Abbreviations HpU-9-H, HpU-9 heavy chain; HpU-9-L, HpU-9 light chain; VIP, vasoactive intestinal peptide... Interestingly, as isolated subunits, both the heavy chain 9-H and the light ch
Trang 1antibody light chain
Emi Hifumi1,2, Kenji Hatiuchi2, Takuro Okuda2, Akira Nishizono3, Yoshiko Okamura1,2
and Taizo Uda1,2
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: uda@pu-hiroshima.ac.jp
(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 Km of 1.6· 10)5m and a kcatof 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.
Trang 2light 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-subb-sub-unit 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 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 pyloriurease, 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 (ambn) (approximately 150 kDa) were also observed (The natural form of this enzyme was a6b6.) 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- and a-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.
Trang 3peptides 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 activa(induc-tion 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.
Trang 4Mass 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 Km of
1.6· 10)5m and kcatof 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 95C) 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
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 C in a 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 (a6b6) 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.
Trang 5because 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
(am bn) 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 intensity¼ 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.
Trang 6SDS⁄ 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
Trang 7The 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 import-
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 37C 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 4C 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 4C 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⁄ Pi (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 4C 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 15C
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 15C 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.
Trang 8for 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 4C
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 ⁄ Pi (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, plasticglass-ware, and buffer solutions used in this
experi-ment were sterilized by heating (180C, 2 h), autoclaving
(121C, 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
25C Five hundred microlitres of the buffer solution
con-taining the purified HpU-9-L (40 lgÆmL)1) was mixed at
25C 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 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)
References
1 Paul S, Volle DJ, Beach CM, Johnson DR, Powell MJ
& Massey RJ (1989) Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody Science 244, 1158–1162
2 Shuster AM, Gololobov GV, Kvashuk OA, Bogomolova AE, Smirnov IV & Gabibov AG (1992) DNA hydrolyzing autoantibodies Science 256, 665– 667
3 Kanyshkova, TG, Semenov DV, Khlimankov D, Yu Buneva VN & Nevinsky GA (1997) DNA-hydrolyzing activity of the light chain of IgG antibodies from milk
of healthy human mothers FEBS Lett 416, 23–27
4 Matsuura K, Yamamoto K & Shinohara H (1994) Ami-dase Activity of human Bence Jones proteins Biochem Biophys Res Commun 204, 57–62
5 Matsuura K & Sinohara H (1996) Catalytic cleavage of vasopressin by human Bence-Jones proteins at the argi-nylglycinamide bond J Biochem 377, 587–589
6 Paul S, Li L, Kalaga R, Wilkins-Stevens P, Stevens FJ
& Solomon A (1995) Natural catalytic antibodies:
Trang 9peptide-hydrolyzing activities of Bence Jones protein
and VL fragment J Biol Chem 270, 15257–15261
7 Lacroix-Desmazes, S, Moreau A, Sooryanarayana
Bon-nemain C, Stieltjes N, Pashov A, Sultan Y, Hoebeke J,
Kazatchkine MD & Kaveri SV (1999) Catalytic activity
of antibodies against factor VIII in patients with
hemo-philia Nat Med 5, 1044–1047
8 Mei S, Mody B, Eklund SH & Paul S (1991) Vasoactive
intestinal peptide hydrolysis by antibody light chains
J Biol Chem 266, 15571–15574
9 Hifumi E, Okamoto Y & Uda T (1999) Super catalytic
antibody [I]: decomposition of targeted protein by its
antibody light chain J Biosci Bioeng 88, 323–327
10 Hifumi E, Okamoto Y & Uda T (2000) How and why
41S-2 antibody subunits acquire the activities to catalyze
decomposition of the conserved sequence of gp41 of
HIV-1 Appl Biochem Biotech 83, 209–220
11 Hifumi E, Mitsuda Y, Ohara K & Uda T (2002) Targeted
destruction of the HIV-1 coat protein gp41 by a catalytic
antibody light chain J Immunol Methods 269, 283–298
12 Uda T, Hifumi E, Ohara K & Yan Z (2000) Catalytic
activity of antibody light chain to gp41: a consideration
of refolding in relation to activation mechanism Chem
Immunol 77, 18–32
13 Hatiuchi K, Hifumi E, Mitsuda Y & Uda T (2003)
Endopeptidase character of monoclonal antibody i41–7
subunits Immunol Lett 86, 249–257
14 Hifumi E, Kondo H, Mitsuda Y & Uda T (2003)
Cata-lytic features of monoclonal antibody i41SL1–2
sub-units Biotechnol Bioeng 84, 485–493
15 Mitsuda Y, Hifumi E, Tsuruhata K, Fujinami H,
Yamamoto N & Uda T (2004) Catalytic antibody light
chain capable of cleaving a chemokine receptor CCR-5
peptide with a high reaction rate constant Biotechnol
Bioeng 86, 217–225
16 Graham DY, Malaty HM, Evans DG, Evans DJ Jr,
Klein PD & Adam E (1991) Epidemiology of
Helicobac-ter pyloriin an asymptomatic population in the United
States Effect of age, race, and socioeconomic status
Gastroenterology 100, 1495–1501
17 Ikeda Y, Fujii R, Ogino K, Fukushima K, Hifumi E &
Uda T (1998) Immunological features and inhibitive
effects on enzymatic activity of monoclonal antibodies
against Helicobacter pylori urease J Ferment Bioeng 86,
271–276
18 Uda T & Hifumi E (2004) Super catalytic antibody and
Antigenase J Biosci Bioeng 97, 143–152
19 Mitsuda Y, Tsuruhata K, Hifumi E, Takagi M & Uda
T (2005) Investigation of active form of catalytic
anti-body light chain 41S-2-L Immunol Lett 96, 63–71
20 Pillet D, Paon M, Vorobiev II, Gabibov AG, Thomas
D & Friboulet A (2002) Idiotypic network mimicry and antibody catalysis: lessons for the elicitation of efficient anti-idiotypic protease antibodies J Immunol Methods
269, 5–12
21 James LC, Roversi P & Tawfik DS (2003) Antibody multispecificity mediated by conformational diversity Science 299, 1362–1363
22 Gao QS, Sun M, Tyutyukova S, Webster D, Rees A, Tramontano A, Massey RJ & Paul S (1994) Molecular cloning of a proteolytic antibody light chain J Biol Chem 269, 32389–32393
23 Sun M, Gao QS, Li L & Paul S (1994) Proteolytic activity
of an antibody light chain J Immunol 153, 5121–5126
24 Paul S, Planque S, Zhou Y-X, Taguchi H, Bhatia G, Karle S, Hanson C & Nishiyama Y (2003) Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120 J Biol Chem 278, 20429–20435
25 Paul S, Karle S, Planque S, Taguchi H, Salas M, Nishiyama Y, Handy B, Hunter R, Edmondson A & Hanson C (2004) Naturally occurring proteolytic antibodies J Biol Chem 279, 39611–39619
26 Lacroix-Desmazes S, Moreau A, Sooryanarayana C, Bonnemain Stieltjes N, Pashov A, Sultan Y, Hoebeke J, Kazatchkine MD & Kaveri SV (1999) Catalytic activity
of antibodies against factor VIII in patients with hemo-philia Nat Med 5, 1044–1047
27 Kolesnikov AV, Kozyr AV, Alexandrova ES, Koralew-ski F, Demin AV, Titov MI, Avalle B, Tramontano A, Paul S, Thomas D, Gabibov AG & Friboulet A (2000) Enzyme mimicry by the antiidiotypic antibody
approach Pro Natl Acad Sci USA 97, 13526–13531
28 Fujii R, Morihara F, Oku T, Hifumi E & Uda T (2004) Epitope mapping and features of the epitope for MAbs inhibiting enzymatic activity of H pylori urease Bio-technol Bioeng 86, 434–444
29 Fujii R, Morihara F, Fukushima K, Oku T, Hifumi E
& Uda T (2004) A recombinant antigen from Helicobac-ter pyloriurease as vaccine against H pylori associated disease Biotechnol Bioeng 86, 737–747
30 Labigne A, Cussac V & Courcoux P (1991) Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity J Bacteriol 173, 1920–1931
31 Ha NC, Oh ST, Sung JY, Cha KA, Lee MH & Oh
BH (2001) Supramolecular assembly and acid resis-tance of Helicobacter pylori urease Nat Struct Biol 8, 505–509