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: 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 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. 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Nat Struct Biol 8, 505–509. E. Hifumi et al. Catalytic features of anti-HpU-9 mAb light chain FEBS Journal 272 (2005) 4497–4505 ª 2005 FEBS 4505 . 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; . 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 ,