Báo cáo khoa học: Specific degradation of H. pylori urease by a catalytic antibody light chain pptx

Specific degradation of H. pylori urease by a catalytic
antibody light chain
Emi Hifumi
1,2
, Kenji Hatiuchi
2
, Takuro Okuda
2
, Akira Nishizono
3
, Yoshiko Okamura
1,2
and Taizo Uda
1,2
1 Prefectural University of Hiroshima, Faculty of Bioscience and Environment, Hiroshima, Japan
2 CREST of JST (Japan Science and Technology Corporation), Saitama, Japan
3 Oita University, Faculty of Medicine, Oita, Japan
Many natural catalytic antibodies have been discov-
ered in the last decade. The first natural catalytic anti-
body was isolated from the serum of an asthma
patient [1], and this antibody enzymatically cleaved
vasoactive intestinal peptide (VIP). Gabibov et al. [2]
and Nevinsky et al. [3] reported antibodies with a cata-
lytic activity to cleave DNA molecules. The antibodies
reported by Gabibov et al. were isolated from serum
samples from autoimmune disease (i.e. SLE) patients
and the ones reported by Nevinsky et al. were isolated
from human milk. These antibodies exhibited catalytic
activities as a whole antibody. A natural catalytic anti-
body from the serum of hemophilia A patients repor-
ted by Kaveri et al. was capable of digesting factor

VIII molecule [7], suggesting a pathological role of this
antibody in vivo. The Bence-Jones proteins, which are
found in the urine of patients with certain diseases,
particularly multiple myeloma, are human light chains
of the antibodies. Matsuura et al. [4,5] and Paul et al.
[6] reported that some of the Bence-Jones proteins had
peptidase activities. These reports revealed that anti-
bodies and their light chains naturally produced in the
patients could have a catalytic activity, although their
antigens remained unidentified. Besides these natural
catalytic antibodies, Paul et al. [8] and Uda et al. [9–
12] successfully produced artificial catalytic antibodies
by immunizing mice with ground state polypeptides
and proteins. The light chain of the catalytic antibody
generated by Paul et al. by itself cleaved the antigenic
peptide VIP [8]. Uda et al. showed the light chain of
41S-2 mAb could cleave the HIV-1 env gp41 molecule.
Uda et al. also succeeded in the generation of catalytic
Keywords
catalytic antibody; light chain; Helicobacter
pylori; urease proteolysis
Correspondence
T. Uda, Faculty of Bioscience and
Environment, Prefectural University of
Hiroshima, Shobara, Hiroshima 727–0023,
Japan
Fax: +81 824 74 0191
Tel: +81 824 74 1756
E-mail:
(Received 29 April 2005, revised 7 July

2005, accepted 18 July 2005)
doi:10.1111/j.1742-4658.2005.04869.x
Catalytic antibodies capable of digesting crucial proteins of pathogenic bac-
teria have long been sought for potential therapeutic use. Helicobacter
pylori urease plays a crucial role for the survival of this bacterium in the
highly acidic conditions of human stomach. The HpU-9 monoclonal anti-
body (mAb) raised against H. pylori urease recognized the a-subunit of the
urease, but only slightly recognized the b-subunit. However, when isolated
both the light and the heavy chains of this antibody were mostly bound to
the b-subunit. The cleavage reaction catalyzed by HpU-9 light chain
(HpU-9-L) followed the Michaelis-Menten equation with a K
m
of
1.6 · 10
)5
m and a k
cat
of 0.11 min
)1
, suggesting that the cleavage reaction
was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L effi-
ciently cleaved the b-subunit but not the a-subunit, indicating that the
degradation by HpU-9-L had a specificity. The cleaved peptide bonds in
the b-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and
M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the
digestion by HpU-9-L was specific. In summary, we succeeded in the pre-
paration of a catalytic antibody light chain capable of specifically digesting
the b-subunit of H. pylori urease.
Abbreviations
HpU-9-H, HpU-9 heavy chain; HpU-9-L, HpU-9 light chain; VIP, vasoactive intestinal peptide.

FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4497
light and ⁄ or heavy chains from mAbs such as i41–7
[13], i41SL1-2 [14], and ECL2B [15]. The light chain
of ECL2B mAb was capable of cleaving a pep-
tide (RSSHFPYSQYQFWKNFQTLK) derived from
CCR5, a chemokine receptor, which plays a crucial
role in HIV infection. In all of these catalytic antibod-
ies, a catalytic triad composed of Asp, Ser, and His
was always identified through molecular modeling.
These studies showed that catalytic antibodies capable
of cleaving molecules of interest could be generated
through the immunization of peptides and ⁄ or proteins.
Helicobacter pylori, a Gram-negative spiral bacteria
infecting about 50% of the world’s population, is an
etiologic agent in a variety of gastroduodenal diseases
and is the only microorganism known to inhabit
the human stomach [16]. H. pylori produces a large
amount of urease, which is a hexamer composed of
noncovalently associated a- and b-subunits. The b-sub-
unit contains the active site, while the a-subunit assists
the catalytic activity. Ammonia generated through the
hydrolysis of urea by urease neutralizes gastric acidity
and forms a neutral microenvironment surrounding
the bacterium within the gastric lumen. Thus the
urease of H. pylori plays a crucial role for its survival
in the strong acidic condition of human stomach.
We set out to generate catalytic antibodies that can
degrade H. pylori urease. As we have reported, we pro-
duced 27 cell clones secreting mAbs against H. pylori
urease. Among them, HpU-9 mAb strongly recognized

the a-subunit of the urease but weakly recognized the
b-subunit [17]. Interestingly, as isolated subunits, both
the heavy chain (HpU-9-H) and the light chain (HpU-
9-L) strongly interacted with the b-subunit, but only
weakly with the a-subunit. In this study, we investi-
gated the binding and catalytic features of HpU-
9 mAb subunits against H. pylori urease in details.
Results
Immunological binding features of HpU-9 mAb
and its heavy and light chains
We have reported that the HpU-9 mAb strongly recog-
nized the a-subunit but not the b-subunit of the
H. pylori urease, purified from the ATCC 43504 strain
[17]. Lane 1 in Fig. 1 shows the result of SDS ⁄ PAGE
(reduced condition with silver staining) of H. pylori
urease purified from the Sydney strain (SS1) used in
this study. The b- and the a-subunits were clearly
observed as a 66.0 (± 2.8) kDa band and a 31.0
(± 0.8) kDa band, respectively. Western blot results
showed that the HpU-9 mAb predominantly reacted
with the a-subunit of the urease, as shown in Fig. 1
(lane 2). In this experiment, the a-subunit dimmer
appeared right below the b-subunit band, whose iden-
tity was confirmed by western blot using HpU-2
monoclonal antibody, although this dimer was only
faintly visible by silver staining (lane 1). Some partly
dissociated forms (a
m
b
n

) (approximately 150 kDa)
were also observed. (The natural form of this enzyme
was a
6
b
6
.) These bands were confirmed to be derived
from urease by western blotting with monoclonal anti-
bodies (HpU-2 and )17) against the a- and the b-sub-
units, respectively [17]. The heavy chain (HpU-9-H:
lane 3) and the light chain (HpU-9-L: lane 4), which
were isolated and purified through reduction and sub-
sequent HPLC fractionation of HpU-9 mAb (see
Experimental procedures for details), reacted strongly
with the b-subunit but only weakly with the a-subunit.
This experiment was repeated to confirm this unex-
pected binding characteristic of these two subunits.
Cleavage test for a peptide
Catalytic antibody light chains can cleave the target pro-
teins in a highly specific manner, and then produce small
Fig. 1. Results of SDS ⁄ PAGE and western blot analysis. Lane 1:
SDS ⁄ PAGE of the urease purified from the Sydney strain (SS1).
The b-anda-subunits of the H. pylori urease were clearly observed
at 66.0 and 31.0 kDa, respectively. Lanes 2–4: western blot analy-
sis. Lane 2: HpU-9 mAb, lane 3: heavy chain (HpU-9-H), lane 4; light
chain (HpU-9-L). The antibody, HpU-9 mAb, specifically reacted with
the a-subunit of the H. pylori urease [the bands at around 150 kDa
are multimers (a
m
b

n
) of the subunits]. In contrast, the heavy chain
(HpU-9-H) and the light chain (HpU-9-L) isolated from the parent
HpU-9 mAb primarily reacted with the b-subunit but only scarcely
with the a-subunit.
Catalytic features of anti-HpU-9 mAb light chain E. Hifumi et al.
4498 FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS
peptides and ⁄ or amino acids by the consecutive reaction
[11,18]. Therefore, the light chain can cleave peptides
with low specificity, suggesting that the light chain pos-
sesses two functional sites, recognition and catalysis.
This means that a peptide with characteristics such as
water-soluble and nonaggregative in a phosphate solu-
tion is preferable rather than the peptide sequence
employed for the investigation whether the peptidase
activity is present in the antibody. Several peptides with
these characteristics such as TPRGPDRPEGIEEEG
GERDRD, EILPGSG, SGNIKYN, and YNEKFKG
have been used for this purpose [11,13,14]. In our clea-
vage test, a synthetic peptide SVELIDIGGNRRIFG
FNALVD(1–21) (residues 183–203 of the urease
a-subunit), was used as a substrate to monitor the pepti-
dase activity of the antibody and its subunits, as we did
not know the epitope of HpU-9 mAb.
RP-HPLC was used to monitor the time course of
the cleavage reaction, as shown in Fig. 2A. The whole
HpU-9 mAb did not show any catalytic activity in this
analysis, which confirmed the result that had previ-
ously been reported [9,11,13–15]. The isolated heavy
chain HpU-9-H, which was prepared by exactly the

same purification steps as those for HpU-9-L, also
failed to cleave the antigenic peptide (mass spectros-
copy detected no fragmented peptides but only the
substrate peptide), though a possibility of very slow
cleavage is not excluded.
In contrast to the whole antibody and the heavy
chain, the isolated light chain was capable of cleaving
this peptide. After the peptide was mixed with HpU-
9-L, it was gradually degraded for about 30 h, at which
point the degradation sped up considerably, and at
68 h, the reaction was complete. This cleavage reaction
showed the typical double-phase reaction profile (induc-
tion and activation phases), as frequently observed in
many catalytic reactions reported to date [9–15,18].
Induced fitting may be a possible cause of this induction
phase [19,20]. After the degradation was complete, the
peptide (final concentration: 80 lm) was replenished in
the reaction system (Fig. 2B). In this case, the induction
phase was not observed and the cleavage was completed
in about 21 h. In this reaction, a fragmented peak was
clearly observed at the retention time of 14 min.
Fig. 2. Time course of the catalytic cleavage of a peptide substrate by HpU-9-L. Peptide (SVELIDIGGNRRIFGFNALVDR); 184.5 lgÆmL
)1
,
HpU-9-L; 20 lgÆmL
)1
. The reaction was conducted at 25 °C in a phosphate buffer (pH 6.5). (A) (—d—) Indicates typical degradation curve for
the peptide with HpU-9-L, exhibiting a double-phase reaction profile. Without HpU-9-L, no degradation was observed. (B) (—m—) Indicates the
reaction profile of HpU-9-L when the peptide was replenished after the peptide initially prepared was completely digested, displaying imme-
diate decomposition of the peptide. The main cleavage site of the peptide was R12-I13. The heavy chain, HpU-9-H, failed to cleave the anti-

genic peptide. The parent HpU-9 mAb also did not show any catalytic activity.
E. Hifumi et al. Catalytic features of anti-HpU-9 mAb light chain
FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4499
Mass spectrometry was used to detect the fragmen-
tation of this peptide at 0-, 50.5-, and 68-h of incuba-
tion. The mass of the main fragmented peak at 50.5 h
(m ⁄ z [M + H]
+
¼ 1328.81) matched with the peptide
SVELIDIGGNRR(1–12), whose theoretical mass was
1328.73. A smaller fragment corresponding to LIDI
GGNRR(4–12) (m ⁄ z [M + H]
+
¼ 1013.36) could be
detected at 68 h. The sequence of the fragmented pep-
tide observed in the replenishment experiment was
identified as SVELIDIGGNRR(1–12). These results
suggest that the cleavage at R12-I13 took place first,
followed by successive cleavages into smaller fragments
such as LIDIGGNRR(4–12). These results, clearly
demonstrated the presence of a catalytic activity in
HpU-9-L.
The kinetic analysis was performed after HpU-9-L
completely digested the peptide substrate as this eli-
minated the slow degradation phase [11,13–15,19]
(Fig. 2B). The cleavage reaction by HpU-9-L obeyed
the Michaelis–Menten equation with a K
m
of
1.6 · 10

)5
m and k
cat
of 0.11 min
)1
. This result indica-
ted that the cleavage reaction must be enzymatic but
does not show cleavage-site specificity.
Cleavage tests for H. pylori urease
The cleavage of H. pylori urease from the Sydney
strain (SS1) by HpU-9-L was monitored by SDS ⁄
PAGE under a nonreduced condition (in order to pre-
vent a possible of protein cleavage through the reduc-
tion by 2-mercaptoethanol at 95 °C) with silver
staining at 0, 4, and 8 h of incubation (Fig. 3A). The
band at 52.2 kDa below the b-subunit (Fig. 3B, lanes
1–3) was an impurity not related to urease. In
Fig. 3A, slight changes compared with the control
(Fig. 3B) in the band pattern were observed even at
0 h of incubation (lane 1: In this case, about 15 min-
utes passed by the application of the sample to the
SDS ⁄ PAGE analysis). The bands (4; 26.5 kDa) and
(5; 16.5 kDa) were faintly observed simultaneously, as
the urease cleavage initiated immediately after mixing.
The band of HpU-9-L (23 kDa) was barely detectable
AB
Fig. 3. Cleavage tests for H. pylori urease by HpU-9-L.urease; 225 lgÆmL
)1
, HpU-9-L; 16 lgÆmL
)1

. The reaction was conducted at 25 ° Cina
phosphate buffer (pH 6.5). Cleavage results were followed by SDS ⁄ PAGE (nonreduced condition) with silver staining. (A) Cleavage of the
urease with HpU-9-L Lanes 1, 2, and 3 show the result of 0, 4 and 8 h of incubation after mixing the H. pylori urease and HpU-9-L. H. pylori
urease is a hexamer composed of noncovalently associated a- and b-subunits (a
6
b
6
). In SDS ⁄ PAGE, the bands of the monomeric b- and
a-subunits of the H. pylori urease appeared at 66.0 and 31.0 kDa, respectively. The new bands (4; 26.5 kDa) and (5; 16.5 kDa) were faintly
observed immediately after mixing (lane 1). At 4 h of incubation (lane 2), the bands of partially dissociated urease (a
m
b
n
) became faint as
well as the band of the b-subunit monomer. In contrast, the intensity of the band (1) (52.2 kDa) became stronger and two new bands (2;
39.2 kDa) and (3; 38.3 kDa) appeared. Bands 4 and 5 became darker, whereas the band of the a-subunit showed little change. At 8 h of
incubation (lane 3), the band of the b-subunit became very faint. Some new bands between bands 1 and 2 became clearer and several
bands around bands 4 and 5 also became darker. The band strength of the b-subunit decreased by 65% after 8 h of incubation, whereas
that of the a-subunit decreased only by 10%. BSA was not degraded even after 7 days. (B) Controls of the cleavage. Lanes 1, 2 and 3 show
the controls (without HpU-9-L) at 0, 4 and 8 h of incubation.
Catalytic features of anti-HpU-9 mAb light chain E. Hifumi et al.
4500 FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS
because of its low concentration. At 4 h of incubation
(lane 2), significant changes of the band pattern were
observed. The bands of partially dissociated urease
(a
m
b
n
) became faint as well as the band of the

b-subunit monomer. In contrast, the intensity of the
band (1; 52.2 kDa) became stronger (the fragmented
b-subunit overlapped on the contaminant protein at
band 1) and two new bands (2; 39.2 kDa) and (3;
38.3 kDa) appeared. The bands (4; 22.5 kDa) and (5;
16.5 kDa) became darker, whereas the band of the
a-subunit showed little change. At 8 h of incubation
(lane 3), this pattern became more prominent. The
band of the b-subunit became very faint. Some new
bands between bands 1 and 3 became clear, and sev-
eral bands around bands 4 and 5 became stronger.
The a-subunit band changed little during this 8 h
incubation. Conversely, the urease hardly degraded
without HpU-9-L during the incubation (Fig. 3B,
lanes 1–3). By densitometric analysis using NIH
Image software, the band strength of the b-subunit
decreased by 65% after 8 h of incubation, whereas
that of the a-subunit decreased only by 10%.
In order to examine substrate specificity, HpU-9-L
was incubated with, BSA, under conditions identical
to those employed for the H. pylori urease. BSA was
not degraded even when incubated for 7 days, show-
ing the cleavage by HpU-9-L was specific to H. pylori
urease.
Analysis of cleavage sites
We characterized the cleavage sites of the urease by
N-terminal amino-acid sequencing of the peptide frag-
ments. From the band (1), a sequence of GLIVT was
detected with the intensity of 2 pmol. As a minor scis-
sile bond, L121-A122 (detection intensity ¼ 0.9 pmol)

in the b-subunit was also identified. Thus, the major
scissile bond was identified at E124-G125 of the b-sub-
unit (Fig. 4A). Combined on the size estimate based
on the mobility in SDA-PAGE, we concluded that
band 1 was the G125-F568 fragment derived from
the b-subunit. Band 5 gave a sequence of MKKIS
(18 pmol), which corresponds to the other b-subunit
derived fragment (M1-E124) cleaved at E124-G125.
On the other hand, band 2 gave three main sequences:
GLIVT (0.9 pmol), AINHA (0.9 pmol), and DVQVA
(0.8 pmol). The first one was identical to the N-ter-
minal sequence of the main band (1), and we con-
cluded that this fragment was produced through
successive digestions of the G125-F568 fragment. The
second one indicated that the peptide was cleaved at
S229-A230 and the third at Y241-D242 of the b-sub-
unit. From band 3, the major scissile bond was identi-
fied as M262-A263 in the b-subunit. Band 4 gave a
sequence of MKLTP (19 pmol), which was identical to
the N-terminal sequence of the a-subunit. Smaller size
of this band indicated this was a fragment generated
by digestion of the a-subunit.
Discussion
The binding analysis of the HpU-9 mAb, HpU-9-L,
and -H yielded unexpected results (Fig. 1). Although
the HpU-9 mAb heterotetramer specifically recognized
the a-subunit of the H. pylori urease, the isolated
heavy and light chains bound mostly to the b-subunit.
This result was confirmed to be reproducible. Initially,
we considered that the denaturation of urease during

B
A
Fig. 4. Cleavage sites of H. pylori urease by
HpU-9-L.The sequence is the H. pylori
urease of SS-1 [30,31]. (A) b-Subunit, (B)
a-subunit. The cleavage sites confirmed by
N-terminal amino-acid sequencing are indica-
ted with red arrows; the blue underlines are
the assumed cleavage sites based on
molecular sizes and sequencing. The main
digestion of the urease by HpU-9-L was initi-
ated by the cleavage of the peptide bond at
E124-G125 of the b-subunit, followed by
successive digestions. HpU-9-L may cleave
several peptide bonds in the b-subunit. We
observed only a slight digestion of the
a-subunit.
E. Hifumi et al. Catalytic features of anti-HpU-9 mAb light chain
FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4501
SDS ⁄ PAGE and western blotting as a possible cause
of this difference. However, we concluded this was
unlikely as we could show that the light chain (HpU-
9-L) was capable of cleaving the intact H. pylori
urease.
James et al. pointed out that a single monoclonal
antibody could take several different structures, and
they could exist simultaneously in an equilibrium state
in a solution. In one case, one of the structures could
specifically bind an antigen, while another could not
[21]. In this study, it was possible that the conforma-

tions of the isolated light and heavy chains were dis-
tinct from that of the intact parent antibody. The
conformation of the light or heavy chains could be
more flexible when they were isolated than when they
existed in the whole antibody. This difference in the
conformation might lead to different binding prop-
erties. A similar difference of molecular recognition
pattern had also been observed with another monoclo-
nal antibody, HpU-2. (This mAb reacted to the a-sub-
unit, but the isolated heavy chain could bind to both
the a- and b-subunits [17]). In general, a light chain
tended to form a dimer, while an isolated heavy chain
easily formed an aggregate. In the reaction system, the
structure of isolated HpU-9-L may be changed, for
instance, to expose hydrophobic patches formally
buried inside the structure. This structural transition
makes HpU-9-L forming multimers and shifting its
recognition character. In our previous experiment of a
catalytic antibody light chain 41S-2-L cleaving gp41 of
HIV-1, the results indicated the formation of multi-
mers in the reaction system [19], and we suspect a
similar process was taking place in this study.
We have already demonstrated that the isolated light
chain (41S-2-L) could specifically bind to HIV-1 env
gp41 protein. However, the heavy chain cross-reacted
with many HIV-1 proteins, while the parent antibody
(41S-2 mAb) was as specific to the gp41 molecule as
41S-2-L was [10,11]. In some cases, a significant
change in the immunological character of the heavy or
light chain could occur, resulting in a different specific-

ity from that of the parent antibody. The conforma-
tional diversity as pointed out by James et al. [21]
might be the cause of the multimer formation by iso-
lated light and heavy chains, leading to the specificity
difference from the whole antibody. However, not all
of the isolated heavy or light chains change their spe-
cificity. In the case of HpU-17 and )20 mAb (a series
of mAbs obtained along with HpU-9) [17], their heavy
or light chains showed the same specificity (to the
b-subunit) as their parent mAbs.
It has been well documented that the light chain of
an antibody could possess a catalytic cleavage activity
against peptides and ⁄ or proteins [4–6,18,22]. HpU-9-L
displayed catalytic ability. In our cleavage assay for
the peptide SVELIDIGGNRRIFGFNALVD(1–21),
HpU-9-L degraded the peptide with a lag phase
(induction phase). We also observed a similar lag
phase in many cleavage reactions by catalytic anti-
bodies [11,13–15,18,19], as well as in proteolysis by
an anti-idiotypic antibody [20]. It was suggested that
some conformational changes caused by events such
as induced fitting might be the reason for this lag
phase. Moreover, the formation of multimers of the
catalytic light chain may contribute to the long lag
phase [19].
Using the intact H. pylori urease, a cleavage test was
also performed. The cleavage sites confirmed by N-ter-
minal amino-acid sequencing were indicated with red
arrows in Fig. 4: The blue underlines were the identi-
fied cleavage sites based on the molecular sizes and the

sequencing results. HpU-9-L cleaved several peptide
bonds in this experiment. Paul et al. also reported a
multisite cleavage by monoclonal catalytic antibodies
[23–25]. In the polyclonal catalytic antibody cleaving
factor VIII reported by Kaveri et al. several peptide
bonds were cleaved [26]. Although a catalytic antibody
usually showed a high recognition specificity, these
results demonstrated that the cleavage could take place
at multiple sites. We observed that the main digestion
of the urease by HpU-9-L was initiated by the cleavage
of the peptide bond at E124-G125 of the b-subunit,
followed by successive digestions. The locations of
these scissile bonds were identified (Fig. 5). The pep-
tide bonds cleaved by HpU-9-L are indicated with
arrows. The scissile bonds were on the loops exposed
to the solution but not on the inner loops. These loca-
tions of the scissile bonds were divided into two
groups. One was group A consisting of L121-A122
(yellow arrow) and E124-G125 (green arrow). Another
was group B consisting of S229-A230 (pink arrow),
Y241-D242 (red arrow), and M262-A263 (blue arrow).
From the amino-acid sequence analysis, the cleavage
at E124-G125 was the most prominent. Therefore, it
appeared that HpU-9-L can access the group A, and
binds the loop on which the peptide bond of E124-
G125 is present. This peptide bond might be cleaved
first, followed by successive cleavages of the peptide
bond such as L121-A122. The group B could be
cleaved either after group A or simultaneously with
group A. The details of these cleavage mechanisms are

not yet clear.
We observed only a slight digestion of the a-subunit,
indicating that HpU-9-L preferentially targeted the
b-subunit over the a-subunit. This observation was in
good agreement with the binding feature of HpU-9-L.
Catalytic features of anti-HpU-9 mAb light chain E. Hifumi et al.
4502 FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS
The molecular modeling result of the antibody struc-
ture suggested that HpU-9-L had a catalytic triad
composed of Asp1, Ser27a and His93. These amino-
acid residues were found at the identical locations in
other catalytic antibodies such as VIPase, i41SL1-2
[14] and ECL2B [15]. As pointed out previously, the
presence of this catalytic triad seemed to indicate
whether the antibody subunit possessed a catalytic
capability [14,15,18]. Friboulet et al. concluded that a
catalytic dyad composed of His and Asp was import-
ant for the esterase activity of their catalytic anti-idio-
typic antibody [27]. In our case of HpU-9-H, no
catalytic triad was observed as it lacked a histidine
residue.
Catalytic antibodies against essential bacterial pro-
teins may lead to a novel therapeutic intervention
method against bacterial infections. The current study
provided the key first step towards such a therapy, and
we are currently following up to investigate the feasi-
bility of this approach.
Experimental procedures
Preparation of H. pylori urease and the mAbs
H. pylori of the Sydney strain (SS1) was cultured on a

Brucella broth agar medium containing 10% (v ⁄ v) fetal
bovine serum at 37 °C for 2–4 days under a microaerobic
environment. The propagated bacteria were suspended in
0.15 m NaCl and harvested by centrifugation at 4000 g for
10 min at 4 °C and the supernatant was decanted out. The
harvested pellet was resuspended in 20 mL 0.15 m NaCl
and centrifuged at 10 000 g for 10 min at 4 °C twice for
washing. Detailed purification methods of the H. pylori
urease from the harvested pellet are described in the litera-
ture [17,28,29]. Finally, only the a- and b-subunits were
detected by SDS ⁄ PAGE analysis with silver staining.
Production of monoclonal antibodies against
H. pylori urease
Balb ⁄ c mice were primed subcutaneously using 100 lg per
mouse of purified H. pylori urease. Monoclonal antibodies
were produced by cell fusion, HAT selection, and cloning
[17].
Purification and separation of the antibody heavy
chain
HpU-9 mAb was purified according to the purification
manual from the Bio-Rad Protein A MAPS-II kit (Nippon
BIO-RAD, Tokyo, Japan). First, 5 mL of ascites fluid con-
taining HpU-9 mAb was mixed with the same volume of a
saturated solution of ammonium sulfate. The precipitate
was recovered by centrifugation and then 5 mL of NaCl ⁄ P
i
(PBS) was added to the precipitate. This process was repea-
ted twice, followed by two dialyses against PBS. An aliquot
of the PBS solution containing HpU-9 mAb was mixed
with the same volume of the binding buffer of MAPS-II.

This mixture was then placed on a bed packed with Affi-
Gel (protein A) for elution of the bound mAb. The eluted
mAb was dialyzed against the buffer, 50 mm Tris ⁄ 0.15 m
NaCl (pH 8.0), twice at 4 °C. The resulting antibody was
ultrafiltered three times by use of Centriprep 10 (Amicon,
Billerica, MA, USA). A total of 5 mg of the antibody was
dissolved in 2.7 mL of a buffer (pH 8.0) consisting of
50 mm Tris and 0.15 m NaCl and reduced by the addition
of 0.3 mL of 2 m of 2-mercaptoethanol for 3 h at 15 °C.
To this solution, 3 mL of 0.6 m iodoacetamide was added,
followed by adjusting the pH to 8 by adding 1 m Tris. The
solution was then incubated for 15 min at 15 °C. The
resulting solution was ultrafiltered to 0.5 mL, after which a
half volume of the sample was injected into HPLC (col-
umn: Protein-Pak 300SW, 7.8 · 300 mm, Nippon Waters,
Tokyo, Japan) at a flow rate of 0.15 mLÆmin
)1
of 6 m
guanidine hydrochloride (pH 6.5) as an eluent. Fractions
Fig. 5. Structure of b-subunit of H. pylori urease [31]. The peptide
bonds cleaved by HpU-9-L are indicated with arrows. L121-A122,
yellow; E124-G125, green; S229-A230, pink; Y241-D242, red;
M262-A263, blue. The scissile bonds lie on the loops exposed to
the solution but they are not on the inner loops. From the amino-
acid sequence analysis, the strongest cleaved bond was
E124-G125. HpU-9-L can access the peptide bond (in group A) that
might be first cleaved, followed by the successive digestion of the
peptide bonds such as L121-A122. The cleavage of peptide bonds
in group B might take place either after group A or simultaneously
with group A. Detailed cleaving mechanisms are not yet clear.

E. Hifumi et al. Catalytic features of anti-HpU-9 mAb light chain
FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4503
for the heavy and light chains were collected, followed by
dilution with 6 m guanidine hydrochloride. These fractions
were dialyzed against PBS by replacing the buffer seven
times for 3–4 days at 4 °C.
Western blot analysis
After SDS ⁄ PAGE (100 lgÆmL
)1
of the urease was applied)
without staining, electrophoresed proteins were transferred
from the gel onto an Immobilon-P poly(vinylidene difluo-
ride) membrane (Millipore Corporation, Billerica, MA,
USA). The poly(vinylidene difluoride) membrane was
blocked with Tris ⁄ NaCl ⁄ P
i
(TBS) containing 3% (v ⁄ v)
skimmed milk and 0.05% (v ⁄ v) Tween-20 and then incuba-
ted with the mAb (0.5 lgÆmL
)1
), and the heavy
(21 lgÆmL
)1
) or light chain (27 lgÆmL
)1
) for 2 h at room
temperature. After washing with TBS containing 0.05%
(v ⁄ v) Tween-20, the membrane was further incubated with
anti-[mouse Ig(G + A + M)] Ig conjugated with alkaline
phosphatase for 2 h at room temperature. Finally, after

several washings with TBS ⁄ Tween, the color was developed
using BCIP ⁄ NBT (Kirkegaard & Perry Laboratories,
Gaithersburg, MD, USA).
Cleavage tests by HpU-9-L
The peptide of SVELIDIGGNRRIFGFNALVD was
synthesized by the Fmoc solid-phase method by use of an
automated peptide synthesizer (Symphony, Protein Tech-
nologies Inc., Tucson, AZ, USA). The purified peptides
were identified by use of an RP-HPLC-equipped mass spec-
trometer (MALDI-TOF-MASS, Bruker ⁄ Autoflex, Bremen,
Germany). The purity of the peptide was over 95% as
determined by HPLC.
To avoid contamination in cleavage assays, most glass-
ware, plasticware, and buffer solutions used in this experi-
ment were sterilized by heating (180 °C, 2 h), autoclaving
(121 °C, 20 min), or filtration through a 0.20-lm sterilized
filter as much as possible. Most of the experiments were
performed in a biological safety cabinet to avoid airborne
contamination. Catalysis reactions using HpU-9-L were
conducted in a 12 mm phosphate buffer (pH 6.5) contain-
ing 7.3% glycerol, 1.8% SDS, and 60 mm Tris ⁄ HCl at
25 °C. Five hundred microlitres of the buffer solution con-
taining the purified HpU-9-L (40 lgÆmL
)1
) was mixed at
25 °C with the same volume of a solution containing
369 lgÆmL
)1
of the peptide in a sterilized test tube. The
reaction was monitored using the RP-HPLC (Jasco, Tokyo,

Japan) under isocratic conditions. The reaction products
were analyzed by using the mass spectrometer.
Cleavage of H. pylori urease (225 lgÆmL
)1
) was conduc-
ted using HpU-9-L (16 lgÆmL
)1
), which was first permitted
to completely decompose the peptide (Fig. 2B), under the
same conditions as the assay described above. Cleavage of
the urease was monitored by SDS ⁄ PAGE with silver stain-
ing. As another control experiment, degradation of BSA
(25 lgÆmL
)1
) was investigated under similar reaction condi-
tions as the above cleavage assay.
Analysis of N-terminal sequence
After 8 h of incubation a reaction sample (1500 lL) was
concentrated 10-fold using an ultrafiltration membrane
(Amicon Ultra-45000MWCO, Millipore). The sample was
then applied to the separation of 12% gel by SDS ⁄ PAGE
at 20 mA in a nonreducing condition. The bands were
transferred for 1 h at 112 mA onto an Immobilon-PQS
poly(vinylidene difluoride) membrane (Millipore) in 0.1 m
Tris ⁄ HCl, 0.19 m Glycine, 5% methanol at pH 8.7. After
being stained with Coomassie Brilliant Blue, visible bands
were cut and subjected to N-terminal sequencing (Auto-
mated Protein Sequencer, Prosize 494 HT, Applied Bio-
systems (Foster City, CA, USA) for the amount of protein
sequenced, ranging from 2 to 40 pmol. For 0.5–2 pmoles of

the fragment, an automatic protein microsequencer (Prosize
494 cLC, Applied Biosystems) was used.
Acknowledgements
This study was supported by Japan Science and
Technology Agency (Creation of Bio-devices and Bio-
systems with Chemical and Biological Molecules for
Medicinal Use) and Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (13450344,
13022261).
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E. Hifumi et al. Catalytic features of anti-HpU-9 mAb light chain
FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4505