Biochem. J. (2008) 416, 109–116 (Printed in Great Britain)
109
doi:10.1042/BJ20080965
Glutamine-181 is crucial in the enzymatic activity and substrate specificity
of human endoplasmic-reticulum aminopeptidase-1
Yoshikuni GOTO*, Hiroe TANJI*, Akira HATTORI† and Masafumi TSUJIMOTO*1
*Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan, and †Department of System Chemotherapy and Molecular Sciences,
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, 606-8501, Japan
ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a
multifunctional enzyme with roles in the regulation of blood
pressure, angiogenesis and the presentation of antigens to
MHC class I molecules. Whereas the enzyme shows restricted
specificity toward synthetic substrates, its substrate specificity
toward natural peptides is rather broad. Because of the
pathophysiological significance of ERAP-1, it is important to
elucidate the molecular basis of its enzymatic action. In the present
study we used site-directed mutagenesis to identify residues
affecting the substrate specificity of human ERAP-1 and identified
Gln181 as important for enzymatic activity and substrate specificity.
Replacement of Gln181 by aspartic acid resulted in a significant
change in substrate specificity, with Q181D ERAP-1 showing a
preference for basic amino acids. In addition, Q181D ERAP-1
cleaved natural peptides possessing a basic amino acid at the
N-terminal end more efficiently than did the wild-type enzyme,
whereas its cleavage of peptides with a non-basic amino acid
was significantly reduced. Another mutant enzyme, Q181E, also
revealed some preference for peptides with a basic N-terminal
amino acid, although it had little hydrolytic activity toward
the synthetic peptides tested. Other mutant enzymes, including
Q181N and Q181A ERAP-1s, revealed little enzymatic activity
toward synthetic or peptide substrates. These results indicate that
Gln181 is critical for the enzymatic activity and substrate specificity
of ERAP-1.
INTRODUCTION
and IL-1 type II receptor and to promote ectodomain shedding of
these receptors [3,16]. Because of its multifunctional properties,
ERAP-1 is also designated PILSAP (puromycin-insensitive
leucine-specific aminopeptidase) [17], ERAAP (ERAP associated
with antigen presentation) [11] and ARTS-1 (aminopeptidase
regulator of TNFR1 shedding) [16]. The data suggest that ERAP1 plays important roles in several pathophysiological processes,
such as immune and inflammatory responses, and is a potential
therapeutic target. To design a molecule targeting ERAP-1, it is
important to clarify its structural features and the molecular basis
of its action.
ERAP-1 is a monomeric zinc-metallopeptidase that shows a
preference for leucine when using synthetic substrates [18]. On
the other hand, it reveals relatively broad substrate specificity
toward natural peptide hormones and is shown to cleave several
hormones such as Ang II (angiotensin II), kallidin, neurokinin A
and neuromedin B [18]. Several antigenic peptides presented to
MHC class I molecules are also processed both in vitro and in vivo
by the enzyme [11,12]. On the basis of a preference for substrates
of a specific length and with a C-terminal hydrophobic amino acid,
the ‘molecular ruler’ mechanism was proposed for the processing
of antigenic peptides by the enzyme [19]. However, the structural
features and enzymatic mechanism of ERAP-1 remain elusive,
although several amino acid residues important for the enzymatic
activity of the M1 family of enzymes in general were identified
by mutational analysis [20,21].
In the present study we searched for an amino acid residue
affecting the enzymatic properties of human ERAP-1. We
Aminopeptidases hydrolyse the N-terminal amino acid of proteins
or peptide substrates. Among them, the M1 family of zinc
aminopeptidases (gluzincins) shares the consensus GAMEN and
HEXXH(X)18 E motifs essential for the enzymatic activity. This
family, which consists of 11 enzymes in humans [1–3], plays
important roles in several pathophysiological processes, such
as angiogenesis, cell-cycle regulation, reproduction, memory
retention, blood-pressure control and antigen presentation to
MHC class I molecules [4–12].
Previously we cloned a cDNA for P-LAP (placental leucine
aminopeptidase)/oxytocinase, a type II membrane-spanning
protein belonging to the M1 family [13]. Subsequently,
we cloned cDNAs encoding ERAP-1 [endoplasmic-reticulum
aminopeptidase-1; as A-LAP (adipocyte-derived leucine aminopeptidase)] and ERAP-2, which is also referred to as L-RAP
(leucocyte-derived arginine aminopeptidase) from adipocyte and
leucocyte cDNA libraries respectively by searching databases
[14,15]. Structural and phylogenetic analyses indicated that these
three enzymes are most closely related among the M1 family
and are now classified into “the oxytocinase subfamily of M1
aminopeptidases” [3].
ERAP-1 is a multifunctional aminopeptidase with roles in the
regulation of blood pressure, angiogenesis and the presentation of
antigens to MHC class I molecules [3,4]. Moreover, this enzyme
was shown to bind to cytokine receptors such as TNFR1 (tumournecrosis-factor type I receptor), IL-6α (interleukin 6 α) receptor
Key words: endoplasmic-reticulum aminopeptidase-1 (ERAP-1),
oxytocinase subfamily, placental leucine aminopeptidase,
site-directed mutagenesis, substrate specificity.
Abbreviations used: A-LAP, adipocyte-derived leucine aminopeptidase; Ang, angiotensin; APA, aminopeptidase A; ER, endoplasmic reticulum; ERAP,
ER aminopeptidase; HEK-293, human embryonic kidney-293; IL, interleukin; LTA4H, leukotriene A4 hydrolase; Leu-NA, L-leucine β-naphthylamide; Lys-NA,
L-lysine β-naphthylamide; L-RAP, leucocyte-derived arginine aminopeptidase; MCA, 4-methylcoumaryl-7-amide; P-LAP, placental leucine aminopeptidase;
TNFR1, tumour-necrosis-factor type I receptor.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
110
Y. Goto and others
identified Gln181 as a key residue for its enzymatic action and
substrate specificity. In particular, replacement of Gln181 by
aspartic acid caused a drastic change in the substrate specificity
of ERAP-1, which further suggested an evolutionary relationship
between ERAP-1 and ERAP-2 [15].
MATERIALS AND METHODS
Molecular modelling of ERAP-1
The published X-ray-crystallographic structure of human
LTA4H (leukotriene A4 hydrolase) [22] and of Thermoplasma
acidophilum TIFF3 (Tricorn-interacting factor F3) [23] were
used as templates for modelling the catalytic site of ERAP1 with the SWISS-MODEL Internet server (http://www.expasy.
org/swissmod/). The structure was displayed using the CueMol
program (http://cuemol.sourceforge.jp).
Site-directed mutagenesis
The cDNAs encoding mutant ERAP-1s were generated by twostep PCR. PCRs were carried out in 0.2-ml-volume tubes with
a 30 μl reaction volume. Sense oligonucleotide primers were
designed to introduce point mutations in ERAP-1 cDNA. First
PCRs were carried out for one cycle at 98 ◦C for 3.5 min, followed
by ten cycles at 95 ◦C for 0.5 min, 55 ◦C for 1 min, and 72 ◦C
for 2 min using Pyrobest DNA polymerase (TaKaRa, Ohtsu,
Japan). Sense primer A (5 -CACCAACCCTAAAAAACCGCCACCATGGTGTTTCTGCCC-3 ), containing a CACC sequence for directional cloning and initiation ATG codon, and
antisense primers complementary to the desired sequences,
were employed for the amplification of upstream fragments.
Downstream fragments with the sequence for hexahistidine tag
at the 3 -end were amplified using mutagenic sense primers
and primer B (5 -GACTGTCGACTTAGTGATGGTGATGGTGATGCATACGTTCAAGCTT-3 ).
The two products of the first PCR were used as templates for
the second PCR. Second PCRs were carried out with primers A
and B for one cycle at 98 ◦C for 3.5 min, followed by 25 cycles
at 95 ◦C for 0.5 min, 55 ◦C for 1 min, and 72 ◦C for 2 min. The
resultant products were inserted into the entry vector, pENTR-DTOPO, using a TOPO-cloning system (Invitrogen). The sequences
of the products were confirmed by automated sequencing on an
Applied Biosystems model 377 apparatus.
Expression and purification of recombinant wild-type and mutant
ERAP-1s in the baculovirus system
Human wild-type and mutant ERAP-1s with C-terminal
hexahistidine tags inserted into pENTR-D-TOPO were
transfected to the destination vector pDEST8 via Gateway LR
reactions (Gateway LR Clonase Reaction Mix; Invitrogen). The
pDEST8 vectors containing ERAP-1 cDNAs were transformed
to competent DH10bac Escherichia coli cells harbouring the
baculovirus genome (bacmid) and a transposition helper vector
(Invitrogen). Subsequently, insect Sf 9 (Spodoptera frugiperda
9) cells were transfected with recombinant bacmids using the
Cellfectin® reagent (Invitrogen). After a 3 day incubation period,
recombinant baculoviruses were isolated and used to infect Sf 9
cells at a multiplicity of infection of 0.1. At 3 days after infection,
the amplified viruses were harvested.
For the production of ERAP-1s, Sf 9 cells were grown at 27 ◦C
in 100 ml of Sf-900III medium (Invitrogen) and 1.5 × 106 cells/ml
were infected at a multiplicity of infection of 1–3. After 3 days,
c The Authors Journal compilation c 2008 Biochemical Society
the culture medium was collected after centrifugation at 5000 g
for 15 min.
The culture medium was loaded on to a hydroxyapatite
(Nacalai Tesque, Kyoto, Japan) column (bed volume 10 ml)
pre-equilibrated with 5 mM phosphate buffer, pH 7.5. After
extensive washing with the same buffer, ERAP-1 was eluted
from the column with 100 mM phosphate buffer, pH 7.5. The
eluate was then loaded on to a Co2+ -chelating Sepharose
(GE Healthcare) column (bed volume 1 ml) pre-equilibrated
with 10 mM phosphate buffer, pH 7.5, containing 0.1 M NaCl.
After extensive washing with 10 mM phosphate buffer, pH 7.5,
containing 0.1 M NaCl and 5 mM imidazole, ERAP-1 was eluted
with 10 mM phosphate buffer, pH 7.5, containing 0.1 M NaCl
and 100 mM imidazole. The ERAP-1-containing fractions were
extensively dialysed against 25 mM Tris/HCl buffer, pH 7.5,
containing 0.125 M NaCl, concentrated with an ultrafiltration
membrane and stored at − 20 ◦C prior to use.
Expression of wild-type and D198Q ERAP-2s in HEK-293 (human
embryonic kidney-293) cells
The full-length version of ERAP-2 or D198Q ERAP-2 with
a C-terminal hexahistidine tag was inserted into the pTargeT
vector (Promega). For transfection experiments, HEK-293 cells
plated in six-well plates were grown to subconfluency in
Dulbecco’s modified Eagle’s medium containing 10 % (v/v) horse
serum. The cells were then mock-transfected or transfected with
either pTargeT/ERAP-2 or pTargeT/D198Q ERAP-2, employing
FuGENE 6 Transfection Reagent (Roche) according to the
manufacturer’s instructions. After 72 h, culture media were
collected by centrifugation at 5000 g for 15 min. Aminopeptidase
activity in the media was measured as described previously [15].
Measurement of aminopeptidase activity of ERAP-1s
Aminopeptidase activities of wild-type and mutant ERAP-1s were
routinely determined by endpoint assay with various fluorogenic
amino acid MCAs (amino acid 4-methylcoumaryl-7-amides)
as substrates. The reaction mixture containing 100 μM amino
acid MCA and 2 μg/ml enzyme in 0.5 ml of 20 mM phosphate
buffer, pH 7.5, containing 10 μg/ml BSA was incubated at 37 ◦C
for 15 min. The reaction was terminated by adding 2.5 ml of
0.1 M sodium acetate buffer, pH 4.3, containing 0.1 M sodium
monochloroacetate. The amount of 7-amino-4-methylcoumarin
released was measured by spectrofluorophotometry (F-2000
instrument; Hitachi) at an excitation wavelength of 360 nm and
an emission wavelength of 460 nm and is given in arbitrary units.
To determine the kinetic parameters, we used the synthetic
fluorogenic substrates Leu-NA (L-leucine β-naphthylamide)
and Lys-NA (L-lysine β-naphthylamide). The reaction mixture
containing various concentrations of substrates and either
wild-type or mutant ERAP-1 in 500 μl of 20 mM phosphate
buffer, pH 7.5, containing 10 μg/ml BSA, was incubated at
37 ◦C for 5 min. The amount of β-naphthylamine released was
then measured by spectrofluorophotometry (F-2000 instrument;
Hitachi) at an excitation wavelength of 335 nm and an emission
wavelength of 410 nm. The kinetic parameters (K m and V max ) were
calculated using CurveExpert 1.3 (http://curveexpert.webhop.
net/) to fit the data directly to the Michaelis–Menten equation.
Cleavage of peptide hormones by recombinant ERAP-1s
Peptide hormones (Peptide Institute, Osaka, Japan; 25 μM
each) were incubated with either wild-type or mutant ERAP-1
Site-directed mutagenesis of endoplasmic-reticulum aminopeptidase-1
Figure 1
111
Molecular modelling of the catalytic pocket of ERAP-1
(A) Modelling of the catalytic site of ERAP-1 using human LTA4H and Thermoplasma acidophilum TIFF3 as templates. Glu320 in the GAMEN motif, His353 , Glu354 , His357 and Glu376 in the HEXXH(X)18 E
motif and Tyr438 are shown as residues in the catalytic site of the enzyme. (B) Alignment of the ERAP-1 amino acid sequence with the sequences of APA [30,31] and other oxytocinase-subfamily
enzymes P-LAP [13] and ERAP-2 [15]. Gaps are inserted into the sequences for optimal alignment. Residues conserved among the enzymes are shaded. Gln181 of ERAP-1 and the corresponding
residue of P-LAP are shown as a white Q on a black background ( ).
(1–4 μg/ml) at 37 ◦C in 20 mM phosphate buffer, pH 7.5,
containing 10 μg/ml BSA. The reaction was terminated by
addition of 2.5 % (v/v) formic acid. The peptides generated
were separated by reverse-phase HPLC on a COSMOSIL
(4.6 mm internal diameter × 250 mm long) column (Nacalai
Tesque) using a Shimadzu HPLC system (AT-10) at a flow
rate of 0.5 ml/min. Peptides generated from Ang II–Ang IV
were eluted isocratically with 19 % (v/v) acetonitrile in 0.09 %
trifluoroacetic acid. Peptides generated from kallidin (Lysbradykinin), Leu-bradykinin, HIV gag 11 and HIV pol 11 antigen
precursors were eluted isocratically with either 22.5 or 17.5 %
acetonitrile in 0.09 % trifluoroacetic acid. The molecular masses
of peptides were determined by MALDI-TOF (matrix-assisted
laser-desorption-ionization–time-of-flight) MS with a REFLEX
mass spectrometer (Bruker-Franzen Analytik), using α-cyano-4hydroxycinnamic acid as the matrix.
Materials
Asp-MCA (aspartic acid 4-methylcoumaryl-7-amide), Gln-MCA
(glutamine 4-methylcoumaryl-7-amide), Bzl-Cys-MCA (benzylcysteine 4-methylcoumaryl-7-amide) and Glu-MCA (glutamic
acid 4-methylcoumaryl-7-amide) and Leu- and Lys-NAs were
purchased from Bachem AG (Bubendorf, Switzerland). Ala-,
Arg-, Leu-, Lys-, Met- and Phe-MCAs and all peptide hormones
were obtained from The Peptide Institute (Osaka, Japan).
Amastatin was purchased from Sigma. Leu-bradykinin, HIV gag
11 and HIV pol 11 antigen precursors were synthesized by RIKEN
Brain Science Institute, Saitama, Japan.
RESULTS
Modelling of the catalytic pocket of ERAP-1
Molecular modelling is a useful way in which to elucidate
the structural features of ERAP-1 in the absence of its crystal
structure, making it possible to identify residues critical to
enzymatic action. The crystal structures for LTA4H and TIFF3
were resolved [22,23], with human LTA4H and Thermoplasma
acidophilum TIFF3 showing 11.8 and 22.2 % overall similarity
to ERAP-1 respectively. Notably, the sequence similarity around
the catalytic pockets of ERAP-1 is 25 and 40 % respectively,
allowing us to model the structure of the catalytic pocket of ERAP1 using these two enzymes as templates (Figure 1A). Molecular
modelling of the catalytic pocket of ERAP-1 suggests that, as
with Asp221 of human APA (aminopeptidase A), a residue that
is critical to the calcium-induced modulation of APA enzymatic
activity [24], Gln181 is located near the catalytic HEXXH(X)18 E
motif and at a possible S1 site of the enzyme. Alignment of
APA and human-oxytocinase-subfamily enzymes indicates that
Gln181 of ERAP-1 occupies the corresponding site, Asp221 , of
APA, further suggesting the importance of this residue for the
enzymatic activity (Figure 1B). We therefore examined the role
of this residue in the enzymatic activity of ERAP-1.
To elucidate the significance of Gln181 to the enzymatic activity
of ERAP-1, wild-type and mutant ERAP-1s carrying a single
substitution of this residue (i.e. Q181E, Q181N, Q181D and
Q181A ERAP-1s) were transiently expressed in a baculovirus
system and purified to homogeneity by SDS/PAGE. All the
enzymes showed a single band with an apparent molecular mass
of ∼105 kDa under reducing conditions (results not shown).
Characterization of the hydrolytic activities of wild-type and mutant
ERAP-1s towards synthetic substrates
Figure 2 shows the enzymatic activity of wild-type and
mutant ERAP-1s towards various synthetic substrates. As
shown previously [14], the wild-type enzyme cleaved Leu-MCA
preferentially, followed by Met-MCA, indicating a restricted
substrate specificity toward synthetic peptides. On the other hand,
the mutant enzymes revealed little activity toward Leu- and
c The Authors Journal compilation c 2008 Biochemical Society
112
Figure 2
Y. Goto and others
Enzymatic activities of wild-type and mutant ERAP-1s toward synthetic substrates
Purified enzymes (2 μg/ml) were incubated with various amino acid MCA substrates (100 μM) at 37 ◦C for 15 min. Relative activities of the enzymes are shown, taking the hydrolytic activity of
wild-type enzyme toward Leu-MCA as 100 %. Each bar shows the mean +
− S.D. (n = 3).
Met-MCAs, indicating that glutamine was the most favourable
residue at position 181 of ERAP-1 for the hydrolysis of these two
substrates.
When the substrate specificity of Q181D ERAP-1 was
examined, an apparent preference for basic amino acids (i.e. Lysand Arg-MCAs) was observed. It should be noted here that only
Q181D ERAP-1 revealed substantial activity toward basic amino
acids, and that other mutant enzymes, including Q181E ERAP1, had little activity towards any synthetic substrates tested (see
below). These results indicate that Gln181 is important for the
enzymatic activity as well as for the substrate specificity of ERAP1 towards synthetic substrates.
Table 1 shows the kinetic parameters of the wild-type and
mutant ERAP-1s using Leu- and Lys-NA as substrates. We could
not obtain parameters by using conventional amino acid MCAs,
because higher concentrations of amino acid MCAs tended to
inhibit the enzymatic activity of ERAP-1. All mutant enzymes
revealed reduced hydrolytic activity toward Leu-NA as compared
with the wild-type enzyme. An increase in K m together with a
decrease in kcat caused a significant reduction in the enzymatic
activity of mutant enzymes toward Leu-NA. When Lys-NA was
employed as a substrate, only Q181D revealed considerable
activity. The affinity of Leu-NA for wild-type ERAP-1 was 8.4fold that for the Q181D mutant and comparable with that of LysNA for the mutant, suggesting a conformational change of the
substrate pocket caused by the replacement of Gln181 by aspartic
acid.
Characterization of the hydrolytic activities of wild-type and mutant
ERAP-1s toward peptide substrates
We next compared the hydrolytic activities of the wild-type
and mutant ERAP-1s toward several natural peptides. Figure 3
shows the cleavage of Ang II–Ang IV by the wild-type, Q181D
and Q181E ERAP-1s. The wild-type enzyme cleaved the N
c The Authors Journal compilation c 2008 Biochemical Society
Table 1 Kinetic parameters of wild-type and mutant ERAP-1s toward
synthetic substrates
Kinetic parameters were determined from the Michaelis–Menten equation. Reactions took place
at 37 ◦C for 5 min. Values are means +
− S.D. (n = 3). Relative k cat /K m values are shown taking
Leu-NA hydrolytic activity of the wild-type enzyme as 100 %.
k cat /K m
Substrate/enzyme
Leu-NA
Wild-type
Q181E
Q181D
Q181N
Q181A
Lys-NA
Wild-type
Q181E
Q181D
Q181N
Q181A
K m (μM)
k cat (s−1 )
(mM−1 · s−1 )
250 +
− 41
1800 +
− 580
2100 +
− 790
2400 +
− 140
3100 +
− 780
19 +
− 1.6
6.8 +
− 1.6
1.9 +
− 0.65
6.2 +
− 1.5
0.52 +
− 0.044
75 +
− 5.6
3.8 +
− 0.27
0.93 +
− 0.042
2.6 +
− 0.59
0.18 +
− 0.062
100
5.1
1.2
3.5
0.20
650 +
− 98
320 +
− 50
320 +
− 42
870 +
− 93
1100 +
− 480
3.6 +
− 0.22
1.9 +
− 0.090
8.2 +
− 0.26
0.64 +
− 0.046
0.10 +
− 0.023
5.6 +
− 0.68
5.9 +
− 0.72
26 +
− 2.5
0.74 +
− 0.030
0.097 +
− 0.023
7.5
7.9
35
1.0
0.13
(%)
terminal amino acid of both Ang II and Ang IV sequentially,
and degradation products were clearly detected within 30 min
(Figures 3A and 3C). By contrast, the Q181D and Q181E ERAP1s showed little activity toward these two hormones. Other mutant
enzymes (i.e. Q181N and Q181A) also had little activity (results
not shown), suggesting that Gln181 of ERAP-1 was required for
the maximal enzymatic activity toward these two hormones.
When Ang III was employed as a substrate (Figure 3B),
sequential release of the N-terminal amino acid from Ang III
to des-[Val-Tyr]-Ang IV by the wild-type enzyme was observed.
The conversion of Ang III into Ang IV by Q181D ERAP-1 was
comparable with that by the wild-type enzyme, and consistent
Site-directed mutagenesis of endoplasmic-reticulum aminopeptidase-1
Figure 3
113
Cleavage of Ang II, Ang III and Ang IV by wild-type, Q181D and Q181E ERAP-1s
Ang II (25 μM) (A), Ang III (25 μM) (B) or Ang IV (25 μM) (C) was incubated with purified enzymes (4 μg/ml) at 37 ◦C for 60 min. The peptides generated were loaded on to an HPLC column and
separated, and their amino acid sequences were determined. Abbreviation: mAU, milli-absorbance unit.
with its preference for basic amino acids. Further degradation
to des-[Val]-Ang IV was barely detectable. In addition, Q181E
ERAP-1 also revealed substantial activity to convert Ang III
into Ang IV. This result indicates that, although Q181E ERAP1 showed little hydrolytic activity toward synthetic substrates,
it retained enzymatic activity toward peptide substrates with an
N-terminal basic amino acid.
To characterize further the N-terminal preference of the wildtype, Q181D and Q181E ERAP-1s, we compared the rate of
cleavage of the N-terminal residue from kallidin (Lys-bradykinin)
and Leu-bradykinin. Because of proline at the second last Nterminal position, we could examine the role of the N-terminal
amino acid in the hydrolytic activity of the enzyme directly. As
shown in Figures 4(A) and 4(B), whereas the wild-type enzyme
cleaved Leu-bradykinin much more quickly than did the Q181D
and Q181E ERAP-1s, its cleavage rate for kallidin was rather
low when compared with that of the mutant enzymes. The wildtype enzyme converted 85 % of Leu-bradykinin into bradykinin
within 5 min in our assay system, but only 33 % of kallidin to
bradykinin, again supporting its preference for non-basic amino
acids. By contrast, the Q181D and Q181E mutants cleaved
kallidin (65 and 45 % conversion respectively) more quickly
than Leu-bradykinin (40 and 35 % conversion respectively).
Therefore, although the data do not precisely reflect the substrate
specificity of the enzymes toward synthetic substrates, it is still
possible to conclude that, although the wild-type enzyme ‘prefers’
non-basic amino acids, the Q181D and Q181E ERAP-1s show a
preference for basic amino acids. In addition, the results also
suggest that dependent only on the N-terminal amino acid of the
substrates, the amino acid at position 181 mediates the hydrolytic
efficiency, and thus determines the substrate specificity, of
ERAP-1.
It is well established that ERAP-1 is the final processing enzyme
of antigenic peptides presented to MHC class I molecules in the
ER [3,11,12]. Therefore, we next examined the cleavage of Nextended precursors of HIV gag 11 and HIV pol 11 antigens to
compare further the hydrolytic activity and substrate specificity
of the wild-type and mutant ERAP-1s. As shown in Figure 4(C),
whereas ERAP-1 removed 49 % of the N-terminal leucine from
the HIV gag antigen precursor within 5 min, only 7 and 11 % was
released by Q181D and Q181E ERAP-1s respectively, indicating
that, as in the case of synthetic substrates, the wild-type enzyme
cleaved N-terminal leucine much more efficiently than did the
mutant enzymes. By contrast, when the HIV pol antigen precursor,
which has arginine at its N-terminal end, was employed, both
mutants cleaved the arginine residue more efficiently than did the
wild-type enzyme (Figure 4D). These results again indicate that
the Q181D and Q181E ERAP-1s cleave N-terminal basic amino
acid residues more efficiently than the wild-type enzyme and that
replacement of Gln181 by aspartic acid or glutamic acid causes
a change of substrate specificity to a preference for basic amino
acids. Taken together, these results suggest that Gln181 of ERAP-1
is crucial in the processing of peptide hormones and antigenic
peptides by determining preferential substrates of the enzyme
and thus mediating various pathophysiological functions of the
enzyme.
c The Authors Journal compilation c 2008 Biochemical Society
114
Y. Goto and others
Figure 5 Effects of Zn2+ and amastatin on the enzymatic activity of wild-type
and Q181D ERAP-1s
Purified wild-type ERAP-1 and Q181D ERAP-1 (2 μg/ml) were incubated with various
concentrations of ZnCl2 (A) or amastatin (B) on ice for 5 min. Aminopeptidase activities were
then measured by using Leu-MCA (100 μM) for the wild-type and Lys-MCA (100 μM) for
Q181D ERAP-1.
K i values of amastatin for the wild-type and Q181D ERAP1s were 41.8 +
− 46 μM respectively. Considering
− 10.3 and 168 +
that amastatin [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoylVal-Val-Asp] is a small peptide having an amino-acid-like residue
at its N-terminus, these results are consistent with the notion that
wild-type ERAP-1 binds to non-basic amino acids with higher
affinity than does the Q181D mutant enzyme.
Figure 4 Cleavage of various substrate peptides by wild-type, Q181D and
Q181E ERAP-1s
Leu-bradykinin (25 μM) (A), kallidin (Lys-bradykinin) (25 μM) (B) or the precursor of HIV
gag antigen (25 μM) (C) was incubated with purified ERAP-1s (2 μg/ml) at 37 ◦C for
5 min. The precursor of HIV pol antigen (25 μM) (D) was incubated with purified enzymes
(2 μg/ml) at 37 ◦C for 60 min. Peptides in the reaction mixture were then separated by
HPLC and their amino acid sequences were determined. Abbreviation: mAU, milli-absorbance
unit.
Effects of aminopeptidase inhibitors on the enzymatic activities of
wild-type and Q181D ERAP-1s
We then compared the effects of Zn2+ and amastatin, both wellknown inhibitors of M1 aminopeptidases, on the enzymatic
activity of the wild-type and Q181D ERAP-1s. As shown in
Figure 5(A), Zn2+ inhibited the enzymatic activities of both
enzymes equally, suggesting that Zn2+ interfered with hydrolytic
processes shared by the M1 family members. On the other
hand, amastatin was about 50-fold less effective towards Q181D
ERAP-1 than towards the wild-type enzyme (Figure 5B). The
c The Authors Journal compilation c 2008 Biochemical Society
Comparison of substrate specificity between the wild-type and
D198Q ERAP-2s
As shown in Figure 1(B), Asp198 of ERAP-2 corresponds to
Gln181 of ERAP-1. To obtain further insight into the role of
these residues in the enzymatic properties, we compared the
substrate specificity of the wild-type and D198Q ERAP-2s toward
synthetic substrates. The enzymes were transiently overexpressed
in HEK-293 cells and secreted into culture medium. Westernblot analysis revealed that the secreted enzymes had molecular
masses of ∼120 kDa (Figure 6A). As shown in Figure 6(B),
whereas wild-type ERAP-2 preferentially cleaved basic amino
acids, as reported previously [15], D198Q ERAP-2 cleaved LeuMCA most efficiently, followed by Met-MCA. In addition, the
mutant enzyme still retained a substantial activity toward (S)-BzlCys-, Arg-, and Lys-MCAs. These results indicate that Asp198 of
ERAP-2 also affects the enzymatic properties of ERAP-2 and
that the substrate specificity of D198Q ERAP-2 towards synthetic
substrates is broad and similar to that of P-LAP rather than ERAP1 [14,25].
Site-directed mutagenesis of endoplasmic-reticulum aminopeptidase-1
115
DISCUSSION
Figure 6 Substrate specificity of wild-type and D198Q ERAP-2s towards
synthetic peptides
HEK-293 cells transiently mock-transfected and transfected with either wild-type or mutant
ERAP-2 were incubated at 37 ◦C for 72 h. The culture medium was then collected and the
cells were incubated further with various amino acid MCA substrates (100 μM) to assess
aminopeptidase activity. (A) Western-blot analysis of wild-type and D198Q ERAP-2s expressed
in HEK-293 cells. Enzymes secreted into medium were detected by using rabbit polyclonal
anti-ERAP-2 antibody. (B) Protein levels of ERAP-2s for the aminopeptidase assay was adjusted
by Western-blot analysis. Differences in activity between either wild-type or mutant ERAP-2
and the mock-transfected HEK-293 cell culture medium are shown as relative values, taking the
wild-type enzyme activity toward Arg-MCA as 100 %. Each bar shows the mean +
− S.D. (n = 3).
Figure 7
Because of their pathophysiological significance, it is important
to elucidate the structural and enzymatic features of the M1 family
of aminopeptidases, including ERAP-1. In the present study we
identified Gln181 as a residue critical to the enzymatic activity and
substrate specificity of ERAP-1. Although Gln181 of ERAP-1 was
required for maximal enzymatic activity toward peptide substrates
having an N-terminal non-basic amino acid, replacement of this
residue with aspartic acid increased the enzyme’s preference for
basic amino acids. This replacement might cause rather restricted
substrate specificity through electrostatic interaction. Cleavage of
peptides with an N-terminal basic amino acid by Q181E ERAP-1
also supported a role for electrostatic interaction between Glu181
and the N-terminal basic amino acid of the substrates. Because of
the length of the side chain carrying a negative charge at position
181, Q181E ERAP-1 might be less active towards the N-terminal
basic amino acid of the substrates than Q181D ERAP-1. Thus
it is plausible that the length, as well as uncharged nature, of
the side chain of Gln181 is critical for the enzymatic properties
of ERAP-1. Considering that ERAP-1 is the final processing
enzyme of antigenic peptides presented to MHC class I molecules,
we speculate that Gln181 has been selected during evolution to
maintain a rather broad specificity of the enzyme toward peptide
substrates.
Figure 7 shows a possible mechanism for the enzymatic action
of ERAP-1. It is apparent that Gln181 is involved in the cleavage of
the N-terminal amino acid of the substrates. However, because it is
unlikely that Gln181 interacts with this amino acid (such as leucine)
directly, we speculate that it is involved in the maintenance of the
catalytic pocket’s structure by bridging with another unidentified
residue and thus contributes to the formation of the S1 site.
Replacement of Gln181 by the acidic amino acids aspartic acid
or glutamic acid might cause a local conformational change of
the substrate pocket, resulting in a rather restricted substrate
specificity through electrostatic interaction between the residue
and N-terminal basic amino acid of the substrates. To elucidate
the molecular basis of the role of this residue in the enzymatic
activity of ERAP-1 definitively, it is essential to determine the
crystal structure of the enzyme.
Phylogenetic analysis suggests an evolutionary relationship
and the recent divergence of ERAP-1 and ERAP-2 from P-LAP
Schematic representation of the catalytic pockets of wild-type and Q181D ERAP-1s
Position 181 of either the wild-type or mutant enzyme presumably acts as the S1 site. Gln181 of the wild-type enzyme contributes to the formation of the catalytic pocket of the enzyme by bridging
with another residue, which causes the broad substrate specificity. Replacement of this residue by aspartic acid may induce a conformational change in the pocket. Through electrostatic interaction,
the mutant enzyme shows a preference for a basic N-terminal amino acid.
c The Authors Journal compilation c 2008 Biochemical Society
116
Y. Goto and others
[3,15]. In fact, the human genes for the oxytocinase subfamily
are located contiguously around chromosome 5q15, and the gene
structure of each member is similar, with exon–intron junctions
well conserved [15,26–28].
One of the most characteristic features of ERAP-2 is a
preference for basic amino acids [15], implying that replacement
of Gln181 by aspartic acid in ERAP-1 resulted in a substrate
specificity similar to that of ERAP-2. It is possible that ERAP-2
acquired its preference for basic amino acids during evolution and
thus expanded the repertoire of antigenic peptides processed in the
ER. Complementary and/or co-operative actions of two enzymes
were reported in the processing of antigenic peptides presented
to MHC class I molecules in the ER [29]. In this context, the
substrate specificity of D198Q ERAP-2 is rather broad and similar
to that of P-LAP. Since glutamine occupies the corresponding
site of both P-LAP and ERAP-1, which cleave Leu-MCA most
efficiently of the synthetic substrates tested, it is most plausible
that glutamine at this site is required for the efficient cleavage
of the substrate. However, considering the difference in substrate
specificity between P-LAP and ERAP-1 [14,25], other residue(s)
may be involved. Taken together, our data suggest that Gln181
of ERAP-1 and the corresponding sites of ERAP-2 and P-LAP
are crucial to the enzymatic activity and substrate specificity, and
further confirm the evolutionarily close relationship between the
members of the oxytocinase subfamily.
In the present study we have shown that Gln181 is important for
the enzymatic activity and substrate specificity of ERAP-1. Both
Q181D ERAP-1 and ERAP-2 with an aspartic acid residue at this
site show a preference for basic amino acids, which might reflect
the evolutionary relationship between ERAP-1 and ERAP-2. Our
data should be useful in clarifying the enzymatic mechanism
of ERAP-1 and ERAP-2 and in developing therapeutic reagents
targeting these enzymes.
This work was supported in part by grants-in-aid from the Ministry of Education, Science,
Sports and Culture of Japan and by a Chemical Biology Research Program grant from
RIKEN.
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