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Originally published In Press as doi:10.1074/jbc.M507545200 on December 14, 2005

J. Biol. Chem., Vol. 281, Issue 7, 3995-4001, February 17, 2006
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Characterization of a Counterpart to Mammalian Ornithine Decarboxylase Antizyme in Prokaryotes*

Yoshihiro Yamaguchi{ddagger}1, Yumiko Takatsuka{ddagger}1, Senya Matsufuji§, Yasuko Murakami§, and Yoshiyuki Kamio{ddagger}2

From the {ddagger}Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, and the §Second Department of Biochemistry, Jikei University School of Medicine, Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan

Received for publication, July 12, 2005 , and in revised form, December 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The degradation of mammalian ornithine decarboxylase (ODC) (EC 4.1.1.17 [EC] ) by 26 S proteasome, is accelerated by the ODC antizyme (AZ), a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryotes, AZ has not been found. Previously, we found that in Selenomonas ruminantium, a strictly anaerobic and Gram-negative bacterium, a drastic degradation of lysine decarboxylase (LDC; EC 4.1.1.18 [EC] ), which has decarboxylase activities toward both L-lysine and L-ornithine with similar Km values, occurs upon entry into the stationary phase of cell growth by protease together with a protein of 22 kDa (P22). Here, we show that P22 is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium LDC by a 26 S proteasome. (iii) S. ruminantium LDC degradation is also enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ. In this report, we also show that P22 is a ribosomal protein of S. ruminantium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells and tissues, turnover of ornithine decarboxylase (ODC)3 (EC 4.1.1.17 [EC] ) is very rapid and highly regulated. The degradation of mammalian ODC catalyzed by 26 S proteasome is accelerated by ODC antizyme (AZ), a trigger protein involved in a specific degradation of eukaryotic ODC (1–3). In prokaryotes, S20, L26, and L34 ribosomal proteins were reported as antizymes in Escherichia coli (4). However, Kashiwagi et al. (5) and Ivanov et al. (6) independently reported that none of the three E. coli ribosomal proteins is a "true antizyme" because of their lack of acceleration activity to E. coli ODC degradation. Therefore, no antizymes or direct counterparts of eukaryote antizymes have been found in prokaryotes so far.

Previously, we found cadaverine covalently linked to the peptidoglycan in Selenomonas ruminantium, a strictly anaerobic bacterium, as an essential constituent of the peptidoglycan to maintain the integrity of the cell envelope of this strain. We also found that cadaverine is synthesized constitutively from L-lysine catalyzed by LDC (EC 4.1.1.18 [EC] ) and transferred to the D-glutamic acid residue of the lipid intermediate for synthesis of the peptidoglycan by a lipid intermediate: cadaverine transferase (7–10). S. ruminantium LDC was purified and characterized, its gene (ldc) was cloned (11–13), and the following findings were reported. (i) S. ruminantium LDC consists of two identical monomeric subunits of 45 kDa each and decarboxylates both L-lysine and L-ornithine with similar Km and Vmax values; its decarboxylating activities toward both substrates were completely prevented by DL-{alpha}-difluoromethyl ornithine, a compound previously shown to be a specific inhibitor of eukaryotic ODC (14). (ii) The amino acid sequence of S. ruminantium LDC is 35% identical and 53–60% similar to those of eukaryotic ODCs; 26 amino acid residues, all of which are implicated either in contributing to pyridoxal phosphate- and substrate-binding domains or in formation of the homodimeric forms of eukaryotic ODCs, are conserved in S. ruminantium LDC (13). (iii) S. ruminantium LDC has a sequence homologous to that of the mouse AZ binding region in mouse ODC. (iv) S. ruminantium LDC is classified as a fold type III protein similar to eukaryotic ODCs but not to bacterial ODC or other LDCs. In addition, S. ruminantium has no typical ODC with decarboxylase activity exclusively toward L-ornithine (12). These findings show that S. ruminantium LDC and eukaryotic ODC resemble each other in both biochemical and biophysical characteristics except for the broad substrate specificity of S. ruminantium LDC.

The production of LDC in S. ruminantium is highly regulated and is strictly linked to the growth phase (i.e. a drastic decrease in LDC activity occurs upon entry into the stationary phase of cell growth, which is due to the rapid degradation of LDC) (12). In the current work, we isolated a new protein of 22 kDa (P22), which is induced in putrescine-grown cells as a regulating factor for the degradation of LDC in S. ruminantium cells by a protease whose activity requires ATP (15). Here, we demonstrate that P22 is a direct counterpart of mammalian ODC AZ for the degradation of S. ruminantium LDC by a protease that requires ATP for its activity. In this study, we also show that P22 is a constituent of the ribosome of S. ruminantium. DNA sequence analysis shows that P22 exhibits 47% identity and 60–66% similarity in amino acid sequence compared with those of the reported ribosomal L10 proteins of bacteria.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Plasmid vectors, pMW119 and pET15b, were purchased from Nippon gene (Tokyo, Japan) and Novagen (Madison, WI), respectively. Restriction enzymes, T4 DNA ligase and TaqDNA polymerase, were from Takara (Otsu, Japan). HiTrap chelating HP column, Hybond ECL membrane, and RedivueTM Pro-mixTM L-[35S] in vitro labeling mix containing 35S-labeled L-methionine and 35S-labeled L-cysteine (37 TBq/mmol), were obtained from Amersham Biosciences. A 26 S proteasome was obtained from Immatics Biotechnologies (Tübingen, Germany). An anti-ubiquitin antibody was from Hokudo (Sapporo, Japan). Creatine kinase and Freund's complete adjuvant were from Wako Pure Chemical Industries (Osaka, Japan). Anti-rabbit or anti-mouse IgG (Fc)-alkaline phosphatase conjugate was a product of Promega (Madison, WI). The Biacore X instrument, the CM5 sensor chip, and the reagents for the surface plasmon resonance experiments were all delivered by Biacore AB (Uppsala, Sweden). ABI Prism 377 DNA sequencer and ABI PRIZM BigDye Terminator Cycle Sequencing Ready Reaction kit were from Applied Biosystems (Foster City, CA).

Bacterial Strains and Culture Conditions—The strains used in the present study included S. ruminantium ssp. lactilytica (16) and E. coli BL21(DE3), which were used as the host strains. Culture media included a tryptone-yeast extract-glucose medium (16) and L broth for the growth of S. ruminantium and E. coli strains, respectively. S. ruminantium was grown under the anaerobic conditions described previously (16).

Cloning of the P22 Structural Gene from Chromosomal DNA of S. ruminantium—The chromosomal DNA of S. ruminantium, which was isolated and purified by standard methods, was digested by various restriction endonucleases, and resultant fragments were analyzed by Southern blot hybridization with the fluorescein-labeled DNA probe 5'-AARGAYCARNNNACNAAYGCNAARGG-3'; this probe was synthesized based on the known N-terminal amino acid sequence, 15KDQXTNAKG23 of the pure P22 protein (in the sequence of the probe, R, N, and Y represent A or G; A, G, C, or T; and C or T, respectively). The screening of a PstI library of the S. ruminantium chromosomal DNA gave a single positive clone from ~100,000 colonies. The positive clone contained an insert of 912 bp whose sequence encoded the N-terminal region but not the full-length P22 protein. Therefore, to obtain the full-length p22 coding sequence, S. ruminantium chromosomal DNA was digested with EcoRI or HindIII, followed by self-ligation with T4 DNA ligase. The self-ligated and circularized DNA fragments were used as templates for amplification of the p22 gene by PCR with two primers (P917-Fw, 5'-ATTTCATCGGAACCAACGAAGTCTGCA-3'; P917-Rv, 5'-TGAACTTCGCGCCGCTGGTGTAACGTA-3') reading outward from the known sequence of the p22 gene. The amplified product (6,041 bp from the EcoRI digest or 4,050 bp from the HindIII digest) was purified by agarose electrophoresis and inserted into the corresponding EcoRI or HindIII sites of the pMW119 vector. The DNA sequencing of the cloned fragments was done using an ABI Prism 377 DNA sequencer with an ABI PRIZM BigDye terminator cycle sequencing ready reaction kit.

Homology Searches, Identification of ORFs, and Multiple Sequence Alignments of the Nucleotide and Amino Acid Sequences—Protein and nucleotide sequences were compared with the sequence data base using the BLAST (version 2.0) programs implemented at the EMBL/GenBankTM/DDBJ nucleotide sequence data base. Open reading frame (ORF) identification and multiple sequence alignments were performed using the GENETYX program (Software development Co., Tokyo, Japan).

Construction of a Plasmid for P22 Expression in a T7 Promoter-dependent Expression System—For expression of recombinant P22 (rP22), a DNA fragment containing only the p22 structural gene was amplified from the chromosomal DNA by PCR using primers 5'-TTTCATATGGCAAATATG ACGAAG-3' and 5'-TTTGGATCCTTATGCGGATTCCTTCTGAGCGCGAACAGCG-3' (single and double underlined sequences represent the NdeI and BamHI restriction sites), which are located upstream and downstream of p22, respectively. The amplified fragment was digested with NdeI and BamHI, and the NdeI-BamHI fragment was inserted into the NdeI-BamHI site of an expression vector pET15b with an N-terminal His tag sequence to construct plasmid pEP22, in which p22 was under the control of the T7 promoter.

Isolation and Purification of a His-tagged Recombinant P22 Protein from E. coli BL21(pE22k)—Cells of E. coli BL21(pE22k) were grown in LB (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl) medium containing ampicillin (100 µg/ml) at 37 °C with shaking to an optical density of 0.6 at 660 nm; isopropyl-beta-D-galactopyranoside was added at a final concentration of 1.0 mM, followed by incubation with shaking for an additional 3 h. The cells were collected by centrifugation at 4 °C and disrupted in a French pressure cell. The cell lysate was centrifuged at 18,000 x g, and the precipitate was dissolved in 50 ml of 20 mM sodium phosphate buffer (pH 6.8, buffer A) containing 6 M urea. The crude extract thus obtained was centrifuged at 200,000 x g for 1 h. The supernatant was applied a 1-ml HiTrap chelating HP column preloaded with nickel sulfate and equilibrated with buffer A. After washing the column extensively with buffer A, His-tagged P22 was eluted from the column with buffer A containing 50 mM imidazole, and the eluate was dialyzed for a total of 36 h at 4 °C against 80 mM ammonium acetate buffer (pH 6.0) containing successive concentrations of urea, at 4, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 M urea, continuously. As a final step, the sample was dialyzed against buffer A at 4 °C for 4 h to obtain a clear sample. The N-terminal amino acid residues of the purified, recombinant P22 preparation were identical with those expected from the expression plasmid sequence.

Preparation of Anti-P22 Antiserum—Mouse antiserum raised against recombinant P22 was prepared by the following method. One ml of the purified P22 (1 mg/ml) was emulsified with 1 ml of Freund's complete adjuvant and injected subcutaneously into a female BALB/C mouse. Two weeks later, a second injection of the recombinant protein emulsified with Freud's incomplete adjuvant was given in the same way, followed by four additional injections every 2 weeks. Two weeks after the seventh injection, the mouse was anesthetized and killed for preparation of antiserum.

Construction of Plasmids for Expression of S. ruminantium LDC and Its Mutants and for E. coli Ribosomal Protein L10—Expression of plasmids for LDC and its mutants and for E. coli ribosomal protein L10 (RplJ) was generated from vector pET15b. The plasmid pELDC to express wild type LDC was constructed from pTLDC (13) by PCR, using the primers 5'-TTTTTTCATATGAAAA ATTTCAGACTTAG-3' and 5'-ATATCCGGATCCATGCATCTGCAGTAATCCAGCTCCTGCA-3' (single and double underlined sequences represent the NdeI and BamHI restriction sites, respectively). The coding sequence for the LDC mutant in which the Lys103, Lys123, and Lys126 residues are replaced by Ala residues was constructed by an overlapping-extension method (17, 18), using three pairs of oligonucleotide primers, KARv (5'-GTCAGCCGCCGCAGCCGCGAGGCCACGGGC-3') and KAFw (5'-GGCCTCGCTGCTGCGGCTGA-3'), KKAAFw (5'-CGGAAATCGACGCGATGGCCGCGGCTGTGCCG-3') and KKAARv (5'-CCCGGCACAGCCGCGGCCATCGCGTCGATTTCCG-3'), and T7Fw (5'-TAATACGACTCACTGTA-3') and T7Rv (5'-GCTAGTTATTGCTCAGCGG-3'). Mutations in each of the amplified products were confirmed by DNA sequencing. The E. coli ribosomal protein L10 gene was amplified from E. coli chromosomal DNA by PCR, using the primers 5'-TTTTTTCATATGGCTTTAAATCTTCAAG-3' and 5'-TTTTTGGATCCTTAAGCAGCTTCTTTCGC-3' (single and double underlined sequences represent the NdeI and BamHI restriction sites, respectively). Recombinant LDC proteins were expressed in E. coli BL21(DE3) by induction at A660 = 0.6 with 1 mM isopropyl-beta-D-galactopyranoside for 3 h and purified by a HiTrap chelating HP column using a linear gradient from 0 to 300 mM imidazole in 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl. LDC and its mutants were eluted in a fraction containing ~150 mM imidazole. Recombinant E. coli ribosomal protein L10 (RplJ) was expressed and purified by the same method as that for rP22 purification.

Assay for rP22 Binding to LDC—Surface plasmon resonance experiments were performed with a Biacore X biosensor system at a sensor temperature of 25 °C. The His-tagged LDC preparation and bovine serum albumin were coupled to different flow cells of a sensor chip (CM5 research grade) via standard N-hydroxysuccinimide and N-ethyl-N-(dimethylaminopropyl) carbodiimide activation. Ethanolamine was then injected to quench the unreacted N-hydroxysuccinimide groups. The mobile phase buffer used was HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P-20). Analytes were injected at various concentrations, and the bound analytes were removed by washing with buffer 180 s after the injection. Data were analyzed with BIAevaluation (version 3.0) software using the global fitting model. Equilibrium dissociation constant values (KD) were calculated from the steady-state resonance unit (1 resonance unit = 1 ng/mm2) binding signal.

Inhibitory Effects of P22 and Mouse AZ on the S. ruminantium LDC Activity—Mouse AZ preparation was purified by the method described previously (19). The purified P22 or mouse AZ preparation was added to a reaction mixture containing 0.1 M Tris-HCl (pH 7.5), 0.1 mM pyridoxal phosphate and either the purified S. ruminantium LDC or a mouse ODC preparation. The mixture was incubated for 10 min at 0 °C, and then the remaining LDC or ODC activity was measured by the method described previously (12, 20).

Measurement of the Degradation Rate of LDC Protein—The rate of degradation of the LDC protein was determined by measuring the decay of radiolabeled LDC. S. ruminantium cells were anaerobically grown overnight at 37 °C in a chemically defined medium (21) lacking methionine. The culture was diluted 100-fold with TYG medium and incubated again at 37 °C. When the cell density reached 5 x 108 or 1.8 x 109 cells/ml, 37 MBq/ml L-[35S]methionine was added to the culture, and the incubation was continued. After 10 min, 2 mM cold L-methionine and chloramphenicol (10 µg/ml) was added to the cultures, and the incubation was continued. At 15-min intervals, the cells were harvested by centrifugation and boiled in 1% SDS in 50 mM sodium phosphate buffer (pH 6.5). After centrifuging at 100,000 x g, the supernatants were diluted 100-fold and incubated with anti-LDC antiserum at 4 °C for 1 h and then shaken with Protein G-Sepharose at 4 °C for 2 h. The mixture was centrifuged at 5,000 x g for 5 min. The precipitate was washed four times each with 1 ml of sodium phosphate buffer, pH 6.8, containing 0.1% Triton X-100 and then resuspended and boiled with solubilizing buffer for SDS-PAGE. After electrophoresis, the gels were dried, and the band corresponding to LDC was visualized by autoradiography. The degradation rate was calculated by measuring the radioisotope incorporated into LDC using Image Gauge (version 3.0) software. The percentage degradation of LDC was calculated from the ratio of the remaining amount of LDC to the amount of LDC at time 0.

Depletion of S. ruminantium P22 from Cell Extracts for Subsequent Protease Assays—The S. ruminantium cells were grown in TYG medium until the cell density reached 1.6–2.6 x 109/ml (at early stationary phase). To remove endogenous P22, S. ruminantium cell extract (200 µg of protein) was incubated in a reaction mixture (200 µl) containing 1 mM dithiothreitol and anti-P22 antiserum in 20 mM potassium phosphate, pH 6.5, at 4 °C for 60 min. Protein G-Sepharose 4B beads (20 µl of a 50% slurry) was then added to the sample followed by incubation for 2 h at 4° C. After centrifugation at 10,000 x g for 5 min, the supernatant was obtained. The removal of P22 from the supernatant was confirmed by immunoassay.

Preparation of 35S-Labeled LDC and Mouse ODC—35S-Labeled LDC and mouse ODC were prepared by the method described by Zhang et al. (22).

LDC or Mouse ODC Degradation Assay in Vitro—Degradation of LDC in the P22-free extract was carried out with or without rP22 in a basal reaction mixture (Mixture A), which contained 10 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, and P22-free extract (136 ng) in a total volume of 50 µl. The reaction mixture was treated with SDS (2%) in the presence of 2-mercaptoethanol (2%) at 100 °C for 5 min and analyzed on 12.5% SDS-PAGE. The proteins on the gel were transferred to a nitrocellulose membrane (Hybond-ECL) and then incubated with anti-LDC antiserum. The antigen-antibody complex was detected with anti-rabbit or anti-mouse IgG (Fc)-alkaline phosphatase conjugate. Bands were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as the substrate. The intensity of the LDC bands was quantitated using the NIH Image 1.61 program. Relative amounts of the remaining LDC protein were expressed as a percentage of the starting amount. For the degradation of S. ruminantium LDC by mouse AZ or RplJ in Mixture A, AZ (500 nM) or RplJ (600 nM) was used. For the degradation of S. ruminantium LDC in 26 S proteasome, which was confirmed to be free from ubiquitin (Ub) using anti-Ub antibody, the basal reaction mixture (Mixture B) contained 40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 0.5 mM ATP, 10 mM phosphocreatine, 5 µg of creatine kinase, 0.1 mM cycloheximide, 50 nM [35S]LDC, and 20 µg of 26 S proteasome in a total volume of 100 µl. Reactions were carried out at 37 °C by incubating Mixture B with AZ (500 nM), P22 (500 nM), AZ plus P22 plus MG132. The reaction mixtures were analyzed by SDS-PAGE and autoradiography. The radioactivity of bands corresponding to LDC was measured and quantified with Image Gauge (version 3.0). The degradation of mouse ODC in S. ruminantium P22-free extracts and in 26 S proteasome was done using 35S-labeled mouse ODC preparation (40 nM).

Preparation of a Ribosome Fraction from S. ruminantium and E. coli—Ribosomes were prepared from S. ruminantium cells at log phase. The cells were suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM NH4Cl, 10 mM magnesium acetate, and 5 mM 2-mercaptoethanol (buffer A) and sonicated. After cell debris was removed by centrifugation at 30,000 x g for 20 min at 4 °C, supernatant was further centrifuged at 200,000 x g for 2 h at 4° C. The precipitate thus obtained was resuspended in the same buffer and layered over an equal volume of a 1.1 M sucrose cushion made up in Tris-HCl (pH 7.5) containing 500 mM NH4Cl, 10 mM magnesium acetate, and 5 mM 2-mercaptoethanol and centrifuged at 100,000 x g for 17 h at 4 °C. The pellet was resuspended in 20 mM Tris-HCl (pH 7.5) containing 100 mM NH4Cl, 1 mM magnesium acetate, and 5 mM 2-mercaptoethanol (buffer B) and then dialyzed against the same buffer for 6 h at 4°C. One hundred fifty µl of the dialyzed sample was layered on 11 ml of 10–30% linear sucrose gradient prepared in buffer B and centrifuged at 50,500 x g for 16 h at 4 °C using a Beckman SW40.2Ti rotor. The contents were fractionated and subjected to Western blotting analysis using anti-P22 antiserum.

Miscellaneous—Protein was measured by the method of Bradford using bovine serum albumin as a standard (23). The N-terminal amino acid sequences of the P22 preparation were determined by the methods described previously (13). SDS-PAGE was carried out by the method described previously (24).

Nucleotide Sequence Accession Number—The nucleotide sequences of the p22 gene and seven open reading frames have been deposited in the GenBankTM/EMBL/DDBJ data bases under accession number AB100500 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nucleotide Sequence of the S. ruminantium P22 Gene and Its Flanking Region—The determined 8.5-kbp sequence included eight ORFs (Fig. 1). ORF1 encoded a 179-amino acid protein of 19,312 Da whose N-terminal sequence matched that determined for the purified P22 protein. The p22 gene, spanning positions 3744–4283 within the cloned genomic sequence, started with an ATG codon and ended with a TAA stop codon. Putative –35 (TTGATA) and –10 (TATTGT) sequences were found 140 and 114 bp upstream of the translation initiation codon of p22, respectively. Eleven base pairs upstream from the ATG codon, a ribosomal binding site consensus sequence (AGGAGG) was found at positions 3727–3733. No termination loop was found downstream of the TAA stop codon.


Figure 1
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  		FIGURE 1.
Restriction maps of the 8.5-kbp fragment from S. ruminantium chromosomal DNA including the p22 gene. The solid and open arrows show the p22 gene and seven other predicted ORFs and the directions of their transcription. A 1.0-kb PstI-PstI fragment used in colony hybridization is shown as horizontal arrows above the gene.

 


Figure 2
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  		FIGURE 2.
Amino acid sequence alignment of S. ruminantium P22 with the ribosomal protein L10 of Gram-positive bacteria. S. ruminantium P22 exhibits ~60% similarity to the ribosomal protein L10 of Gram-positive bacteria, including C. perfringens, S. aureus, and B. halodurans. On the consensus line below the aligned sequences, identical amino acids are indicated by asterisks, and conserved residues are indicated by dots.

 
Functional Analysis of the p22 Gene Product—To prove that p22 is the gene encoding S. ruminantium P22, the cloned sequence was expressed in E. coli; the recombinant P22 (rP22) was purified, and its characteristics were compared with those of the earlier P22 protein preparation (15). E. coli BL21(pE22) expressed P22 at a final concentration of ~2% of its total protein. The purified rP22 protein had characteristics identical to those of the native P22 protein, as judged by the apparent molecular mass (22 kDa) determined by SDS-PAGE and its degradation-promoting activity for S. ruminantium LDC. The degradation of LDC in P22-free extract was not observed (Fig. 3A). Thus, we concluded that ORF1 is the p22 gene of S. ruminantium.

Amino Acid Sequence Homologies of P22 with Bacterial Ribosomal Protein L10—The amino acid sequence of S. ruminantium P22 showed 47% identity and ~60–66% similarity to bacterial ribosomal L10 proteins from Clostridium perfringens, Staphylococcus aureus, and Bacillus halodurans (25–27) (Fig. 2). In addition, the two ORFs (ORF2 and ORF3 in Fig. 1) just upstream of p22, consisting of 423 and 678 nucleotides, which are tentatively called protein 2 and protein 3, respectively, and one ORF (ORF4 in Fig. 1), consisting of 366 nucleotides, which is tentatively called protein 4, just downstream of the p22 gene were found. The amino acid sequences of proteins 2–4 exhibited 75, 62, and 67% overall identity with those of L11, L1, and L7/L12, respectively, of the aforementioned three bacteria (25–27). ORF5, -6, -7, and -8 encoding 473-, 179-, 124-, and 347-amino acid proteins, which are tentatively called proteins 5, 6, 7, and 8, respectively, were also identified. The amino acid sequences of proteins 5, 6, and 8 exhibited 59, 23, and 29% overall identity with the transcription anti-termination protein NusG, multidrug efflux transporter NorM, and E. coli AidB regulator AbrB, respectively (25, 28, 29). ORF7 encoded a hypothetical protein of unknown function.


Figure 3
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  		FIGURE 3.
Degradation of S. ruminantium LDC and mouse ODC in vitro. A, the degradation of LDC in P22-free extracts by the rP22 preparation. Mixture A (see "Experimental Procedures") was incubated at 37 °C in the presence (open circles) or absence (closed circles) of the purified P22 preparation (500 nM). Relative amounts of the remaining LDC protein were expressed as a percentage of the starting amount. B, the degradation of S. ruminantium LDC in P22-free extracts by the purified mouse AZ preparation. The P22-free extract was incubated in Mixture B (see "Experimental Procedures") by P22 (500 nM) (open circles), AZ (500 nM)(closed circles), RplJ (600 nM)(open triangles), or nothing (closed triangles). The amount of the remaining LDC protein was expressed as a percentage of the starting amount. C, the degradation of S. ruminantium LDC in 26 S proteasome. The reaction was carried out at 37 °C by incubating Mixture B with AZ (500 nM)(open circles), P22 (500 nM)(closed circles), AZ plus P22 plus MG132 (closed triangles), or no additional proteins (open triangles). D, the degradation of mouse ODC in P22-free extracts and in 26 S proteasome. The 35S-labeled ODC preparation (40 nM) was incubated in S. ruminantium P22-free extracts (open symbols) or in 26 S proteasome (closed symbols) with AZ (circles), P22 (triangles), or neither (squares) as described in the legend for A and C. The labeled proteins remaining at the indicated period of incubation were expressed as a percentage of the starting amount.

 
Degradation of S. ruminantium LDC in a P22-free Cytoplasmic Fraction by Mouse AZ—Previously, we reported that the purified P22 protein strongly enhances LDC proteolysis, which requires ATP in cell-free extracts of S. ruminantium (15) and suggested that P22 is a mouse AZ-like enhancer for degradation of S. ruminantium LDC/ODC because of many similarities between LDC degradation and that of mouse ODC degraded by 26 S proteasome in the presence of mouse AZ. To test this proposal, the following experiment was done. Cytoplasmic fractions from the S. ruminantium cells, which were grown without putrescine, were treated with anti-P22 antiserum and assayed for LDC degradation activity in a reaction mixture containing 1 mM ATP, 10 mM MgCl2, 1 mM dithiothreitol, and 86 nM recombinant LDC protein. The following results were obtained (Fig. 3B): (i) no LDC degradation was observed in P22-free extract, (ii) LDC degradation occurred upon the addition of the purified rP22 to the P22-free extract, (iii) the degradation of a G350D mutant of LDC (13), which forms only monomers, was enhanced by rP22 more efficiently than that for the native LDC, and (iv) the addition of a purified recombinant mouse AZ preparation instead of rP22 in the P22-free extract enhanced LDC degradation to the same extent as did rP22. The rP22 protein could not be replaced by a purified preparation of E. coli ribosomal protein L10 in enhancing LDC degradation. Thus, we conclude that mouse AZ as well as P22 are trigger proteins for the degradation of S. ruminantium LDC in our cell-free system. The data also suggest that P22 and mouse AZ function similarly in the degradation of LDC.


Figure 4
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  		FIGURE 4.
Inhibition of LDC and ODC activities by P22 and mouse AZ. To measure the inhibition of LDC (open circles) and mouse ODC activities (closed circles) by P22 and AZ, the LDC and ODC preparations were incubated on ice with the indicated amounts of the mouse AZ or P22 preparations. After 10 min of incubation, 5 mM L-ornithine or 5 mM L-lysine was added, and each enzyme activity was determined by measuring the amount of cadaverine or putrescine formed from L-lysine or L-ornithine, respectively.

 
Degradation of S. ruminantium LDC by 26 S Proteasome in the Presence of either P22 or Mouse AZ—As a next step, we examined LDC degradation in 26 S proteasome free from Ub and Ub-conjugating enzymes. As shown in Fig. 3C, the degradation of S. ruminantium LDC in 26 S proteasome occurred to the same extent as in the cell-free system of S. ruminantium. The degradation of LDC required ATP and either mouse AZ or P22. No degradation products of LDC in the reaction mixture were detected as visible bands on the polyacrylamide gel in SDS-PAGE, suggesting that rapid and complete proteolysis of LDC occurred under the conditions used in this study. These findings showed that S. ruminantium LDC was degraded specifically by the proteasome in an AZ- or P22-dependent manner. We examined whether or not MG132, which is known to be an inhibitor of the proteasome, inhibits LDC degradation in 26 S proteasome. As shown in Fig. 3C, MG132 clearly inhibited the degradation of LDC in the presence of either P22 or AZ. It is of interest to note that S. ruminantium LDC was degraded in the presence of anti-Ub antibody (Hokudo) in 26 S proteasome. Thus, we conclude that the degradation of LDC in 26 S proteasome is catalyzed by the proteasome and depends on AZ or P22 without ubiquitination.

Degradation of Mouse ODC Promoted by P22 as Well as Mouse AZ—The degradation of mouse ODC was examined in P22-free extract and 26 S proteasome. As shown in Fig. 3D, P22 exhibited a strong stimulating activity for the degradation of mouse ODC both in P22-free extract and 26 S proteasome. Moreover, the mouse AZ preparation accelerated the degradation of mouse ODC in our P22-free extract as well.

P22 Binding to LDC and Inhibition of LDC Activity in Vitro—The amino acid residues from Val117 to Met140 in mouse ODC were identified as the antizyme-binding region, in which basic Lys121, Lys141, and Arg144 residues have been suggested to be pivotal for AZ binding to mouse ODC (30). In S. ruminantium LDC, basic residues Lys103, Lys123, and Lys126 correspond to the putative antizyme-binding residues in mouse ODC (13). Molecular interactions of P22 or mouse AZ with S. ruminantium LDC were examined using BIAcore by the method described under "Experimental Procedures." Both P22 and mouse AZ bound to S. ruminantium LDC with KD values of 8.5 x 10–11 and 10.8 x 10–11 M, respectively. However, they did not bind to S. ruminantium LDC mutated in the AZ binding region, in which three Lys residues, Lys103, Lys123, and Lys126 were replaced by Ala residues, resulting in no degradation of the mutant LDC in S. ruminantium P22-free extract (Table 1). The kcat/Km ratio (catalytic efficiency) of the mutant LDC was same as that of wild type LDC (data not shown). This result suggest that no major conformational alterations occurred in the mutant enzyme. These results suggest a similar mechanism for LDC degradation during entry into the stationary phase of S. ruminantium cells compared with mouse ODC degradation. We also examined whether LDC and mouse ODC activities are inhibited by P22 and mouse AZ. Indeed, both P22 and AZ inhibited LDC/ODC activities of S. ruminantium LDC in our cell-free system (Fig. 4). In addition, P22 completely inhibited mouse ODC activity in vitro (data not shown). These characteristics of P22 are consistent with those of mouse AZ. Thus, we conclude that P22 and mouse AZ are equivalent in function.


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  		TABLE 1
Kinetic constants of the interaction of LDC (wild type) and LDC mutant (K103A/K123A/K126A) with the purified P22 or mouse AZ preparation

Surface plasmon resonance was performed at 25 °C using a BIACORE X instrument. One hundred resonance units each of purified LDC preparation and bovine serum albumin were immobilized on different flow cells of a CM5 sensor chip using amine-coupling chemistry. The purified P22 or AZ preparation at a concentration range of 0–100 µM was injected at a constant flow rate of 25 µl/min. ND, not detected.

 
Correlation between Appearance of P22 in the Cytoplasmic Fraction and LDC Degradation during Cell Growth of S. ruminantium—Previously, we found that the production of P22 is induced in putrescine-grown cells of S. ruminantium (15). Fluctuations in the amount of P22 during cell growth were examined. Sonicated extracts from the cells taken from the culture at various times as noted in Fig. 5 were prepared and centrifuged at 200,000 x g for 60 min to remove ribosome and membrane fractions. The amount of LDC and P22 were measured quantitatively by immunoblotting analysis using anti-LDC antiserum and anti-P22 antiserum, respectively. As shown Fig. 5, P22 dramatically appeared at early stationary phase and increased in amount up to 30 min, and thereafter it decreased rapidly and disappeared 1 h later. This rapid degradation of P22 was accompanied by a decrease in LDC.

Degradation Rate of LDC during Cell Growth—To determine the half-life of LDC protein, S. ruminantium cells taken at exponential or early stationary phase were incubated at 37 °C with [35S] methionine. After further incubation for 10 min in the presence of an excess amount of cold methionine and chloramphenicol, the cells were harvested at various times as noted in Fig. 6, and the amount of labeled LDC protein in the cells was measured as described under "Experimental Procedures." On the basis of the calculated degradation rate of the radioactive LDC, the half-life of LDC in the cells at exponential phase was determined to be 60 min, whereas at early stationary phase, it was shortened to 10 min.


Figure 5
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  		FIGURE 5.
Fluctuations in LDC and P22 levels during growth of S. ruminantium. S. ruminantium cells were grown in 1 liter of TYG medium at 37 °C, and the levels of cell growth (closed circles), amount of LDC (triangles), and P22 levels (open circles) were measured. Ten ml of the culture was removed and centrifuged. The cells were suspended in 20 mM potassium phosphate buffer and sonicated. After removing cell debris and ribosome fraction, the cytoplasmic fractions were treated with cold 5% trichloroacetic acid, resuspended in loading buffer, and analyzed by SDS-PAGE using 12.5% polyacrylamide gel. LDC and P22 proteins on the gel were assayed immunologically with anti-LDC or anti-P22 antibodies.

 
Identification of P22 as a Ribosomal Protein of S. ruminantium—To prove that P22 is a constituent of ribosomes in S. ruminantium, the ribosome fraction was prepared from S. ruminantium cells, and the ribosomal proteins were analyzed by Western blotting using anti-P22 antiserum. As shown in Fig. 7, P22 was detected in 50 S fraction but not in the 30 S fraction. P22 was also detected in only the 50 S fraction of the ribosome preparation from E. coli BL21(pHP22) in which S. ruminantium P22 was expressed (data not shown). Thus, we conclude that P22 is a constituent of the ribosomal 50 S subunit in S. ruminantium.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the previous study, we identified P22 as a stimulating factor for the degradation of LDC, which was catalyzed by ATP-dependent protease(s) in S. ruminantium (15). In this study, we cloned p22 and characterized the gene product. DNA sequence analysis of p22 revealed that the amino acid sequence of P22 is 47% identical and 60–66% similar to those of the reported ribosomal L10 proteins of bacteria. The p22 gene cluster of S. ruminantium consisted of 4 ORFs, which encode tentatively designated proteins 2–4. The amino acid sequences of proteins 2–4 exhibited 75, 62, and 67% overall identity with those of L11, L1, and L7/L12, respectively. The order of the genes is identical to that of E. coli (i.e. L11-L1-L10-L7/12) (31). P22 was found in the 50 S subunit peak from the S. ruminantium ribosomal fraction. These findings clearly indicate that the P22 is ribosomal protein L10 in S. ruminantium. Previously, two ribosomal proteins, S20/L36 and L34, in E. coli were reported to be "E. coli antizyme proteins" on the sole basis of their inhibition of E. coli ODC activity (4). It was also reported that various other ribosomal proteins isolated from E. coli inhibited ODC activity (5). In contrast, P22 is distinguished from E. coli antizymes by the following evidence. (i) P22 not only inhibits S. ruminantium LDC/ODC activities but also accelerates LDC/ODC degradation in our cell-free system and in the 26 S proteasome. (ii) E. coli ribosomal protein L10 did not promote degradation of S. ruminantium LDC/ODC in our cell-free system.

Table 1 shows that neither P22 nor mouse AZ binds to the S. ruminantium LDC triple mutant with alanine residues replacing Lys103, Lys123, and Lys126 residues, located at the interface between its two identical monomers. Our results showed that both P22 and mouse AZ could bind to the antizyme-binding region in LDC. The association of LDC with P22 is very tight, with a KD = of 8.5 x 10–11 M; this value is similar to the dissociation constant for the interaction of ODC with mouse AZ, with a KD = of 1.4 x 10–11 M (32). Moreover, S. ruminantium LDC is clearly inhibited by mouse AZ, and conversely, P22 inhibited mouse ODC. This tight interaction should prevent the reassociation of the LDC subunits into active homodimers.


Figure 6
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  		FIGURE 6.
Turnover of LDC during cell growth of S. ruminantium. Turnover of LDC at either the midlogarithmic phase (open circles) or stationary phase (closed circles) of S. ruminantium was measured as described under "Experimental Procedures."

 


Figure 7
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  		FIGURE 7.
Presence of P22 in ribosomal 50 S subunit from S. ruminantium. The ribosomal fraction from S. ruminantium was prepared, and 50 and 30 S subunits were separated as described under "Experimental Procedures" (A). Selected fractions (fraction numbers 19–51) were analyzed by immunoblotting for P22 (B).

 
Compared with mouse ODC, S. ruminantium LDC lacks 31 residues at its C terminus (13). This region in mammalian ODC has been demonstrated to be essential for the extremely fast degradation of the protein (33). However, we found that LDC is degraded rapidly in the presence of AZ or P22 in 26 S proteasome as well as in our degradation system (Fig. 3, B and C), suggesting that P22 fulfills an AZ-like role in the 26 S proteasome. How do the 26 S proteasome and bacterial protease(s) recognize the LDC-P22 complex? Previously, Perrson et al. (34) reported that a Crithidia fasciculata ODC, which lacks the C-terminal domain of mammalian ODC, is degraded rapidly by the 26 S proteasome and that this degradation is Ub-dependent but AZ-independent. Ub conjugation plays a well documented role in protein degradation, but our LDC might not become a substrate of the ubiquitin pathway, because anti-Ub did not affect LDC degradation in 26 S proteasome (data not shown). These data suggest that S. ruminantium LDC contains a structural domain, which might be exposed for P22 or AZ binding and recognized by the 26 S proteasome or S. ruminantium protease(s). Although the function of the ATP requirement for protease activity in our S. ruminantium cell-free system is not yet clarified, ATP may be required by a chaperone-associated ATPase or may act as a component of an ATP-dependent protease.

As shown in Fig. 5, P22 dramatically appeared at early stationary phase and thereafter decreased with a gradual slope over 1 h. We showed that the half-life of LDC in early stationary phase (10 min) was shorter than in midlog phase (60 min). Based on these data, P22 might be induced by putrescine or another polyamine(s) in early stationary phase and degraded together with LDC. In mammalian cells, the expression of AZ requires a specific +1 translational frameshift. In contrast, the p22 gene does not require a ribosomal frameshift for its expression. Recently, it has been reported that AZ expression also leads to degradation of the cell cycle regulatory protein cyclin D1 (35). It remains to be determined whether or not P22 can contribute to the degradation of other enzyme proteins in S. ruminantium.

Finally, we propose the following mechanism for LDC regulation in S. ruminantium. During entry into the stationary phase of S. ruminantium cells, the {alpha}-carboxyl group of the D-glutamic acid residue of the peptidoglycan is saturated with cadaverine and putrescine (9, 36). Therefore, excess free putrescine might accumulate constitutively in the cells. The accumulation of free putrescine or another polyamine in S. ruminantium would prompt P22 induction and binding to LDC for forming inactive LDC-P22 complexes, accelerating the degradation of LDC in S. ruminantium.


   FOOTNOTES
 
* This work was supported in part by the Noda Institute for Scientific Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a postdoctoral fellowship from Japan Society for the Promotion of Science. Back

2 To whom correspondence should be addressed. Tel.: 81-22-717-8779; Fax: 81-22-717-8780; E-mail: ykamio{at}biochem.tohoku.ac.jp.

3 The abbreviations used are: ODC, ornithine decarboxylase; AZ, antizyme; ORF, open reading frame; rP22, recombinant P22; Ub, ubiquitin; LDC, lysine decarboxylase. Back


   ACKNOWLEDGMENTS
 
We thank Leslie B. Poole for careful reading and helpful comments on the manuscript. We also thank Dr. H. Taira of Iwate University for useful suggestions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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