadis, Andrea Amalfitano,
Emmanuel Saridakis, Irene M. Mavridis and Efstratios
Stratikos
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The Journal of Immunology is published twice each month by
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 189:2383-2392; Prepublished online 25
July 2012;
doi: 10.4049/jimmunol.1200918
http://www.jimmunol.org/content/189/5/2383
The Journal of Immunology
A Common Single Nucleotide Polymorphism in Endoplasmic
Reticulum Aminopeptidase 2 Induces a Specificity Switch
That Leads to Altered Antigen Processing
Irini Evnouchidou,*,1 James Birtley,*,1 Sergey Seregin,† Athanasios Papakyriakou,*
Efthalia Zervoudi,* Martina Samiotaki,‡ George Panayotou,‡ Petros Giastas,x
Olivia Petrakis,{ Dimitris Georgiadis,{ Andrea Amalfitano,† Emmanuel Saridakis,*
Irene M. Mavridis,* and Efstratios Stratikos*
ytotoxic T lymphocyte responses are driven by the recognition of antigenic peptides presented on the cell surface by specialized receptors on the MHC. MHC class I
molecules present peptides originating from the proteolytic degradation of intracellular (direct presentation) or endocytosed
(cross-presentation) proteins (1, 2). Although the proteolytic cascade leading to the generation of these peptides is initiated in the
cytosol by the proteasome, the last few steps of antigenic peptide
customization occur in the endoplasmic reticulum (ER) or in
specialized endosomal vesicles (3, 4). There, specialized amino-
C
peptidases trim extended precursors to the mature antigenic epitopes so that the correct-length peptides can be loaded onto nascent
MHC class I molecules (5). Aminopeptidase-mediated antigenic
peptide customization has been shown during the last few years to
be a key, indispensable component to antigenic epitope generation
(6).
At least two intracellular aminopeptidases, ER aminopeptidases
1 and 2 (ERAP1 and ERAP2, respectively), are localized in the ER
and cooperate to trim N-terminally extended peptide precursors to
generate mature antigenic epitopes (7–9). A third homologous
*National Center for Scientific Research Demokritos, 15310 Athens, Greece;
†
Department of Microbiology and Molecular Genetics, Michigan State University,
East Lansing, MI 48824; ‡Institute of Molecular Oncology, Alexander Fleming Biomedical Sciences Research Center, 16672 Vari, Greece; xDepartment of Biochemistry, Hellenic Pasteur Institute, 11521 Athens, Greece; and {Laboratory of Organic
Chemistry, Chemistry Department, University of Athens, 15771 Athens, Greece
formed modeling and computational analysis. E.Z. purified and characterized the
inhibitor. M.S. and G.P. designed and performed the mass-spectrometric analysis.
P.G. collected and processed the x-ray diffraction data. O.P. and D.G. designed and
synthesized the inhibitor. E. Saridakis, J.B., and I.M.M. solved and interpreted the
crystal structure. E. Stratikos conceived the project, designed experiments, interpreted results, and wrote the manuscript together with input from other authors.
1
Atomic coordinates and structure factors have been deposited in The Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/pdb/
home/home.do) under Protein Data Bank ID 4E36.
I.E. and J.B. contributed equally to this work.
Received for publication March 28, 2012. Accepted for publication June 26, 2012.
This work was supported by the European Commission (FP7-Marie Curie-IAPPTOPCRYST, 217979), the Hellenic Rheumatology Society, the National Institutes of
Health (Grant R01 AR056981 to A.A.), the Osteopathic Heritage Foundation, the Greek
Secretariat for Science and Technology (action “Aristeia” Grant 986 to E. Stratikos), the
Special Account for Research Grants of University of Athens, the action “Supporting
Postdoctoral Researchers” from Greek Secretariat for Research and Technology (to A.P.),
and the graduate fellowship program of National Centre for Scientific Research Demokritos (to I.E.); and support for crystallographic data collection was provided by European
Molecular Biology Laboratory-Hamburg at the Doppel-Ring Speicher storage ring,
Deutsches Elektronen-Synchrotron, Germany and the Swiss Light Source, Paul
Scherrer Institut, Villigen, Switzerland (Research Infrastructures Grant 226716).
I.E. performed the biochemical characterization of the variants, designed experiments, and interpreted data. J.B. generated the ERAP2K variant and crystallized
the protein. S.S. and A.A. designed and performed the cell-based assay. A.P. perwww.jimmunol.org/cgi/doi/10.4049/jimmunol.1200918
Address correspondence and reprint requests to Dr. Efstratios Stratikos, National
Center for Scientific Research Demokritos, Patriarhou Gregoriou and Neapoleos
Street, Agia Paraskevi Attikis, 15310 Athens, Greece. E-mail addresses: stratos@rrp.
demokritos.gr and [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ER, endoplasmic reticulum; ERAP, ER aminopeptidase; kcat, catalytic turnover rate; MS, mass spectrometry; R-AMC, L-arginyl-7amido-4-methyl coumarin; RP-HPLC, reverse phase HPLC; SNP, single nucleotide
polymorphism; SR, specificity ratio; TFA, trifluoroacetic acid.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading
onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in
ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to
autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease
etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the
enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster
compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids.
These effects are primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate. X-ray
crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N
terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This
specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal
residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support
the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in
humans. The Journal of Immunology, 2012, 189: 2383–2392.
2384
AN ERAP2 SNP CHANGES THE SPECIFICITY OF THE ENZYME
activity and specificity of the enzyme up to two orders of magnitude, altering its Ag processing profile, as well as its ability to
complement ERAP1-mediated Ag processing. Structural and
biochemical analysis suggests that these effects are due to changes
in the catalytic site of the enzyme that affect transition-state stabilization. To our knowledge, these are the largest functional
changes described for a common coding polymorphism in the Ag
processing machinery. Our results provide mechanistic insight to
the hypothesized causal link between this ERAP2 SNP and disease. Furthermore, our results support the hypothesis that allelic
variability in Ag processing enzymes is an important component
of immune system variability in natural populations.
Materials and Methods
Peptides
Peptides WLRRYLENGK, RLRRYLENGK, LSLYNTVATL, KSLYNTVATL, LSRHHAFSFR, RSRYWAIRTR, LSRYWAIRTR, LRSRYWAIRTR, YKRFEGLTQR, LKRVVINKDT, and ASRHHAFSFR were
purchased from JPT Peptide Technologies (Berlin, Germany). Peptide
KSLYNTVATL was purchased from Invitrogen. All peptides were purified
by reverse phase HPLC (RP-HPLC) and were .95% pure.
Protein expression and purification
Construction of the N392K ERAP2 variant (ERAP2K) was performed using
the Quikchange II XL site-directed mutagenesis kit (Agilent Technologies,
Santa Clara, CA) on the pFastbac1-ERAP2N vector (described in Ref. 14).
The primers used for the variant construction were: 59-GGA ATG ATA TCT
GGC TTA AGG AGG GTT TTG CAA AAT AC-39 (sense) and 59-GTA
TTT TGC AAA ACC CTC CTT AAG CCA GAT ATC ATT CC-39 (antisense). DNA sequencing was performed to validate successful mutagenesis.
Overexpression of ERAP2N in insect cell cultures has been described
previously (14, 15). The pFastbac1-ERAP2K vector was used to generate
recombinant baculovirus according to the directions of the bac-to-bac
baculovirus expression system (Invitrogen), as described before (17).
Human recombinant ERAP2K was produced in Hi5 cells postinfection
with recombinant baculovirus carrying the ERAP2 gene with a C-terminal
6-His tag. The cell supernatant was harvested 4 d postinfection by centrifugation and subjected to Ni-NTA affinity chromatography purification
as described previously (15, 17). In brief, after binding in the presence of
buffer containing 1 mM imidazole for 2 h, protein was eluted from the
column using step gradient of 3, 5, 10, 20, 40, and 150 mM imidazole
solutions (Supplemental Fig. 1A). The fractions with highest enzymatic
activity and purity were collected, dialyzed to remove the imidazole, and
stored at 280˚C in aliquots in the presence of 10% glycerol, until needed.
Each aliquot was thawed once, used in assays immediately, and then
discarded. Both ERAP2 variants were overexpressed and purified in parallel for purposes of comparing enzymatic activities.
Enzymatic assays
The activity of human recombinant ERAP2 was measured by following the
time-dependent increase of the fluorescence signal at 460 nm of the fluorogenic
substrate L-arginyl-7-amido-4-methyl coumarin (R-AMC; Sigma). Excitation
was set at 380 nm. Fluorescence was followed for 5 min using a Quantamaster-4 fluorometer (Photon Technology International, Lawrenceville, NJ),
and the resulting time slope was used to calculate the rate of hydrolysis of the
substrate. ERAP2 specificity for small fluorogenic substrates was analyzed
using a library of fluorogenic compounds described before (14, 32). A total of
10 mM of each substrate was incubated with 8 ng ERAP2 (total volume, 140
ml; ERAP2 concentration, 0.52 nM) at 25˚C, and fluorescence was recorded
for 5 min with excitation set at 380 nm and emission at 460 nm using a Tecan
Infinite M200 microplate fluorescence reader. Inhibition data were fit to
a simple binding model V = Vo * X/(Ki + X) 2 Vo, where V is the rate of
substrate hydrolysis, X the inhibitor concentration, Ki the inhibition constant,
and Vo is the rate of hydrolysis in the absence of inhibitor. Inhibition by the
DG001 peptide was found to proceed to ,100%, and data were fit to a partial
inhibition model V = Vo * X/(Ki + X) 2 Vo + B, in which the parameter B is
the maximal inhibition attained after saturation.
Synthesis, purification, and characterization of pseudopeptide
transition-state analogs
The L-C[PO(OH)CH2]-LAFKARAF peptide (DG001) was synthesized by
Fmoc-solid–phase peptide synthesis using the multipin technology as
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aminopeptidase, insulin-regulated aminopeptidase, has been recently implicated in a separate endosomal pathway of crosspresentation in specialized cells (4). Of these three, the better
characterized one, ERAP1 has been shown to play the role of an
antigenic peptide editor, influencing the antigenic peptide repertoire and the adaptive immune response in vivo (6, 10–12).
ERAP1 achieves peptide editing by both generating many mature
epitopes from elongated precursors and by destroying others by
further trimming them to smaller peptides that cannot bind onto
MHC class I molecules (8, 13). Less is known about the function
of ERAP2, although it has been proposed that it acts as an accessory aminopeptidase, complementing ERAP1 specificity to
allow the trimming of the vast number of different sequences in
the antigenic peptide repertoire (9, 14, 15). ERAP1 has some key
functional properties that make it suitable for antigenic peptide
precursor trimming: it is specialized for relatively large peptides
and can trim peptides depending on their length and internal sequence (16, 17). Furthermore, ERAP1 can be regulated by its own
substrates and products, suggesting a complex and poorly understood landscape of the regulation of Ag processing (18, 19).
ERAP2 and insulin-regulated aminopeptidase, although less
studied to date, appear to share some of those properties, but
also have key differences that may underlie distinct biological
roles (9, 13, 15, 16).
Several large-scale, population-wide genetic studies have linked
specific amino acid coding single nucleotide polymorphisms
(SNPs) in ERAP1 and ERAP2 with predisposition to human
disease (reviewed in Ref. 20). SNPs in ERAP1 and ERAP2 have
been linked with predisposition for development of the chronic inflammatory disease ankylosing spondylitis, a disease with a strong
hereditary autoimmune component (21–23). The original hypothesis that this link is mediated by the Ag processing function of
ERAP1/ERAP2 was later supported by the demonstration of interplay between ERAP1 and ERAP2 SNPs and more importantly
with the MHC class I allele HLA-B27 (24–26). Recently, ERAP1/
ERAP2 haplotypes have been linked with resistance to HIV infection (27). More specifically, the ERAP2 SNP rs2549782 that
codes for the variation N392K has been linked with resistance to
HIV infection in homozygous individuals and with development
of pre-eclampsia (27–29). ERAP1 and ERAP2 polymorphisms
have been proposed to be the result of a long-standing balancing
selection, indicating significant functional consequences of both
polymorphic states (27, 30). Preliminary results regarding the
effects of coding ERAP1 polymorphisms have pointed toward
changes in both enzyme activity and specificity that may influence
the antigenic peptide repertoire (19, 26, 31). Although these
changes in antigenic peptide processing have been of a relatively
low magnitude (generally up to 2-fold), it has been hypothesized
that they can be sufficient in altering chronic immune and/or inflammatory responses (19, 26).
In this study, we characterized the ERAP2 SNP rs2549782 that
codes for the amino acid variation N392K. This particular polymorphism constitutes an attractive model for understanding the role
of polymorphic variation in Ag processing for three reasons: 1)
both polymorphic states are almost equally distributed in the human
population, presumably as a result of host–pathogen balancing
selection (27); 2) it is the main polymorphism in ERAP2 that has
been associated with disease in virtually all genetic studies; and 3)
its structural proximity to the enzyme’s active site makes it reasonable to expect at least some perturbation in enzymatic activity
(15). We used a combination of biochemical, cell-based, structural, and computational approaches to understand the effects of
this polymorphic variation to ERAP2 function. We demonstrate
that the polymorphic variation at position 392 affects both the
The Journal of Immunology
previously described (33). Peptide Fmoc-AFKARAF was developed on
trityl alcohol lanterns (surface: polystyrene, loading 15 mmol) using the
Fmoc-solid–phase protocol (34). Pmc and Boc protecting groups were
used for the side-chain functional groups of Arg and Lys, respectively.
After the deprotection of the N-terminal Ala, the phosphinic dipeptide unit
was introduced by shaking the lantern-anchored peptides with a solution of
1.3 equivalent of phosphinic block BocLeu-C[PO(OAd)CH2]-LeuOH,
DIC (2 equivalents), and HOBt (2 equivalents) in dichloromethane during 24 h. The phosphinic block was synthesized based on a previously
described protocol (35). The crude peptide was obtained after treatment of
lanterns with trifluoroacetic acid (TFA)/dichloromethane/H2O/triisopropylsilane
80/15/2.5/2.5 for 2 h, and purified by RP-HPLC using a Chromolith C-18
semiprep column (Merck) and a 5250% acetonitrile gradient in water
containing 0.05% trifluoroacetic acid. Eluted peaks were collected, lyophilized, and characterized by mass spectrometry (MS).
Analysis of peptide trimming activity on RP-HPLC
Analysis of peptide trimming activity by mass spectroscopy
Trimming of ASRHHAFSFR peptide by ERAP2 was followed by mass
spectroscopy because the product SRHHAFSFR is poorly separated by
HPLC. Reactions were set up as described earlier with the exception that
they were stopped by adding 1% formic acid. The reactions were initially
cleaned up using pipette tips packed with reverse-phase material (C18MB,
OMIX, VARIAN). The samples were dried completely in a speed-vac,
dissolved in 100 ml of 0.1% formic acid and 2% acetonitrile, and sonicated for 1 min. The samples were analyzed by direct infusion nanoelectrospray on an LTQ Orbitrap XL mass spectrometer at a resolving
power of 60,000 at m/z 400. The scan range was set to 350–450 m/z. MS/
MS spectrum was acquired to confirm the sequence of the peptides. The
triply charged peptides were monitored. Reaction progress was evaluated
by measuring the relative peak intensity of the substrate and product
peptide, respectively. All reactions were evaluated at ,30% completion to
ensure linearity of the rate measurement.
Cell-based Ag presentation assay
Construction of plasmids, encoding for ERAP2N and ERAP2K variants
(rs2549782, GenBank accession no. NM_022350.3), was performed as
previously described for ERAP1 alleles (7, 19) in pTracerCMV-RFP,
where the GFP/Zeo fusion protein was replaced with red fluorescent
protein ORF. In brief, ERAP2N ORF was subcloned into pcDNA3.1
(Invitrogen, Carlsbad, CA). pcDNA-ERAP2K was generated using Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA),
using the following primers (AAT mutated into AAG codon): ERAP2_MUT
(forward), 59-GGAATGATATTTGGCTTAAGGAGGGTTTTGCAAAATAC39; ERAP2_MUT (reverse), 59-GTATTTTGCAAAACCCTCCTTAAGCCAAATATCATTCC-39.
pcDNA-based ERAP2 containing plasmids were digested with SalI and
XhoI, and subcloned into pShuttleCMV. pShuttle-ERAP2 plasmids were
digested with NotI and XbaI, and subcloned into pTracerCMV-RFP. Integrity of ERAP2N and ERAP2K genes, cloned into pTracerCMV-RFP, was
confirmed by sequencing of whole-length ERAP2 ORF using the following
primers: ERAP2cl.end_SEQ (forward), 59-GAACTTGGCAGCTCTCCTTCATGC-39; ERAP2cl.Start_SEQ (reverse), 59-CTGACTGAAGGGTGGCATTCGTG-39; ERAP2-SEQ (reverse), 59-GGTTTGGAGATAGGGCGGGATG-39; ERAP2-SEQ (forward), 59-CAACCCAGGCACGCATGGC-39.
HeLa cells, stably expressing H-2Kb, HLA-B27, and Herpesvirus protein ICP47 (TAP1 blocker), HeLa-Kb-B27/47 were described previously
(8). To construct plasmids expressing HLA-B27–restricted peptides (and
their N-terminal precursors), we subcloned nucleotide sequences corre-
sponding to ASRHHAFSFR and SRHHAFSFR peptides into pTracerCMV
(Invitrogen, Carlsbad, CA). All peptides were designed to contain the ERsignal sequence MRYMILGLLALAAVCSA (from the Ad2E3gp19K
protein) upstream of the peptide sequence (7). HeLa-Kb–B27/47 were
transiently transected with HLA-B27–specific peptide and either ERAP2Nor ERAP2K-expressing plasmids for 48 h. Surface expression of HLA-B27
in transfected cells was measured by flow cytometry, exactly as described
previously (19). Statistical analysis was completed using one sample t test
([mean ratio 2 1]/SE) to test whether Ag presentation in ERAP2 transfected cells deviated from mock transfections performed in parallel.
Quantitation of protein expression in HeLa cells
HeLa-Kb-B27/47 cells were transfected with pTracer-CMV-RFP-ERAP2N
(or ERAP2K). At 24 h after transfection, protein was harvested in lysis
buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl) containing
1% Triton X-100 with protease inhibitors. Lysed samples were then
centrifuged at maximum speed (16,000 3 g) for 15 min at 4˚C, after which
the protein concentration of the supernatant was determined using BCA
method. Equivalent concentrations of protein samples (5–20 mg) were run
on 8% polyacrylamide gels and transferred onto nitrocellulose membranes.
Blots were then probed with the mouse anti-hTubulin monoclonal primary
Ab (Sigma-Aldrich, St. Louis, MO) at 1:30,000 dilution for 20 min and
primary polyclonal mouse-anti-hERAP2 (Abcam, Cambridge, MA) at
1:500 dilution for 3–16 h. After incubations, membranes were probed with
fluorescent Abs (IRDYE800 conjugated goat anti-mouse IgG (Rockland,
Gilbertsville, PA) for 45 min at 1:3300 dilution. Blots were scanned and
bands were quantified using Licor’s Odyssey scanner (models 2.1 or 3.0).
For data analysis, the fluorescence of ERAP2 bands was normalized to
Tubulin bands before quantification.
X-ray crystallography
Crystallization of ERAP2K was performed using conditions identical to the
ones described previously for ERAP2N (15). During the last stages of
concentration, DG001 peptide was added in the protein solution at a protein/peptide ratio of 1:4; the mixture was incubated at room temperature
for 4 h, and the sample was further concentrated to 6 mg/ml. Single
crystals were obtained as for ERAP2N using the Morpheus (36) protein
crystallization screen (Molecular Dimensions, Newmarket, U.K.) by the
sitting drop vapor diffusion technique: by mixing 200 nl ERAP2K (6 mg/
ml in 150 mM NaCl, 0.01% NaN3, 25 mM HEPES pH 7.0) with 100 nl
precipitant using an Oryx4 nano-drop crystallization robot and incubating
against 50-ml reservoir. Initial crystals appeared after 2–3 d. The crystal
used for data collection was grown in a 2-ml drop at 4˚C, using the condition 10% PEG 8000, 20% ethylene glycol, 20 mM each of glycine, DLalanine, DL-serine, DL-lysine, and Na-glutamate, 31 mM MES, and 69 mM
imidazole at pH 6.5. X-ray diffraction data were collected at 100 K using
synchrotron radiation at the XO6DA beamline (Swiss Light Source, Paul
Scherrer Institut, Villigen, Switzerland). Diffraction data up to 3.2-Å resolution were processed by MOSFLM (37) and scaled with the SCALA
software (38). Five percent of reflections was flagged for Rfree calculations.
The crystal is isomorphous with that of ERAP2N, space group P21, a = 74.3
Å, b = 134.4 Å, c = 127.4 Å, and b = 90.8˚ (Supplemental Table I). The
structure was determined by the isomorphous replacement method using
the ERAP2N coordinates (Protein Databank ID, 3SE6). Phenix.refine (39)
was used for structure refinement imposing 2-fold noncrystallographic
symmetry restraints. All but 16 pairs of residues were restrained according
to noncrystallographic symmetry. Alternating cycles of restrained refinement and manual fitting/building with Coot (40) resulted in an R factor and
Rfree of 21.36 and 26.12%, respectively. Residual electron density (.3s) at
the active site resembled that of ERAP2N and was interpreted to belong to
a lysine (Lysa) and MES, present at high concentrations in the crystallization mixture (15). A set of three cycles of refinement with Lysa and MES
included resulted in final R factor and Rfree of 20.87 and 26.08%.
Besides the two crystallographically independent protein molecules, the
asymmetric unit includes a total of 14 sugar residues and 106 water
molecules. A lysine and an MES molecule were modeled on residual
electron density close to the zinc atom of the active site of each protein
molecule in the dimer. The carbohydrate moieties were found attached to
the following Asn N-glycosylation sites: A85, A103, A219, A405, A650,
B85, B219, and B431. All occupancies of protein and carbohydrate atoms,
as well as water molecules, were set to one, except for the disordered amino
acid side chains and the two MES molecules. Two amino acid side chains in
one of the molecules (A) were disordered (Arg366 and Lys839) and refined
with two alternative conformations. There was no assignable density before residue 54 of chain A and residue 55 of chain B. Also missing were
residues 127–129, 503–527, and 570–580 of chain A and 126–132, 503–
531, and 571–582 of chain B. Figures were made using Pymol (41).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Analysis of digestion products after incubation of peptides with ERAP1 or
ERAP2 was performed by RP-HPLC on a Chromolith C-18 analytical column
(Merck) as previously described (17). In brief, 20 mM of each peptide was
incubated at 37˚C with 100–4000 ng (7.3–293.6 nM) ERAP1 or ERAP2 in 50
mM HEPES pH 7.0, 100 mM NaCl buffer in a total volume of 125 ml for 15
min to 1 h. After incubation, the reactions were terminated by adding 100 ml
0.1% trifluoroacetic acid and stored at 220˚C until analysis. Before analysis,
samples were centrifuged for 5 min at 10,000 3 g to remove precipitated
protein. HPLC elution was performed using a 5–50% acetonitrile gradient,
while following the absorbance at 220 nm. To calculate initial reaction rates,
we performed several experiments for each peptide to achieve conditions
under which the reaction was ,30% complete. All comparisons of the activity of ERAP1 and the two ERAP2 variants were performed in parallel.
Product peaks in the HPLC chromatogram were identified by running peptide
controls and by MS as described previously (13).
2385
2386
AN ERAP2 SNP CHANGES THE SPECIFICITY OF THE ENZYME
Atomic coordinates and structure factors have been deposited in the
Protein Data Bank as entry 4E36.
Computational methods
Results
ERAP2K is less active in hydrolyzing small fluorogenic
substrates
Small fluorogenic substrates such as R-AMC have been used
previously to characterize the activity and specificity of M1
aminopeptidases (14). To compare the enzymatic activity of the
two ERAP2 variants, we measured the rate of hydrolysis of RAMC by ERAP2N and ERAP2K. The two variants had significantly different activity, with R-AMC being hydrolyzed at 78 6 1
mmol product/mol enzyme sec21 by ERAP2N and 4.8 6 3 mmol/
mol sec21 by ERAP2K. Such a difference in activity is at least 10fold higher compared with previously characterized ERAP1
polymorphisms (19, 31). To investigate whether this change in
catalytic activity was due to changes in substrate recognition or
catalytic turnover rate (kcat), we performed a standard Michaelis–
Menten analysis (Fig. 1). According to this analysis, the polymorphic state at position 392 of ERAP2 affected substrate affinity
by ,2-fold (ERAP2N: KM = 30 6 9 mM; ERAP2K: KM = 17 6 5
mM) but affected kcat to a large degree, lowering the kcat constant
by ∼18-fold (ERAP2N: kcat=104 6 9 3 1023 sec21; ERAP2K: kcat =
5.8 6 0.4 3 1023 sec21). This large change in catalytic efficiency
brought about by a common polymorphism is, to our knowledge,
the largest described for this class of enzymes to date.
FIGURE 1. (A) Michaelis–Menten analysis of R-AMC hydrolysis by
ERAP2N (filled circles) and ERAP2K (open circles) variants. (B) Doublereciprocal plot of the data.
Trimming of antigenic peptide precursors
Because the trimming of small fluorogenic substrates does not
adequately describe the in vivo function of the enzyme, we tested
the trimming of antigenic peptide precursors by the two ERAP2
alleles. ERAP2N was able to efficiently trim the leucine residue
from the precursor peptide with the sequence LSRHHAFSFR and
generate the mature epitope SRHHAFSFR (from the aggrecan
protein, a critical component for cartilage structure and function
of the joints, normally presented by the HLA-B2705 MHC class I
allele; Fig. 2A). This result was rather surprising given the postulated role of ERAP2 in removing positively charged amino acids
(9, 14) and may be because of other factors including the recognition of side chains distal from the N terminus allowing the
processing of peptides with suboptimal N termini, a property
established for ERAP1 (19) (also see later). In contrast with
ERAP2N, ERAP2K was much less efficient in processing this
antigenic peptide precursor and generated the mature epitope with
a 37-fold slower rate (Fig. 2A, 2B). This change in trimming rate
was not due to substrate recognition because the KM for this
peptide was similar for both alleles (33 6 9 versus 19 6 5 mM) as
calculated by competition titrations (Fig. 2C). We conclude that
the difference in peptide processing kinetics is due to a change in
the kcat of the enzyme for this particular substrate (Fig. 2D).
ERAP2 has been described previously as an accessory aminopeptidase that “assists” ERAP1 in trimming positively charged
residues from antigenic peptide precursors, residues for which
ERAP1 has reduced activity (9, 14). To test whether ERAP2 allelic variation affects the trimming of a peptide precursor with
a positively charged N terminus, we measured the kinetics of
trimming of a peptide with the sequence KSLYNTVATL, a precursor to the epitope SLYNTVATL (derived from the HIV-1 p17
Gag protein and presented by the HLA-A0201 MHC class I allele). In sharp contrast with the SRHHAFSFR epitope, SLYNTVATL was produced equally effectively by both ERAP2 alleles
(Fig. 3). This surprising result suggested that the change in kcat
between ERAP2N and ERAP2K may be substrate dependent for
larger, more physiologically relevant, peptidic substrates.
To test the generality of changes in substrate specificity between
the two ERAP2 alleles, we measured the trimming rates for eight
additional antigenic peptide precursors in vitro. These precursors
were designed based on the protein sequence upstream of antigenic
peptides that are known to be generated by the action of intracellular aminopeptidases. The N-terminal amino acid was in every
case either a positively charged residue or a hydrophobic residue.
The trimming rates for all precursors tested are compared in Fig. 4.
In most cases, there was a significant difference between the
trimming rates for the two alleles; these differences were much
more potent for precursors with a hydrophobic N-terminal amino
acid. For example, the precursors RSRYWAIRTR and KSLYNTVATL were processed with almost identical rates by both ERAP2
alleles. In contrast, the precursor WLRRYLENGK was processed
with a rate difference of 165-fold (330 6 34 pmol nmol21 sec21
for ERAP2N versus 2.0 6 0.04 pmol nmol21 sec21 for ERAP2K).
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Continuum electrostatic calculations were performed using the finite difference Poisson–Boltzmann method, implemented in Delphi v5 (42, 43). The
individual residue contributions to the electrostatic interaction energies were
calculated as described previously (44, 45). In brief, hydrogen atoms and
missing side chains of the two x-ray structures (ERAP2K and ERAP2N) were
built and optimized with AMBER v10 (46). Energy minimizations were
performed in implicit solvent with the Generalized Born GBn method (47) by
applying harmonic constraints of 100 Kcal mol21 Å22 to heavy atoms so that
their crystallographic positions were marginally perturbed. Atomic radii and
charges were taken from the param22 parameter set of the CHARMM program (48), except for Zn that was assigned a charge of +2.0 and a radius of
1.4 Å. The interior of the enzyme and water were modeled with dielectric
constants of 4 and 80, respectively, and boundary conditions were approximated by the sum of Debye–Hückel potential of all the charges. The ionic
strength of the solution was set to 0.15 M, the solvent probe radius to 1.4 Å,
and the ion exclusion radius to 2.0 Å. Calculations were performed using the
linearized PB equation until the maximum change of the grid potential
converged to within 1024 kBT, where kB is the Boltzmann constant and T is
the absolute temperature. Each calculation was performed 10 times with the
protein translated systematically on the grid, and 10 separate times by
varying the percentage fill of a lattice with resolution of 3.0 grids/Å. The
reported values are the average of 20 runs, with the SD being ,1%, representing the uncertainty caused by the granularity of the grid.
To investigate whether the change in catalytic turnover was
substrate specific, we used a previously characterized collection of
fluorogenic substrates bearing all-natural amino acids as their
scissile N termini (14, 32). Consistent with previous observations
on the specificity of ERAP2, both polymorphic states presented
a highly similar pattern of N-terminal selectivity with only positively charged side chains processed efficiently (Supplemental Fig.
1B). This finding suggests that the N392K polymorphism affects
the catalytic efficiency but not the specificity of ERAP2, at least
toward these small fluorogenic substrates.
The Journal of Immunology
2387
We concluded from this limited screen that the polymorphic
variation in position 392 of ERAP2 can affect peptide specificity,
primarily for peptides that carry a hydrophobic amino acid as their
N terminus. This is in sharp contrast with what we discovered for
small nonpeptidic substrates (Fig. 1, Supplemental Fig. 1), in
which case, we could only demonstrate a difference of catalytic
turnover between the two alleles but not in specificity. This apparent paradox may signify fundamental differences between
recognition of small versus larger natural substrates. Such a distinction has recently been described for ERAP1 and has been
hypothesized to constitute the main mechanism for efficient large
peptide trimming necessary for its biological function (18).
In an effort to isolate the importance of the N-terminal residue of
the peptide substrate in the trimming rate by the two ERAP2 alleles,
we compared the trimming of identical peptide backbones that only
differ in the N-terminal residue. The ratio of the trimming rate of
the peptide carrying an N-terminal extension of a positively
charged amino acid versus the trimming rate of the same peptide
carrying a hydrophobic N-terminal extension defines a specificity
ratio (SR) that indicates the preference of ERAP2 for positive
versus hydrophobic N termini. The calculated SR value for three
antigenic epitopes is shown in Fig. 5A. In all cases, ERAP2K
showed a clear preference for positively charged residues at the N
terminus of the peptide (SR . 1, maximal SR = 92), although the
magnitude of this value depended on the remaining peptidic sequence, indicating that the whole peptide sequence is important
for substrate recognition (as has been demonstrated for ERAP1;
see Ref. 17). In addition, in all cases, the SR value for ERAP2N
was significantly lower, indicating that the strong preference for
positively charged N termini is not shared by this allele that can
efficiently process both hydrophobic and positively charged N
termini. These differences in specificity are highly significant and
can lead to qualitative differences in trimming between the two
alleles. As shown in Fig. 5B, trimming of the precursor RSRYWAIRTR is essentially identical for both alleles, whereas trimming of the precursor LSRYWAIRTR (same sequence but with
different N terminus) is drastically different, with ERAP2K essentially failing to produce detectable amounts of mature epitope
under the specific experimental conditions. Overall, these results
suggest that the polymorphism at position 392 of ERAP2 can
induce changes in specificity when trimming natural peptidic
substrates.
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FIGURE 2. (A) RP-HPLC chromatograms of products of the enzymatic digestion of peptide LSRHHAFSFR (substrate) by the two ERAP2 variants. The
produced epitope SRHHAFSFR (product) is indicated. Two characteristic reaction conditions are shown, using 200 ng (14.7 nM, molar ratio peptide to
enzyme = 1361) of enzyme for 15 min or 1 h and 2 mg (147 nM, molar ratio peptide to enzyme = 136) enzyme for 1 h. (B) Epitope generation rate for each
ERAP2 variant. (C) The rate of hydrolysis by each ERAP2 variant of the fluorogenic substrate R-AMC was plotted versus the concentration of competing
substrate LSRHHAFSFR, and data were fit to a simple competition model to allow the calculation for the affinity for the competing substrate (56). (D)
Turnover number (kcat) and specificity constant (kcat/KM) for the production of the SRHHAFSFR epitope by the two ERAP2 variants.
2388
AN ERAP2 SNP CHANGES THE SPECIFICITY OF THE ENZYME
ERAP2N can supplement ERAP1-mediated trimming of
hydrophobic N termini
ERAP2 has been proposed to act as an accessory aminopeptidase
to ERAP1, assisting it in trimming peptide sequences for which
ERAP1 has suboptimal affinity such as peptide carrying positivecharged N-terminal amino acids (9). Our finding that ERAP2N can
excise hydrophobic N termini much more efficiently than
ERAP2K prompted us to investigate how well ERAP2 activity
compares with ERAP1. We measured the rate of generation of
the SRHHAFSFR epitope from a precursor carrying an Nterminal leucine residue, by ERAP1, ERAP2N, and ERAP2K
(Fig. 6A). Because ERAP1 has been reported to also be able to
FIGURE 4. Trimming rates of model epitope precursors by ERAP2N
(black bars) and ERAP2K (white bars). A total of 20 mM of each indicated
epitope precursor was incubated with an appropriate amount of ERAP2 to
produce a limited amount of mature epitope as described in Materials and
Methods. Error bars correspond to SD calculated from three separate
experiments.
destroy antigenic epitopes, we measured the rate of destruction
of the same epitope by quantitating smaller peptides accumulated after treating it with each of the three enzymes (characteristic chromatograms of epitope generation and destruction
are shown in Supplemental Fig. 2). All three enzymes generated
the epitope faster than they destroyed it, suggesting that they
have the inherent ability to accumulate it (13). This effect was
more pronounced for ERAP1 and ERAP2N, because both
enzymes generated the epitope at least 100-fold faster than they
destroyed it. Of the three enzymes, ERAP1 was by far the most
efficient in generating the mature epitope, although ERAP2N
was also competitive. Comparing the relative rate of ERAP1- with
ERAP2N-mediated epitope generation revealed that ERAP2N can
account for ∼16% of the ERAP1 activity for this peptide, whereas
the ERAP2K activity is negligible compared with that of ERAP1
(Fig. 6B). Taken together, these findings suggest that ERAP2N, but
not ERAP2K, can supplement ERAP1 in trimming hydrophobic N
termini.
The substrate specificity of ERAP1 has been shown to correlate
well with Ag processing and presentation in cultured cells, suggesting that in vitro specificity differences are relevant to in vivo Ag
processing (19, 49). To extend these findings to ERAP2 and its
allelic content, we used a cell-based Ag presentation assay that has
been previously established for ERAP1 (8). We modified this assay
to report the effects of ERAP2 allele transfection by cotransfecting
with a plasmid coding for the ERAP2 enzyme carrying the desired
polymorphism and a minigene encoding a miniprotein that after
signal sequence cleavage gives rise to the antigenic peptide precursor ASRHHAFSFR (technical limitations of this assay necessitate the existence of an N-terminal alanine residue; Supplemental
Fig. 3). Removal of the N-terminal alanine residue from this peptide generates the mature epitope that can bind onto the nascent
MHC class I allele HLA-B27 constitutively produced by this cell
line. We measured the changes in epitope presentation on transfection of ERAP2N or ERAP2K compared with control transfected
cells that perform processing only by endogenous ERAP1/2.
ERAP2N was able to enhance SRHHAFSFR epitope generation
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FIGURE 3. (A) RP-HPLC chromatograms of production of the
SLYNTVATL epitope from a single residue extended precursor carrying
a positively charged amino acid (KSLYNTVATL). (B) Trimming rate of
peptide KSLYNTVATL by the two ERAP2 variants. (C) R-AMC competition titrations for the KSLYNTVATL peptide as in Fig. 2 reveal similar
affinity for both ERAP2 variants.
FIGURE 5. (A) Specificity index (defined as the trimming rate for
a peptide with a positively charged N terminus divided by the trimming
rate for the same peptide carrying a hydrophobic N terminus) for three
different antigenic epitope templates using the two ERAP2 variants. The yaxis is broken to enhance visibility of large numerical differences. (B) RPHPLC chromatograms of trimming of the precursor RSRYWAIRTR by
ERAP2N and ERAP2K. Notice the similar amount of epitope generation.
(C) RP-HPLC chromatograms of trimming of the precursor LSRYWAIRTR by ERAP2N and ERAP2K. Notice the absence of a visible epitope peak produced by ERAP2K.
The Journal of Immunology
2389
FIGURE 6. (A) Epitope generation versus epitope destruction: trimming
rates of epitope precursor LSRHHAFSFR (gray bars) and mature epitope
SRHHAFSFR (white bars) by ERAP1, ERAP2N, and ERAP2K. (B)
ERAP2N can trim the precursor LSRHHAFSFR with a rate corresponding
to ∼16% of that of ERAP1, whereas ERAP2K is much less efficient. Bars
represent the ratio of ERAP2/ERAP1 trimming rates for each ERAP2
variant. (C) HeLa cells, stably expressing HLA-B27, as well as TAP1 ERtransporter blocker (ICP47), were transfected with an ERAP2 variant and
an ER-targeted miniprotein that after signal sequence cleavage gives rise to
an HLAB27-specific peptide precursor with the sequence ASRHHAFSFR.
HLAB27 cell-surface translocation was followed by flow cytometry using
an MHC-specific Ab. (D) In vitro trimming rate of the ASRHHAFSFR by
ERAP2 alleles. MIF, mean intensity of fluorescence.
Atomic basis for different trimming between the two ERAP2
alleles
In an effort to understand the atomic basis of the functional differences between the two alleles, we solved the crystal structure of
the ERAP2K allele at 3.2 Å and compared it with the recently
determined crystal structure of ERAP2N (Fig. 8, Supplemental
Table I) (15). Asn392 in ERAP2N is highly conserved among homologous aminopeptidases (Fig. 8A). Although the DG001
to a greater degree than ERAP2K (Fig. 6C). In vitro digestion of the
same peptide by ERAP2 showed that ERAP2N generated the mature epitope ∼2-fold faster than ERAP2K, consistent with the enhanced epitope presentation seen in the cell-based assay (Fig. 6D).
This finding suggests that ERAP2 allelic variation can affect Ag
presentation in cells. Interestingly, the relative effect of ERAP2N is
of a magnitude similar to the effects of ERAP1 allelic variation
described before using a similar assay, suggesting that ERAP2 allelic variation can be of equal importance regardless of the accessory role of this enzyme (19).
Transition-state analogs exert distinct inhibitory effects on the
two ERAP2 alleles
Because the difference in catalytic efficiency between ERAP2N and
ERAP2K was due to differences in the kcat of the enzyme, suggesting changes in the recognition of the transition state, we hypothesized that such differences may also apply in the recognition
of transition-state analogs. Phosphinic pseudopeptide transitionstate analogs have been described before as potent aminopeptidase inhibitors (50). We tested the ability of a phosphinic pseudopeptide of the sequence L-C{PO2CH2}-LAFKARAF (DG001
peptide) carrying a hydrophobic side chain at its N terminus to
FIGURE 7. Inhibition of ERAP2N and ERAP2K by a transition-state
analog (DG001) (A) and a substrate analog (amastatin) (B). The rate of
hydrolysis of the fluorogenic substrate R-AMC was followed in the presence of increasing concentrations of inhibitor for each enzyme. Experimental data were fit to a simple binding model accounting for full or partial
inhibition (see Materials and Methods). The calculated constants of inhibition (Ki) are shown.
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inhibit each ERAP2 allele (a more thorough characterization of
this class of inhibitors will be described elsewhere [D. Georgiadis,
E. Zervoudi, and E. Stratikos, manuscript in preparation]). This
compound carries a phosphinic pseudopeptide bond between the
first two amino acids, resembling the tetrahedral intermediate of
the cleavage pathway for the first peptide bond (50, 51). The sequence of this peptide was designed based on known specificity
determinants for ERAP1 and ERAP2 (14, 17). The enzymatic
hydrolysis of R-AMC by each ERAP2 allele was measured as
a function of inhibitor concentration in the solution (Fig. 7A). The
DG001 peptide was found to inhibit ERAP2 with good affinity but
displayed partial inhibition (Fig. 7A). Strikingly, the DG001
peptide had a much higher affinity for ERAP2N (Ki = 54 6 8 nM)
than for ERAP2K (Ki = 524 6 107 nM; Fig. 7A). This 10-fold
difference in inhibitor affinity is consistent with the lower activity
of ERAP2K for substrates with a hydrophobic N terminus and
further supports the hypothesis that the difference between the two
alleles lies in the recognition of the transition state. In contrast
with these results, inhibition of the two ERAP2 alleles by the
substrate analog amastatin was nearly identical (Fig. 7B). This
distinction between a transition-state analog and a substrate analog further supports the idea that the differences between the two
ERAP2 alleles lie in the mechanism of transition-state stabilization and not substrate affinity. Overall, the differential recognition
of a transition-state analog pseudopeptide by the two ERAP2
alleles indicates differences in the catalytic mechanism and suggests that ERAP2 allelic variation should be taken into account for
any pharmacological approaches that involve modulation of ERAP2
trimming activity.
2390
AN ERAP2 SNP CHANGES THE SPECIFICITY OF THE ENZYME
Structural differences between ERAP2N and ERAP2K were also
evident in the region capping the S1 pocket (Fig. 8D): 1) the NZatom of Lysa forms a strong salt bridge with the carboxylic group
of Asp198 (2.8 Å); and 2) Glu177 assumes a different conformation,
away from Lysa, interacting strongly via H-bonding (2.6 Å) with
the carboxylic group of Asp888 of domain IV. A water molecule
is found close to the previous location of the side chain of Glu177,
H-bonding both to NZ of Lysa and Asp198. These observations
suggest subtle changes in the stabilization/recognition properties
of the S1 pocket that may underlie changes in specificity. Furthermore, the observed interaction between Glu177 and Asp888
observed only in the case of ERAP2K suggests differences in the
interactions between domains II and IV. Reorientation of the relative positions of domains II and IV has been previously reported
to be crucial for the catalytic mechanism of the highly homologous ERAP1 (18).
Discussion
pseudopeptide was present in the crystallization mixture, the residual electron density was not significantly different from that of
ERAP2N and, as a result, the residual electron density was interpreted to belong to a lysine present at high concentrations in the
crystallization mixture. The two structures were found to be
overall identical with only minor structural deviations (root mean
square deviation = 0.251 Å for homologous residues). Superimposition of key catalytic or specificity residues in ERAP2K and
ERAP2N is shown in Fig. 8. Lys392 in ERAP2K assumes a distinct
conformation compared with the Asn residue in ERAP2N and
approaches the N terminus of the bound ligand (Lysa), making
interactions with the Zn-coordinating Glu393 residue and residues
Glu337 and Glu200, both key residues that stabilize the N terminus
of the substrate (distances 2.6, 2.9, and 2.6 Å, respectively; Fig.
8B). The NZ-atom of Lys392 in ERAP2K is located in the position
of a water molecule in ERAP2N and is very close (3.7 Å) to the N
terminus of the bound ligand (Lysa). Computational analysis of the
electrostatic free energy of interaction of the Lysa with ERAP2
suggests that the ERAP2K allele introduces a highly unfavorable
electrostatic interaction between Lys392 and the N terminus of the
bound Lysa (Fig. 8C). This unfavorable interaction may interfere
with transition-state stabilization, resulting in reduced catalytic
efficiency for ERAP2K.
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FIGURE 8. Crystal structure of ERAP2K allele suggests the structural
basis for changes in activity. (A) Sequence alignment of ERAP2 and homologous aminopeptidases showing conservation of the polymorphic
residue 392 (in bold); the adjacent aminopeptidase motif (HELAH) is also
indicated. (B) Key catalytic residues in the ERAP2K structure are shown in
stick representation and superimposed with equivalent residues in ERAP2N
(3SE6). Note the relative positioning of Lys392 with respect to the catalytic
Glu residues, as well as the N terminus of the bound ligand (Lysa).
Electron density 2|Fo|2|Fc| at 2s is indicated around Lys392. (C) Pairwise
electrostatic interaction energies (DΕinter in Kcal/mol) calculated between
the polymorphic residue 392 and the Lysa ligand. The reported values are
the average of the 20 runs with an SD ,1%, which represents the uncertainty because of the granularity of the grid. (D) Superimposition of
ERAP2 residues that cap the S1 specificity pocket of the enzyme. |Fo|2|Fc|
electron density (at 2.5s) calculated in the absence of the ligand is shown
around Lysa. 2|Fo|2|Fc| electron density at 2s is indicated around Glu177.
Two main ERAP2 SNPs have been associated with disease and
proposed to be the result of balancing selection possibly through host–
pathogen interactions (27, 30). SNP rs2248374 is a “loss-of-function” variant because it leads to RNA instability and loss of ERAP2
expression (30). This variant leads to a clear functional defect,
namely, reduced surface MHC class I expression in B cells, but in
contrast with ERAP1 SNPs is not associated with the inflammatory
disease ankylosing spondylitis (52). We show in this article that the
second ERAP2 SNP, rs2549782, which codes for a single amino acid
switch near the enzyme’s active site, is not a loss-of-function variant,
but rather a “change-in-function” variant that leads to substratespecific changes in enzymatic activity, essentially allowing the enzyme to alter its specificity profile for peptidic substrates. This
unique SNP-related functional change along with the association of
this SNP with several human diseases, most notably with resistance
to HIV infection, supports the hypothesis that it has arisen by host–
pathogen balancing selection and is a component of the immune
system variability in natural populations.
The functional consequences of the ERAP2 allelic variation
described in this article come in sharp contrast with the effects
described for coding SNPs in ERAP1, which have been of much
lower magnitude (19, 26, 31). It may, at first view, be difficult to
understand how such large differences in activity between ERAP2
alleles are so common in the population (the two ERAP2 alleles
are almost equally represented in the human population) (27). It is
possible that the proposed “accessory” nature of ERAP2-mediated
Ag processing can account for this apparent discrepancy. If, indeed, ERAP1-dependent Ag processing is the dominant activity in
the cells, large changes in ERAP2 activity may be easier to tolerate without severely compromising the function of adaptive
immunity. Interestingly, the magnitude of Ag presentation changes
we report in the cell-based Ag presentation assay is similar between ERAP1 SNPs (as reported in Ref. 19) and between the two
ERAP2 variants analyzed in this study. The accessory role of
ERAP2 may also present the immune system with an opportunity
for evolving new specificities that can modify the antigenic peptide repertoire without severely damaging Ag processing. However, because disease-related immune responses can be dominated
by a very small number of antigenic epitopes, it is possible that
even an accessory aminopeptidase activity, such as ERAP2, can be
dominant in some cases. Unfortunately, the lack of ERAP2 in
rodents (although ERAP2 has been hypothesized to have been
present in a primate-rodent common ancestor) has limited in vivo
experimentation efforts regarding the role of ERAP2 in adaptive
immunity in humans.
The Journal of Immunology
individuals, a variability that can complement the well-established
variability in Ag binding and presentation by MHC class I alleles.
We furthermore propose that ERAP2N and ERAP2K are treated
as distinct aminopeptidase activities in Ag processing when evaluating epitope generation in humans. The importance of polymorphic
variation in gene products that regulate the Ag processing and
presentation pathway is only now emerging as a key component of
the natural variability of the adaptive immune response. A systematic analysis of the functional consequences of these naturally
occurring polymorphisms in key components of this pathway can be
a valuable tool in both establishing diagnostic tools for disease
predisposition and for developing individualized immunotherapies.
Disclosures
The authors have no financial conflicts of interest.
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Modifications of enzyme activity or specificity by mutation can
be a useful evolutionary tool for altering metabolic or regulatory
pathways, and cancer cells have been shown to use this strategy
to gain growth advantages (53). In a notable example of such
a change in function, histone methyltransferase EZH2 mutants
increase histone methylation in human lymphomas (54). Similar
change-in-function mutations in components of the adaptive immune response may be the result of positive selection of individuals that present enhanced responses to specific pathogens
during human development. HIV resistance of individuals with
a Lys392/Lys392 ERAP2 phenotype could represent an example of
such positive selection. In this context, the continuously evolving
human adaptive immunity can enhance its polymorphic responses
to extend away from the well-established variability in MHC
binding specificity for antigenic peptides into cellular pathways
that are responsible for the generation of those peptides.
Although biochemical and structural analysis suggest a specific
mechanism of reduced activity of the ERAP2K allele through reduced stabilization of the N terminus of the peptide, it is less clear
how these effects lead to altered specificity. One possibility is that
the recognition of substrates by the S1 specificity pocket may be
different between the two alleles. ERAP2 recognizes in its S1
pocket positively charged side chains (specifically Lys and Arg)
by forming strong electrostatic interactions with residue Asp198
(15, 55). Comparison of the local environment of the S1 specificity pocket of the two ERAP2 variants shows changes in atomic
interactions that could affect the energetics of substrate catalysis:
1) the NZ-atom of the bound Lysa in ERAP2K is coming to a
closer proximity to Asp198 compared with ERAP2N by 0.7 Å; 2)
Glu177 assumes a different conformation, away from Lysa, interacting strongly via H-bonding (2.5 Å) with the carboxylic group
of Asp888, leading to a novel interaction with domain IV; and 3)
a water molecule is found, close to the previous location of the
side chain of Glu177, to interact with Asp198 and Lysa via H-bonds
(2.8 and 3.0 Å, respectively). Although the importance of Glu177–
Asp888 interaction is difficult to evaluate in terms of substrate
recognition or catalytic efficiency of the enzyme, it should be
noted that structural rearrangements between domains II and IV
have been demonstrated to be important for catalysis in the homologous ERAP1 (18). An alternative or complementary explanation may lie in the suboptimal recognition of hydrophobic side
chains by S1 pocket or ERAP2 that leads to unfavorable interactions (14). It is possible that these unfavorable interactions can
lead to altered transition-state stabilization in the case of ERAP2K
(in which the stabilization of the N terminus is already reduced),
making the enzyme more sensitive to optimal S1 pocket occupation and, as a result, more specific. Interestingly, ERAP2K is not
the only M1 aminopeptidase with a lysine at position 392; the
homologous enzyme aminopeptidase N also has a lysine residue at
that location, suggesting that this may be a more general strategy
for controlling the stringency of S1 specificity in this family of
aminopeptidases (50). The observation, however, that the changes
in specificity appear only for the larger, physiologically relevant
peptides and not for small, fluorogenic substrates suggests differences in the mechanism of recognition between the two types
of substrates. A similar phenomenon has also been described for
the highly homologous ERAP1 (17, 18).
In summary, we demonstrate that a common coding polymorphism in the Ag processing aminopeptidase ERAP2 significantly
alters its trimming function and provides atomic-level insights on the
mechanism behind this effect. The nature and magnitude of the
observed functional changes in combination with the wide genetic
distribution of this variation suggest that this polymorphic variation
constitutes an integral part of the variability of Ag processing in
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26.
AN ERAP2 SNP CHANGES THE SPECIFICITY OF THE ENZYME