Abstract:
The present invention identifies the apoptosis-inducing protein of  Helicobacter pylori,  analyzes the structure and provides the amino acid sequence of the protein and the nucleotide sequence encoding the amino acid sequence. Furthermore, the present invention provides inhibiting agents of an apoptosis-inducing activity of the bacterium for the treatment of disease ascribed to the bacterium and a vaccine to prevent the infection of the bacterium.  
     The present invention revealed that the apoptosis-inducing protein of  Helicobacter pylon  is a protein complex composed by association of two kinds of proteins, which comprise a part of the amino acid sequence encoded by γ-glutamyl transferase gene (HP1118). The present invention is said protein and a vaccine comprising said protein.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to an apoptosis-inducing protein from  Helicobacter pylori  and, more specifically, relates to two novel proteins constituting the apoptosis-inducing protein.  
       PRIOR ART  
       [0002]      Helicobacter pylon  infects persistently human stomach and has been implicated in the pathogenesis of gastritis and gastric ulcer.  Helicobacter pylori  infection induces apoptosis in gastric epithelial cells, which plays a significant role in the development of the pathological outcomes. Conventionally, an eradication therapy using antibiotics has been applied to the  Helicobacter pylori  infection, however, antibiotics-resistant strains have been emerging and at times the eradication therapy by antibiotics is unsuccessful. Consequently, a new methodology of treatment and prevention has been desired.  
         [0003]     A method for inhibiting the apoptosis-inducing activity of  Helicobacter pylori  is a very promising method. By neutralizing the apoptosis-inducing activity of the bacterium, apoptosis in gastric epithelial cells could be inhibited. Therefore, it is highly possible to suppress the development of gastritis and gastric ulcer.  
         [0004]     To date, as the apoptosis-inducing substances produced by the bacterium, Cag A protein, urease, lipopolysaccharide (LPS), and vacuolus toxins have been studied (WO98/32467 etc.), however, the contribution of these substances in  Helicobacter pylori -mediated apoptosis is rather small and the presence of other unknown factor has been expected for induction of the apoptosis (K. Shibayama et al., Infection and Immunity vol. 69, No. 5, 3181-3189 (2001)).  
         [0005]     On the other hand, the total genome of  Helicobacter pylori  was determined in 1997 by genome project (Tomb J. F., et al., Nature Vol. 388,539-547,1997) and the whole nucleotide sequence has been registered in a database. However, the nucleotide sequence encoding the proteins, which have an apoptosis-inducing activity, has not been specified.  
       PROBLEMS TO BE SOLVED BY THE INVENTION  
       [0006]     The objective of the present invention is to identify the apoptosis-inducing protein of the bacterium, to analyze the structure and to provide the amino acid sequence of the protein and the nucleotide sequence encoding the amino acid sequence. Further objective of the present invention is to provide inhibiting agents of an apoptosis-inducing activity of the bacterium for the treatment of the disease ascribed to the bacterium and a vaccine to prevent the infection of the bacterium.  
       MEANS TO SOLVE THE PROBLEMS  
       [0007]     The inventors of the present invention succeeded in purifying and in identifying the protein with the apoptosis-inducing activity from  Helicobacter pylori  and accomplished the present invention. The apoptosis-inducing protein was found to be composed of two proteins. Analysis of the amino acid sequence of these two proteins and retrieval from the database indicated that both these proteins are encoded by the HP1118 gene. The gene is reported to encode γ-glutamyl transferase (Chevalier C., et al., Molecular Microbiology, vol. 31 1359-1372, 1999). However, the apoptosis-inducing activity of the protein has not examined in their report or in any other reports. Furthermore, γ-glutamyl transferase is a ubiquitous enzyme found throughout bacteria and various biological species, and functions to incorporate amino acids into cytoplasm. However, it has not been reported that the enzyme has the apoptosis-inducing activity.  
         [0008]     In other words, the present invention demonstrated for the first time that a protein complex formed by association of two proteins, either of which is constituted by a part of the amino acid sequence encoded by γ-glutamyl transferase gene (HP1118), has an apoptosis-inducing activity of  Helicobacter pylori.    
         [0009]     Namely, the present invention is an apoptosis-inducing protein of  Helicobacter pylori  comprising two kinds of proteins having an amino acid sequence of SEQ ID NO: 1 or 2, or two kinds of proteins having an amino acid sequence of SEQ ID NO: 1 or 2 wherein one or several amino acids with respect to the amino acid sequence of SEQ ID NO: 1 or 2 is/are deleted, substituted or added, and having an apoptosis-inducing activity in human gastric adenocarcinoma cell line.  
         [0010]     Furthermore, the present invention is a protein comprising either of the following proteins:  
         [0011]     (a) a protein having an amino acid sequence of SEQ ID NO: 1.  
         [0012]     (b) a protein having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of SEQ ID NO: 1, and having an apoptosis-inducing activity in human gastric adenocarcinoma cell line by association with the protein comprising the amino acid sequence of SEQ ID NO: 2.  
         [0013]     Moreover, the present invention is a protein comprising either of the following proteins:  
         [0014]     (a) a protein having an amino acid sequence of SEQ ID NO: 2.  
         [0015]     (b) a protein having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of SEQ ID NO: 2, and having an apoptosis-inducing activity in human gastric adenocarcinoma cell line by association with the protein comprising the amino acid sequence of SEQ ID NO: 1.  
         [0016]     In addition, the present invention is the apoptotic-inducing protein of  Helicobacter pylori  comprising said two proteins.  
         [0017]     Still furthermore, the present invention is a vaccine comprising one of said proteins, wherein a part of the amino acid sequence of SEQ ID NO: 1 and 2 is deleted, substituted or added, and said protein has little or no apoptosis-inducing activity in gastric adenocarcinoma cell line and possesses a defensive effect against the infection of  Helicobacter pylori.    
         [0018]     The defensive effect of the infection could be examined by the following methods: The animal model for  Helicobacter pylori  infection has been established using Mongolian gerbil. Candidate protein for vaccination is administered to Mongolian gerbils by oral administration, transnasal administration, intramuscular injection or subcutaneous injection to induce immune response. Subsequently, live  Helicobacter pylori  are administered orally and the infection established is examined. If defensive effect against the infection is positive in the animal test, then the effect in human is examined in the same way.  
         [0019]     Still moreover, the present invention are respective genes encoding said two kinds of proteins and a method for distinguishing between pathogenic strains and nonpathogenic strains of  Helicobacter pylori , comprising a step of amplifying at least one of the whole or a part of said genes. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]      FIG. 1  shows the cytotoxic activity (the absorbance at 450 nm) of each fraction separated by a cation exchange column (HiTrap SP column, Pharmacia) in Example 1. The lower the value is, the fewer the viable cells are.  
         [0021]      FIG. 2  shows the cytotoxic activity (the absorbance at 450 nm) of each fraction separated by a hydrophobic column (HiTrap Phenyl column, Pharmacia) in Example 1. The lower the value is, the fewer the viable cells are.  
         [0022]      FIG. 3  shows the cytotoxic activity (the absorbance at 450 nm) of each fraction separated by a gel filtration column (Pharmacia) in Example 1. The lower the value is, the fewer the viable cells are.  
         [0023]      FIG. 4  shows the cytotocic activity (the absorbance at 450 nm) of each fraction separated by a cation exchange column (Mono S column, Pharmacia) in Example 1. The lower the value is, the fewer the viable cells are.  
         [0024]      FIG. 5  shows Native-PAGE profile of the purified apoptosis-inducing protein from  Helicobacter pylori.    
         [0025]      FIG. 6  shows SDS-PAGE profile of the purified apoptosis-inducing protein from  Helicobacter pylori.    
         [0026]      FIG. 7  shows the cytotoxic activity (the absorbance at 450 nm) of the purified apoptosis-inducing protein from  Helicobacter pylori  examined with AGS cells and HeLa cells. The lower the value is, the fewer the viable cells are.  
         [0027]      FIG. 8  shows the cytotoxic activity (the absorbance at 450 nm) of the purified apoptosis-inducing protein from  Helicobacter pylori  examined with KATO III cells. The lower the value is, the fewer the viable cells are.  
         [0028]      FIG. 9  shows the rate (%) of apoptotic cells of AGS cells induced by purified apoptosis-inducing protein from  Helicobacter pylori.    
         [0029]      FIG. 10  shows the amino acid sequence of the protein (567 amino acid residues) transcribed from the HP1118 gene and the amino acid sequences of subunit 1 (SEQ ID NO: 1) and subunit 2 (SEQ ID NO: 2) of the apoptosis-inducing protein from  Helicobacter pylori.    
         [0030]      FIG. 11  shows the schematic diagram of the apoptosis-inducing protein from  Helicobacter pylori.   
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0031]     Since the protein complex of the present invention, composed by thee association of two kinds of proteins, has the apoptosis-inducing activity of  Helicobacter pylori,  it can be used as the target molecule for the treatment of gastritis and stomach ulcer induced by  Helicobacter pylori.    
         [0032]     For example, the protein complex can be prepared either by introducing the genes encoding two subunits into expression vectors, e.g. in  E. coli,  separately and by mixing the subunits expressed independently, or by introducing a gene encoding both two subunits into an expression vector, e.g. in  E. coli  and by expressing these subunits at the same time.  
         [0033]     After purification, the protein complex can be used to seek medical agents inhibiting an apoptosis-inducing activity. The apoptosis-inducing activity can be assayed by a conventional method for examining the apoptosis of gastric adenocarcinoma cell line.  
         [0034]     The protein of the present invention can be applied to the development of a vaccine inhibiting the apoptosis-inducing activity of  Helicobacter pylori.    
         [0035]     For example, an animal could be immunized by an oral administration, transnasal administration or subcutaneous injection of the purified said protein complex as an antigen. Then, the animal is infected by the bacterium by an oral administration and is examined whether a defensive effect against the bacterium infection could be established. When the defensive effect in the animal is positive, the effect could be examined for humans. Furthermore, if there are common antigens between said protein complex and substances produced by human cells, the amino acid sequence of the common antigen could be examined and removed from said antigenic protein complex. A defensive effect against the infection could be examined by using the prepared protein, from which the common antigenic part is removed. Finally, the protein thus prepared, with a defensive effect, could be used as a vaccine.  
         [0036]     Moreover, a mutation could be introduced into the gene encoding the apoptosis-inducing protein of  Helicobacter pylori  and the protein without the apoptosis-inducing activity, which is prepared from the modified gene, could be used as a vaccine.  
         [0037]     Still furthermore, a novel  Helicobacter pylori  strain without the pathogenic activity could be prepared by disrupting the gene encoding the apoptosis-inducing protein of  Helicobacter pylori  and could be used as a vaccine.  
         [0038]     Based on the nucleotide sequence of the gene encoding the apoptosis-inducing protein of  Helicobacter pylori  according to the present invention, it is possible to prepare PCR primers to amplify the gene. The PCR primers could be used to differentiate pathogenic  Helicobacter pylori  from nonpathogenic one.  
         [0039]     The following examples illustrate this invention, however, it is not intended to limit the scope of this invention.  
       EXAMPLE 1  
       [0040]     In this example, purification and identification of the apoptosis-inducing protein of  Helicobacter pylori  were conducted.  
         [0041]     In this example, the apoptosis-inducing activity was measured as the cytotoxic activity in human gastric adenocarcinoma cell line AGS (ATCC CRL 1739) using Quick Cell Proliferation Assay Kit (Cat.BV-K301-10) (BioVision). The cell viability (surviving cell), from which an apoptosis-inducing activity is calculated, is based on the activity of intracellular mitochondrial dehydrogenase activity as a marker enzyme and is measured by the absorbance at 450 nm after 1 hr incubation at 37° C. with the substrate of the enzyme. The absorbance indicates the cell viability. The more cytotoxic, the less the absorbance is according to the mesurement.  
         [0042]     First of all,  Helicobacter pylori  strain 26695 (ATCC CTL 700392) was grown in 1000 ml of BHI medium (Becton Dikkinson Co., Brain Heart Infusion N0. 237500) supplemented with 10% serum. After harvest by centrifugation, the bacteria were suspended in PBS (Phosphate buffered saline, physiological saline (0.9% NaCl) adjusted to pH 7.4 with 50 mM sodium phosphate) and washed by centrifugation. The bacterial pellet was resuspended in 50 ml of PBS and disrupted by a French press at 120 Mpa. After low speed centrifugation at 5000×g for 10 min to remove the unbroken bacteria, membrane fraction was sedimented by centrifuging at 100,000×g for 2 hrs. The membrane fractions were suspended in 50 mM phosphate buffer (pH 6.0) containing 1 M NaCl and again centrifuged at 100,000×g for 2 hrs. During the procedures, the apoptosis-inducing protein was separated from membrane fractions and was solubilized in the supernatant fraction.  
         [0043]     The membrane fractions were suspended in 50 mM phosphate buffer (pH 6.0) containing 1 M NaCl and again centrifuged at 100,000×g for 2 hrs. During the procedures, the apoptosis-inducing protein was separated from membrane fractions and was solubilized in the supernatant fraction.  
         [0044]     From the supernatant fraction, the apoptosis-inducing protein was purified in the following way.  
         [0045]     In the beginning, the supernatant fraction was dialyzed against 250 mM NaCl-containing buffer, loaded in a cation exchange column (HiTrap SP column, Pharmacia), and fractionated by eluting with the gradient of 250-500 mM NaCl. Then, each fraction was assessed on the apoptosis-inducing activity.  
         [0046]      FIG. 1  shows the result. Fractions 10 to 12 contained the activity. These fractions were collected and used for the subsequent purification steps.  
         [0047]     Then, the collected fraction containing the activity was loaded in a hydrophobic column (HiTrap Phenyl, Pharmacia) and fractionated by eluting with the gradient of 20-10% ammonium sulfate. Then, each fraction was assessed on the apoptosis-inducing activity.  FIG. 2  shows the results. Fractions 8 to 12 contained the activity. These fractions were collected and used for the following purification steps.  
         [0048]     Then, the collected fraction containing the activity was loaded in a gel-filtration column (Pharmacia), fractionated by 50 mM phosphate buffer (pH 6.0) containing 500 mM NaCl and each fraction was assessed on the apoptosis-inducing activity.  FIG. 3  shows the results. Fractions 12 to 14 contained the activity. These fractions were collected and used for the following purification steps.  
         [0049]     Then, the collected fraction containing the activity was dialyzed against 250 mM NaCl-containing buffer, loaded in a cation exchange column (MonoS column, Pharmacia) and fractionated by eluting with the gradient of 250-500 mM NaCl. Then, each fraction was assessed on the cytotoxic activity.  FIG. 4  shows the results. Fractions 7 and 8 contained the activity. These fractions were collected.  
         [0050]     The purified protein obtained by the above procedures was analyzed by a native PAGE under acid condition. As shown in  FIG. 5 , a single band was observed. The single band was subsequently analyzed by 2D SDS-PAGE. As shown in  FIG. 6 , the band was separated into two bands (their molecular weight were about 40 kDa and 22 kDa).  
         [0051]     In native gel, which is free from strong detergent such as SDS in acryl amide, the three dimensional structure of the protein is preserved. Observation of the single band indicates that the protein is composed of a single complex in its native state.  
         [0052]     On the other hand, in SDS-PAGE, the three dimensional structure of the protein is disrupted by SDS and multiple protein subunits are separated. The result that a single band in native PAGE is separated into two bands in SDS-PAGE indicates that the protein complex is composed of the two protein subunits.  
         [0053]     In other words, the apoptosis-inducing protein from  Helicobacter pylori  is a protein constructed by an association of two kinds of subunits.  
       EXAMPLE 2  
       [0054]     In this example, the apoptosis-inducing activity of the protein purified in example 1 was examined in human gastric adenocarcinoma cell line AGS (ATCC CRL 1739), in human gastric carcinoma cell line KATO III (Human Science Research Resources Bank, Cat No. JCRB0611 (ATCC, HTB-103)) and in human uterine cervix cancer-derived HeLa cell (Human Science Research Resources Bank, Cat No. JCRB9004).  
         [0055]     The cytotoxic effect was assayed using WST-1 kit (Roche Diagnotics). The results are shown in  FIGS. 7 and 8 .  
         [0056]     As shown in these figures, the protein purified in example 1 has the cytotoxic activity in all the above cell lines.  
         [0057]     Also, the induction of apoptosis in AGS cells by the protein was examined: Trypsinized apoptotic and control cells were collected and stained with a fluorescent dye (Hoechst 33342), which stains cellular nuclei. Since the nuclei of apoptotic cells are highly condensed and fragmented, apoptotic cells are easily differentiated from control cells under fluorescence microscopy. Cells were put on a slide glass, the number of apoptotic cells and control cells were counted and the percentage of apoptotic cells in total cells was enumerated. At least total 500 cells were counted for each experiment.  
         [0058]     The results are shown in  FIG. 9 . It was confirmed that the cytotoxic effect of the protein against AGS cells was mainly ascribed to apoptosis.  
       EXAMPLE 3  
       [0059]     The amino acid sequencing of N-terminal end of the two subunits purified in example 1 indicated that both subunits were encoded by the HP1118 gene.  FIG. 10  shows the amino acid sequence of two subunits (subunits 1 and 2) together with that of the HP1118 gene. More specifically, the subunit 1 (SEQ ID NO: 1) and the subunit 2 (SEQ ID NO: 2) comprise amino acids 27-379 and 380-567, respectively, of the amino acid sequence encoded by the HP1118 gene.  
         [0060]     As above described, the apoptosis-inducing protein of  Helicobacter pylori  is a protein complex composed of two protein subunits as shown in  FIG. 11 : one (subunit 1) is produced by the cleavage between 26 th  and 27 th  and between 379 th  and 380 th  of the amino acid residues of the protein transcribed from the HP1118 gene and the other (subunit 2) is produced by the cleavage between 379 th  and 380 th  of the amino acid residues of said protein.