Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Article abstract of DOI:10.1134/S1068162008030126

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of complexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 ± 0.4 and 2.5 ± 0.4 nM, respectively, for recombinant tACE and 4.7 ± 0.5 and 1.6 ± 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

Full text of DOI:10.1134/S1068162008030126

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 3, pp. 323–328. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © I.A.Naperova, I.V. Balyasnikova, M.N. Petrov, A.V. Vakhitova, V.V. Evdokimov, S.M. Danilov, O.A. Kost, 2008, published in Bioorganicheskaya Khimiya,
2008, Vol. 34, No. 3, pp. 358–364.
Characteristics of Monoclonal Antibody Binding
with the C Domain of Human Angiotensin Converting Enzyme
I. A. Naperovaa, I. V. Balyasnikovab, M. N. Petrova, A. V. Vakhitovaa, V. V. Evdokimovc,
S. M. Danilovb, and O. A. Kosta,1
a Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
b Faculty of Anesthesiology, Illinois University, 108 Henry Administration Bldg, Urbana, IL 61801, Chicago, Unites States
c Research Institute of Urology, Rosmedtekhnology, Moscow
Received July 3, 2007; in final form, November 16, 2007
Abstract—Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin con-
verting enzyme (ACE) to human testicularACE (tACE) (corresponding to the C domain of the somatic enzyme)
was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of com-
plexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 0.4 and 2.5 0.4 nM, respectively, for recombinant
tACE and 4.7 0.5 and 1.6 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with
tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding
epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram dem-
onstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human
and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and
Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616
located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas
mAbs 1B8 and 3F10 contain Pro730.
Key words: angiotensin converting enzyme, C domain, monoclonal antibodies
DOI: 10.1134/S1068162008030126
INTRODUCTION
Localization of antibody binding epitopes on the
ACE surface would provide identification of new func-
tional regions of the enzyme molecule. Determination
of binding of sheep polyclonal antibodies to human
somatic ACE using potential linear antigen determi-
nants (synthetic linear hexapeptides) showed that about
70% of these peptides actually comprise antibody bind-
Angiotensin converting enzyme (ACE, peptidyl
dipeptidase, EC 3.4.15.1), a Zn2+ dependent metal-
lopeptidase belonging to the class of zincins, is one of
the major regulators of blood pressure and content of
vasoactive peptides in organism.2 This enzyme is also
involved in neuropeptide metabolism and immune and ing epitopes and are located on the surfaces of both C
reproductive functioning [1, 2].
and N domains of ACE protein globule, sometimes giv-
ing clusters [5]. Using monoclonal antibodies, a region
on the ACE N domain surface responsible for the ACE
carbohydrate-controlled dimerization, which may be
related to ACE proteolytic cleavage from the cell sur-
face, was determined [6]. Determination of binding
epitopes for two mAbs to theACE N domain along with
available kinetic data provided the demonstration of
conformational changes in the enzyme molecule upon
the ligand (substrate or ligand) inhibitor binding [7].
The knowledge of binding epitopes for some mAbs
allowed the development of detection methods of
amino acid residue mutations in the perimembrane
enzyme region, which lead to a considerable increase in
ACE shedding from the cell surface [8, 9]. In addition,
mAbs to human ACE recognizing conformational
epitopes on the surface of the ACE N domain and
Two ACE isoforms are synthesized in mammalian
cells. The ACE somatic form is composed of two
homologous domains (N and C) within a single
polypeptide chain, each of which contains a catalytic
active site. A shorter testicular ACE form (tACE) syn-
thesized in testicles corresponds to the C domain (with
exception of 36 aa from the N terminus). Now crystal
structures of single domains have been published [3, 4],
but the structure of a full size enzyme remains
unknown.
1
Corresponding author; phone: +7 (495) 939-3417; fax: +7 (495)
939-5417; e-mail: kost@enzyme.chem.msu.ru
Abbreviations: ACE, angiotensin converting enzyme; tACE, tes-
ticular ACE; mAb, monoclonal antibody; ELISA, enzyme-linked
immunosorbent assay; Hip, hippuryl.
2
323

324
NAPEROVA et al.
Fluorescence, relative units
mulation. The reaction time was taken, so that the
1.4
hydrolysis percentage did not exceed 10% and fluores-
cence values exceeded the background five times or
more.
1.2
1.0
0.8
0.6
0.4
0.2
1
2
The curves showing the dependence of tACE com-
plex formation with the immobilized mAb 1E10 on the
enzyme amount at various mAb concentrations are
given in Fig. 1. One can see that the curves correspond-
ing to the selected mAb concentrations have a quasi lin-
ear region up to the enzyme concentration of
40 mU/ml, which corresponds to 0.3 µg/ml. In further
experiments, both tACE preparations were used within
the range of 5–20 mU/ml (0.04–0.15 µg/ml).
3
The mAb concentration of 3 µg/ml was chosen for
further studies, because, in this case, the fluorescence
signal was rather intensive and considerably (by 10–
20 times) exceeded that of the background. This con-
centration also helped to avoid additional consumption
of antibodies (10 µg/ml versus 3 µg/ml, practically,
threefold difference). Since it turned out that mAb
1E10 is relatively weakly bound to tACE (see below),
the conditions chosen for these antibodies were also
suitable for other mAbs with the same or better binding
to the enzyme.
0
20
40
60
80
100
Activity of tACE, mU/ml
Fig.1. Dependence of tACE complex formation with mAb
1E10 on the concentration of the enzyme at mAb concentra-
tions (1) 10, (2) 3, and (3) 1 µg/ml. Antibodies 1A10 were
bound to the polyclonal antibodies to mouse immunoglob-
ulins preliminarily sorbed in microplate wells and the
enzyme at varied concentrations was added into the wells.
The mAb–tACE complex formation was monitored by
measuring the ACE enzymatic activity.
Analysis of mAb Binding to tACE
sequential epitopes on the denatured C domain are suc-
cessfully used for the quantitative determination of
both ACE forms [10] and enzyme inhibitors [11] in
solution using ELISA, as well as ofACE on the cell sur-
face using flow cytometry [12].
Whereas the epitope mapping of the ACE N domain
has been rather complete [7, 11, 13, 14], binding
epitopes for only two mAbs, 1B3 and 5C8, recognizing
C domain, have been found so far [9], the mAb 5C8
binding only to the denatured enzyme.
Relative binding of mAb to ACE C domain was
evaluated by determination of activities of tACEs (both
recombinant and spermatozoid) bound to various plate-
adsorbed mAbs. It turned out that fluorescence values
obtained upon tACE binding to mAbs 1B3, 1E10,
3F11, and 4E3 were significantly lower than those
bound to the 1B8, 2B11, 2H9, and 3F10 mAbs (Fig. 2).
A relatively low fluorescence signal of ACE binding to
the first mAb group could result from either lower bind-
ing constants to these mAbs or antibody-induced inhi-
bition of tACE activity.
With the goal to evaluate the reason for a low fluo-
rescence signal upon tACE binding to some antibodies,
we analyzed 1B3, 1E10, 3F11, and 4E3 mAbs in solu-
tion for the inhibitory activity toward tACE. It turned
out that a considerable excess of 4E3 antibodies
(mAb/tACE 4E3 = 300/1) decreased the tACE activity
nearly by 40%, whereas a similar excess of other anti-
bodies did not affect the enzymatic activity. Thus, we
can presume that monoclonal 4E3 antibodies display
small inhibitory activity and all mAbs of this group are
characterized by relatively low binding constants to
tACE, antibodies 1B8, 2B11, 2H9, and 3F10 being
more tightly bound to the enzyme.
We describe in this work a comparative study of
binding of a panel of eight mAbs to tACE and the iden-
tification of some amino acid residues of the enzyme
critical for binding.
RESULTS AND DISCUSSION
A comparison of binding of various mAbs to tACE
was achieved by immunoprecipitation. For optimiza-
tion of conditions of all further experiments, we used
spermatozoid tACE and mAb 1E10.
For immunoprecipitation, polyclonal antibodies to
mouse immunoglobulins, then mAbs, and finally tACE
were successively adsorbed in microplate wells. In this
case, we chose the concentration of polyclonal antibod-
ies of 10 µg/ml, which was enough for saturation of the
plate wells [14]; mAb and tACE concentrations were
varied. The amount of the resulting mAb–tACE com-
plex in microplate wells was determined by the enzy-
matic activity of bound ACE. As a substrate, Hip-His-
We determined dissociation constants of the com-
plexes of two effectively binding mAbs 1B8 and 2H9
with both tACE preparations using immunoprecipita-
tion.
For mAbs 1B8 and 2H9, the obtained values were
Leu was used; its hydrolysis rate was determined fluo- 4.7 0.5 and 1.6 0.3 nM for spermatozoid and 2.3
rimetrically according to the product (His-Leu) accu- 0.4 and 2.5 0.4 nM for recombinant tACE, respec-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

CHARACTERISTICS OF MONOCLONAL ANTIBODY
325
Fluorescence, relative units
tively. The difference in dissociation constants of the
mAb complexes with two tACE preparations could be
determined by both differences in glycosylation of the
native and recombinant forms and a possibility of keep-
ing the hydrophobic anchor in the native spermatozoid
tACE.
(a)
1.2
1.0
0.8
0.6
0.4
0.2
Competition of Monoclonal Antibodies
for Binding to tACE
We evaluated the binding of tACE–competing anti-
body complexes to the tested antibodies by ELISA to
analyze the mAb capacity to compete with each other
for tACE binding. If the tACE–competing antibody
complex binds to the tested antibodies with the effi-
ciency close to that of the free enzyme, binding
epitopes for the two antibodies do not overlap. In the
case if the tACE–competing antibody complex does not
bind to the tested antibodies, one can claim that the
binding epitopes for these antibodies are overlapped to
a large degree. If the binding of the tACE–competing
antibody complex to the tested antibodies is less effec-
tive than that of the free enzyme, one can propose that
the binding epitopes for these antibodies are partially
overlapped.
We believe that epitopes of two antibodies consider-
ably overlap if the formation of tACE–competing anti-
body complex is inhibited by more than 75%; i.e., the
overlapping degree of these epitopes exceeds 75%. The
inhibition of tACE binding lower than 25% implied
weak competition of two antibodies. If a degree of
binding inhibition was lower 10%, we considered it
within an experimental error and believed that mAb did
not compete for binding to tACE.
0
(b)
1.2
1.0
0.8
0.6
0.4
0.2
0
Monoclonal antibodies
We determined the capacity to compete with each
other for binding to tACE for each mAb pair; both
mAbs from each pair were used both as competing and
tested. The obtained results are shown in Fig. 3‡.
Fig. 2. Relative binding of (a) recombinant and (b) sperma-
tozoid tACE to the mAb against the ACE C domain.
bound to these enzymes practically with the same effi-
ciency. The analysis of amino acid sequences of human
and chimpanzee ACE showed that their C domains dif-
fer by three amino acid residues, Lys677, Pro730, and
Gly1269, with Gly1269 being located in the transmem-
brane region (Fig. 4b). Since we used in this work
human and chimpanzee enzymes lacking a membrane
anchor, the Gly1269 residue was not involved in the
complex formation with the tested mAb. Thus, only
two amino acid residues differing in human and chim-
panzee ACE C domains (Lys677 and Pro730) are
responsible for binding of three mAbs: 1B8, 3F10, and
1E10.
An analysis of the data obtained resulted in a dia-
gram clearly demonstrating competitive and noncom-
petitive mAb–tACE binding (Fig. 3b). Based on the
obtained results, we can state that there are three immu-
nologically important regions on the ACE C domain
surface, where mAb epitopes are located. One region
contains five mAb binding epitopes (1E10, 2B11, 2H9,
3F11, and 4E3), the second contains two (1A8 and
3F10), and the third one, one mAb (1B3). At the same
time, a considerable (mAb 1E10 and 4E3, 1B8 and
3F10) or partial (mAb 1E10 and 2B11, 3F11 and 2H9)
competition occurs within the groups for binding to
ACE C domain.
An analysis of the surface of human ACE C domain
([3], PDB: 1O8A) showed that Lys677 and Pro730 are
located at the opposite sides of the enzyme globule. The
binding epitopes for 1B8 and 3F10 antibodies are
Comparative Binding of Monoclonal Antibodies to
Human and Chimpanzee ACE
A comparison of mAb binding to somaticACE from nearly completely overlapped, whereas mAb 1E10
human and chimpanzee blood revealed three antibodies does not compete with mAbs 1B8 and 3F10 for tACE
(1E10, 1B8, and 3F10) that could bind to human, but binding. Therefore, we can conclude that Lys677 is
not to chimpanzee ACE (Fig. 4‡). Other antibodies responsible for binding of mAb 1E10, while Pro730 is
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

326
NAPEROVA et al.
also partially weakened the mAb 2H9 binding to tACE
(‡)
Tested antibodies
(by 50% if compared with tACE lacking the D616L
substitution) but did not affect binding of other mAbs to
the C domain. Thus, we can presume that Asp616 com-
prises binding epitopes for mAb 1E10 and 2H9,
although this mAb does not play an essential role for
binding of the latter. Since the Asp616 residue on the
ACE C domain surface is located close to Lys677, we
can propose that the mAb 1A10 binding epitope con-
tains Lys677 rather than Pro730, whereas binding
epitopes for mAbs 1B8 and 3F10 contain Pro730.
100
50
0
100
50
0
100
50
0
100
50
0
100
50
EXPERIMENTAL
0
100
50
Antibodies. Preparation of monoclonal antibodies
to the ACE C domain was described in [9]. Polyclonal
antibodies to murine immunoglobulins were from
Pierce.
0
100
50
0
Enzymes. Human recombinant and spermatozoid
tACEs were used. The plasmid for recombinant tACE
and tACE produced in Chinese hamster ovary cells
were described in [15]. Culture medium containing a
soluble form of recombinant tACE was used in this
work. Human tACE were also obtained from spermato-
zoids of healthy donors; they were centrifuged at
2000 g, twice resuspended in physiological solution,
and centrifuged again. The residue was dissolved in
0.05 M phosphate buffer, pH 7.5, containing 0.15 M
KCl, 1 µM ZnSO4 (buffer A), and 0.1% Triton X-100
and stirred overnight at +4°ë. After centrifugation at
10000 g, dry ammonium sulfate was added to the
supernatant in small portions up to 35% saturation, the
mixture was stirred for 30 min, centrifuged at 10000 g,
ammonium sulfate was added up to 80% saturation, and
the mixture was stirred under the same conditions and
centrifuged. The resulting residue was dissolved in
buffer A and purified by affinity chromatography up to
the electrophoretically homogenous state using the
method described in [16].
100
50
0
Competing antibodies
(b)
4E3
1B3
1E10
2H9
1B8
2B11
3F11
3F10
Fig. 3. Determination of mAb competing properties for
binding to human tACE. (a) An inhibition degree (%) with
competing mAbs of the recombinant tACE binding to the
tested mAbs. A mixture of tACE and competing mAbs at a
100-fold excess was incubated before evaluation of binding
of the resulting complex to microtitrator cells covered with
the tested antibodies; (b) a diagram demonstrating a com-
petitive and noncompetitive binding of the panel of mono-
clonal antibodies to tACE. Competitive binding is shown by
overlapping circles; noncompetitive binding, with nonover-
lapping circles.
In single experiments, human and chimpanzee sera
as well as the mutant form D616L of C domain of
human ACE were used (unpublished data).
ACE activity was determined by the initial hydrol-
ysis rate of Hip-His-Leu (Sigma, United States) in 0.05 M
Tris buffer, pH 8.3, containing 0.3 M KCl (buffer B) at
37°ë. The accumulation of the reaction product His-
Leu was fluorimetrically measured using o-phthalalde-
hyde [17]. Background fluorescence was measured
under the same conditions, with buffer A used instead
of the enzyme. Standard fluorescence was measured as
the background one, with the reaction product His-Leu
at known concentrations used instead of the substrate.
The amount of the enzyme capable of hydrolysis of
1 µmol of the substrate for 1 min under the conditions
was taken as an enzyme activity unit (U).
a component of binding epitopes of mAbs 1B8 and
3F10 or vice versa. For determination of the amino acid
residue composing any binding epitope, we studied the
mAb interaction with the mutant ACE C domain
(D616L), in which Asp616 located close to Lys677 on
the ACE C domain surface was replaced by Leu. This
substitution proved to dramatically affect the mAb
The enzymatic activity was measured in the absence
and in the presence of antibodies after the preliminary
1E10 binding and practically completely inhibited it; it incubation of tACE with mAb at various concentrations
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

CHARACTERISTICS OF MONOCLONAL ANTIBODY
327
(‡)
2.5
2.0
1.5
*
*
*
1.0
0.5
0
*
*
*
(b)
6 71
681
691
70 1
71 1
YGTQARKFDV NQLQNTT I KR I I KKVQDLER AAL PAQELEE YNKI LLDMET
h uma n
c h i mp
AA L PAQELEE
YGTQARRFDV NQLQNTT I KR I I KKVQDLER
YNKI LLDMET
7 21
7 31
7 41
7 51
7 61
h uma n
c h i mp
TYSVAT VCHP NGSCLQ LE PD LTNVMATSRK YEDL LWAWEG WRDKAGRA I L
TYSVAT VCHT NGSCLQ LE PD LTNVMATSRK YEDL LWAWEG WRDKAGRA I L
1 23 1
WLL L FLG I A L LVATLGLSQR L FS I RHRSLH RHSHGPQFGS EVELRHS
EVELRHS
1 24 1
12 5 1
1 2 6 1
12 7 1
h uma n
c h i mp
WLL L FLG I A L LVATLGLSQR L FS I RHRSLH RHSHGPQFDS
Fig. 4. Identification withinACE C domain of amino acid residues functionally important for binding to mAbs by comparative anal-
ysis of mAb binding to human and chimpanzee ACE. (a) Comparative binding of somatic human and chimpanzee ACE to various
antibodies; (b) a comparison of primary amino acid sequences of human and chimpanzee ACE [http://cn.expasy.org/
enzyme/3.4.15.1]. Amino acid residues different for human and chimpanzee ACE are shaded.
up to a 1000-fold excess for 2 h at 37°ë for the deter- tion (0.15 µg/ml, 50 µl) in buffer A was added. The
mination of tACE inhibition affected by mAb.
mixture was incubated for 2 h at 37°ë, and the wells
were washed again. For developing antibody-bound
tACE, a solution of 5 mM Hip-His-Leu (100 µl) in
buffer B was added into each well and incubated at
37°ë for a certain time period. The amount of the
formed product was determined fluorimetrically using
His-Leu solutions at the known concentration as a stan-
dard.
Kinetic parameters of spermatozoid tACE–cata-
lyzed hydrolysis of Hip-His-Leu substrate were
determined in buffer B at 37°ë by varying the substrate
concentration within the range of 0.1–2 mM at the con-
stant enzyme concentration of 0.5 nM. The content of
tACE active molecules was preliminarily determined
by lysinopril titration [18]; the kinetics of the enzy-
matic reaction obeyed to the Michaelis–Menthen equa-
Competition of mAbs for tACE binding. Poly-
tion. The activity was determined as described above; clonal antibodies to murine immunoglobulins were
the hydrolysis time was chosen in the way, so that the sorbed in microplate wells as described above and the
hydrolysis degree of the substrate did not exceed 10%. mAbs tested for the antibody–antibody competition
The data were analyzed in [S]/v –[S] coordinates [19], and tACE preliminarily incubated with a 100-fold
where [S] is an initial substrate concentration, and v is excess of all competing mAbs were successively added
the rate of enzymatic hydrolysis. Kinetic constants into the wells. The tACE complex formation with the
were kcat 319 22 s–1, and Km 1.9 0.1 mM, which is tested antibodies was compared in the presence and
in good agreement with the earlier data [20].
absence of competing mAbs.
The binding of tACE to mAb. Polyclonal antibod-
Dissociation constant of tACE complexes with
ies to mice immunoglobulins (50 µl, 10 µg/ml of each) mAb 1B8 and 2H9. Polyclonal antibodies to murine
were loaded into a 96-well polystyrol plate and incu- immunoglobulins were sorbed in microplate wells, fol-
bated at 4°ë overnight. The solution was removed and lowed by successive addition of mAbs 1B8 or 2H9 and
the wells were washed with buffer A containing 0.05% 0.5–5 nM (0.05–0.5 µg/ml) tACE, and the fluorescence
Tween 20. A solution of mAbs in buffer A (3 µg/ml, of the product of enzymatic hydrolysis of the Hip-His-
50 µl) was added into each well and incubated at 37°ë Leu substrate was determined at a fixed time. Initial
for 1 h. The wells were washed again with bufferA con- concentrations of the active enzyme were calculated
taining 0.05% Tween 20 and recombinant tACE solu- using the Michaelis–Menthen equation and the rate of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 3 2008

328
NAPEROVA et al.
the enzymatic reaction determined under our condi-
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Dissociation constants of tACE–mAb complexes
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Cb
----- = – Cb + [mAb]0
Ka Ka
1
1
-----
-----
(2)
Cf
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ACKNOWLEDGMENTS
London: Portland, 1999.
This work was supported by the Russian Foundation
of Basic Research, project no. 07-04-01581.
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