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Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201-aa 338 of beta-subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease-specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138-immunized mice than in control mice. Furthermore, the expression of interferon-gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non-eradicated group. The combination of Th1- and Th2-mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori., ((c) 2008 Wiley Periodicals, Inc.)

Helicobacter pylori has to counteract acidity during colonization in the stomach. The most important region for the enzymic activity of H. pylori urease, consisting of 138 aa (ureB138), was determined by a comparison of the homology of amino acid sequences, and a structural analysis, between urease of H. pylori and various other species. This region was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which was cleaved by PreScission protease between the GST moiety and ureB138. The ureB138 protein was then purified by gel filtration. The polyclonal antibody (pAb) induced by immunization with the purified ureB138 could suppress urease activity by about 50 %, while the pAb against the H. pylori urease did not show any inhibitory effect at all. Immunohistochemical analysis indicated that the ureB138-specific pAb specifically recognized the H. pylori infecting human gastric tissues. The effects of vaccination of recombinant ureB138 against infection by this organism were also examined. Specific IgG and IgA antibodies against H. pylori urease were induced in the serum of mice immunized with ureB138. A reduction in the number of colonizing H. pylori was observed in mice treated with ureB138 compared to ones treated with BSA and infection control mice. In the protected mice, severe gastritis characterized by marked infiltration of mononuclear cells was noted compared with the gastritis observed in unprotected mice. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in ureB138-vaccinated mice than in non-vaccinated mice. This indicates that local IgA antibody and severe post-immunization gastritis correlate well with the protection of mice against H. pylori infection.

The HpU-2 monoclonal antibody (mAb) raised against Helicobacter pylori urease mainly recognized the alpha-subunit of the urease. On the other hand, the heavy chain of HpU-2 mAb (HpU-2-H) isolated from the parent mAb recognized both the alpha- and beta-subunit, in which the beta-subunit was recognized more strongly than the beta-subunit. HpU-2-H cleaved a peptide, SVELIDIGGNRRIFGFNALVD, which is the epitope sequence recognized by HpU-2 mAb, showing a double-phase reaction profile at 25 degrees C in a phosphate buffer. After an induction time of 24h, the cleavage of the peptide was initiated by HpU-2-H at a high rate and it was completed at 80 h of incubation. By mass spectroscopy, two main fragmented peptides, SVELIDIGGNRR and SVELIDIGGNRRIFG, were identified. In addition, many small peptide fragments were produced by successive cleavage of the fragmented peptides. Cleavage tests for H. pylori urease by HpU-2-H revealed that the beta-subunit of the urease was cleaved first and completely decomposed at 20 h of incubation. Cleavage of the alpha-subunit started after the complete decomposition of the beta-subunit. These cleavage results were in good agreement with the immunological features of HpU-2-H. The irrelevant proteins, BSA and HSA, were hardly cleaved by HpU-2-H.

Antigenase has an ability to decompose the antigen peptide or protein. We have produced some monoclonal antibodies(HpU mAbs series) for H. pylori urease. Out of them, the light chain of HpU-9 mAb possesses a catalytic triad composed of Asp, Ser and His, which acts as a catalytic site against the antigen, based on the structural analysis of molecular modeling. HpU-9-L belongs to the germline cs1 which inherently encodes the catalytic triads in the sequence, indicating that HpU-9-L must be an antigenase. As expected, HpU-9-L showed the specific degradation against the beta-subunit of the urease. The heavy chain of HpU-2 also exhibited the specific degradation of the urease. These antigenases will be used for the medicinal application.

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria 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 antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-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 x 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 efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-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 preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum., (Copyright 2004 Wiley Periodicals, Inc.)

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit., (Copyright 2004 Wiley Periodicals, Inc.)

By immunizing ground-state peptides or proteins, we can produce super catalytic antibodies possessing serine protease-like characteristics. The unique feature of super catalytic antibodies is their ability to decompose a target molecule that is being killed. The authors have succeeded in preparing super catalytic antibodies that destroy (i) the HIV-1 envelope protein gp41, (ii) chemokine receptor CCR5 peptide, and (iii) Helicobacter pylori urease, etc. Some of them can degrade antigens at high catalytic reaction rates. Regarding their Km and kcat, super catalytic antibodies show intermediary values between that of enzymes (high Km and kcat) and that of antibodies (low Km and kcat [=0]). The catalytic function of an antibody mostly resides in its light chain. From mouse Vκ germline analysis, it became clear that super catalytic antibodies are generated from some discrete germlines such as bb1, cr1, cs1, bl1, bj2 and bd2. In these Vκ germlines, at least one catalytic triad composed of three amino acid residues, namely, Asp1, Ser27a and His93, is encoded. Namely, the antibody light chains (super catalytic antibodies) generated from the germlines are inherently able to enzymatically decompose antigens. Thus, such antibody light chains can be referred to as antigenase (antigen-decomposing enzyme) and may have arisen during the evolution of antibodies to acquire a higher ability than that of enzymes for developing a sophisticated self-defense system for survival. [Copyright &y& Elsevier]