Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Correspondence
Nicola D. Walker
Nicola.Walker{at}agresearch.co.nz
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ABSTRACT |
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The GenBank accession number for the Prevotella albensis dipeptidyl peptidase type IV (DPP-IV) sequence reported in this article is AJ310187.
Present address: AgResearch Ltd, Grasslands, Private Bag 11008, Palmerston North, New Zealand.
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INTRODUCTION |
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Four species of ruminal Prevotella have been described, differentiated on the basis of phenotypic and genetic diversity (Avgutin et al., 1994
, 1997
), but molecular phylogenetic analysis would suggest that many more species are present in the rumen (Wood et al., 1998
). Prevotella (Pre.) albensis is one of the recognized subgroups of Prevotella found in the rumen; its type strain is M384T (Avgu
tin et al., 1997
). Ion-exchange chromatography has revealed that this organism possesses at least four different DPPs, distinguished by their substrate and inhibitor specificity (Wallace et al., 1997
). One of these peptidases displayed similar characteristics to mammalian DPP-IV: it hydrolysed recognized test substrates for DPP-IV and was inhibited by serine protease inhibitors. DPP-IV appears to have a specialized role in hydrolysing proline-containing peptides, with a wide distribution in both prokaryotes and eukaryotes (McDonald & Barrett, 1986
; Misumi & Ikehara, 1998
). The aim of this work was to clone the gene from Pre. albensis encoding DPP-IV and to characterize its properties both biochemically and genetically. This is the first report of the cloning and functional expression of a DPP from a ruminal bacterium.
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METHODS |
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Escherichia coli TOP10 (Invitrogen) and its derivative clones were grown in LB broth containing 50 µg kanamycin ml-1 at 37 °C. Clone DPP-IV-2B contained the complete dpp-IV gene and its flanking regions and the control clone contained the 16S rRNA gene from Pre. albensis. Plasmid DNA was isolated using Wizard SV plasmid preparation columns (Promega).
PCR amplification of genomic DNA with degenerate primers based on regions of amino acid identity and cloning of PCR products.
Amino acid sequences of DPP-IVs from Porphyromonas (Por.) gingivalis and Chryseobacterium (formerly Flavobacterium) meningosepticum were aligned and two conserved regions of homology, DWVYEEE (located approximately 190 aa from the N terminus) and GWSYGG (located approximately 130 aa from the C terminus), were identified. These regions were approximately 1200 bp apart. Degenerate primers were designed by reverse translation: DPP-IV Sen1 and DPP-IV Rev1 (Table 1). Oligonucleotides were obtained from Cruachem. Thirty-six cycles of DNA amplification were performed using a denaturation temperature of 94 °C for 40 s, an annealing temperature of 50 °C for 2 min and an amplification temperature of 72 °C for 3 min. A final extension step of 72 °C for 7 min was included. The resulting amplification product was checked by electrophoresis prior to cloning and was approximately 1200 bp in size, which was in accordance with the expected size. The PCR product was purified using Wizard resin (Promega) and cloned into TOPO TA vector following the manufacturer's guidelines (Invitrogen).
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Specific primers (DPP-IV N-term and DPP-IV C-term) were designed on the flanking regions of the dpp-IV gene (Table 1) and used to amplify a sample of genomic DNA to obtain a segment of DNA that contained the complete gene. Thirty-six cycles of DNA amplification were performed using a denaturation temperature of 94 °C for 1 min, an annealing temperature of 50 °C for 2 min and an amplification temperature of 72 °C for 5 min. A final extension step of 72 °C for 7 min was included. The resulting PCR product was cloned into TA vector as before. The size of the inserts was tested in 10 clones and the N- and C-terminal regions of the clones were sequenced to check the orientation and nature of the insert. Clones which possessed inserts of the correct size were screened for DPP-IV activity (see below) and one, DPP-IV-2B, was selected for further study.
Phylogenetic analysis and KyteDoolittle plot.
DPP-IV and related sequences were aligned via the internet using CLUSTAL W (Thompson et al., 1994) with the aln option as the preferred output option. A phylogenetic tree was constructed by selecting the PHYLIP output option from CLUSTAL W. The output was analysed using programs from the PHYLIP package, version 3.57c (Felsenstein, 1989
). Programs used from this package were SEQBOOT, PROTPARS and CONSENSE. The final treefile produced by the CONSENSE program was viewed using TREEVIEW (Page, 1996
). A hydrophilicity profile was determined via the internet using a KyteDoolittle plot (Kyte & Doolittle, 1982
) (http://bioinformatics.weizmann.ac.il/hydroph/plotfft_hydroph.html). The N-terminal 50 aa sequence was analysed for the presence of a putative signal peptide using the SignalP predictor service (http://www.cbs.dtu.dk/services/SignalP) (Nielsen et al., 1997
).
Preparation of cell-free extracts.
A fresh overnight culture of E. coli clone DPP-IV-2B in LB medium containing 50 µg kanamycin ml-1 was harvested by centrifugation (27 500 g, 4 °C, 15 min), washed twice in 25 mM potassium phosphate buffer and resuspended in 5 ml of the same buffer. The cells were then sonicated at 30 µm (Soniprep 150; http://www.reallabware.com), for a total sonication time of 10 min on ice, in 30 s bursts with 30 s cooling intervals. Cell debris and intact cells were removed by centrifugation (15 000 g, 4 °C, 15 min) and the supernatant was retained on ice as the crude extract. Crude extracts were prepared in the same way for the control E. coli TOP10 strain which contained a 16S rRNA gene insert from Pre. albensis and for an overnight culture of Pre. albensis grown in M2 medium.
Purification of cloned DPP-IV.
Aliquots (0·5 ml) of crude extract from the control and DPP-IV-2B E. coli clones were applied to a Poros HQ anion exchange column (Perseptive Biosystems) and eluted with 20 mM Tris/HCl, pH 8·0, at 3 ml min-1 using a salt gradient of 01·0 M NaCl. Absorbance (at 280 nm) was monitored continuously. Fractions were tested for their peptidase activities. Several runs were performed and active fractions were pooled. These were then used to characterize the cloned enzyme.
Gel filtration was used to assess the molecular mass of the native enzyme. Three millilitres of active fraction were applied to a 100x1·0 cm column of Sephacryl-300 HR (Amersham Pharmacia Biotech). The column was eluted at 0·25 ml min-1 with 50 mM potassium phosphate buffer, pH 7·0.
Measurement of peptidase activities, effect of inhibitors and protein determination.
Peptidase activities were measured in crude extracts and purified enzyme using chromogenic and fluorogenic synthetic peptides (Wallace et al., 1997) or reverse phase HPLC analysis (Wallace & McKain, 1989
). Acetylation of GlyProGly2 was performed by dissolving 20 mg peptide in 1 ml distilled water and chilling this solution on ice for 30 min. Acetic anhydride (200 µl) was added and the mixture was stirred for 1 h. It was then frozen at -70 °C and freeze-dried. The acetylated peptide was then reconstituted in distilled water. The degree of N-terminal modification was determined using ninhydrin (Moore & Stein, 1954
) and demonstrated that the peptide was completely acetylated. The effect of inhibitors on peptidase activity was determined using purified enzyme by methods used previously (Wallace et al., 1997
). The protein content of the cell extract was measured using the Folin reagent following alkaline digestion (Herbert et al., 1971
) with bovine serum albumin as standard.
Determination of optimum pH, optimum temperature, temperature stability and Km of DPP-IV.
The pH and temperature activity profiles were determined using crude extract from the clone DPP-IV-2B, by the rate of hydrolysis of GlyPro-p-nitroanilide (GlyPro-pNA). The test buffers were: 0·1 M potassium citrate/phosphate buffer pH 2·6, 3·4, 4·2, 5·0 and 5·8; 0·2 M potassium phosphate buffer pH 6·2, 6·6, 7·0, 7·4 and 7·8; and 0·2 M Tris/HCl buffer pH 7·2, 8·0, 8·8 and 9·0. The optimum temperature of DPP-IV was determined from initial reaction rates at temperatures between 22 and 70 °C. Temperature stability was determined by incubating aliquots of the sample at 22, 30, 37, 40, 42, 45, 49, 55, 60, 63, 65 or 70 °C for 1 h before being analysed for enzyme activity.
To determine the MichaelisMenten constant, Km, the activity of the crude extract against GlyPro-pNA was determined as before, except that the concentration of GlyPro-pNA in the incubation mixture was adjusted to 0·018·0 mM. The results were plotted by linear regression on a double-reciprocal plot of 1/V against 1/S where the intercept on the horizontal axis is -1/Km.
Influence of medium composition on DPP-IV activity.
A defined minimal medium (DM) was used to determine the influence of peptides and amino acids on DPP-IV activity. DM was based on the basal medium of Pittman & Bryant (1964), with added 0·1 % ammonium chloride and 0·0045 % methionine. Trypticase (1 %, w/v) or casein acid hydrolysate (1·55 %, w/v) was added to DM to determine the influence of peptidase substrates and products on DPP-IV activity. Pre. albensis M384T was grown to mid-exponential phase, then the crude extract was prepared as described for clone DPP-IV-2B. Peptidase activity was measured using GlyPro-pNA.
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RESULTS |
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The deduced sequence of 730 aa (Fig. 1) contained the characteristic catalytic domain GWYSGG found in all known DPP-IVs and the conserved region DWVYEEE which was used to design the other degenerate primer. The putative DPP-IV had a calculated molecular mass of 83 kDa and a theoretical pI of 7·56. The calculated G+C content of this sequence was 39 mol%. A KyteDoolittle hydropathy plot of the sequence was constructed (not shown), which indicated that the protein was hydrophilic in nature apart from the N-terminal region. Further SignalP analysis of the N-terminal sequence confirmed this to be a putative signal sequence, with a predicted cleavage site between residues 19 and 20.
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Purification of DPP-IV was carried out by anion-exchange chromatography. The DPP-IV activity was separated from the background aminopeptidase activities of E. coli, and much of the protein was also removed (Fig. 3). The fraction that contained the highest DPP-IV activity also had activity against LysAla-MNA. Active DPP-IV fractions were pooled. Only one band, of 80 kDa, was detected on an SDS-polyacrylamide gel stained with Coomassie blue and the preparation appeared to be homogeneous. Twenty-two synthetic peptides were tested for hydrolysis by the pooled fractions. Only six were hydrolysed. GlyPro-pNA, LysPro-MNA and GlyPro-MNA were hydrolysed most rapidly [900, 377 and 167 nmol min-1 (mg protein)-1, respectively], while the rates of hydrolysis of ValAla-pNA, LysAla-MNA and Ala2-pNA were lower [52, 44 and 16 nmol min-1 (mg protein)-1, respectively]. p-Nitroanilides of Ala, Arg, Asp, Glu, Gly, Leu, Lys, Met, Pro, Tyr, Val and GlyArg were not hydrolysed, nor were MNA esters of Leu, ArgArg, GlyArg or LeuVal. Thus, the DPP-IV-like enzyme did not break down aminopeptidase substrates or the test substrates for DPP-I (GlyArg-MNA) and DPP-III (ArgArg-MNA).
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The effect of different inhibitors on activity against GlyPro-MNA and LysAla-MNA was measured (Table 2). Only diprotin A, PMSF and dichloroisocoumarin inhibited DPP-IV activity.
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Influence of medium composition on DPP-IV activity in Pre. albensis
DPP-IV activity in defined medium, containing no added amino acids except methionine, was 12·0 (SD 1·5) nmol min-1 (mg protein)-1. In defined minimal medium supplemented with trypticase, activity increased to 21·0 (SD 3·8) nmol min-1 (mg protein)-1. Cells grown in medium containing added amino acids gave activity of 26·1 (SD 3·8) nmol min-1 (mg protein)-1.
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DISCUSSION |
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A hydropathy plot identified an N-terminal region that was hydrophobic in nature, suggesting that it was associated with the cell membrane, and sequence analysis revealed a likely signal sequence. Wallace et al. (1997) found that DPP-IV was solubilized by sonication of Pre. albensis, but the enzyme may have been displaced from the membrane or from the periplasm. Other DPP-IVs are membrane-bound (Kiyama et al., 1998
; Misumi & Ikehara, 1998
), so the same may be true for the Pre. albensis enzyme. A membrane location may place the enzyme in close proximity to the peptide transporters that translocate the peptide across the membrane, thus placing the enzyme in an ideal location to hydrolyse its substrate.
The apparent molecular mass of the native enzyme was around 240 000 Da, in comparison with a molecular mass of 83 000 Da derived from the amino acid sequence. The cause of this difference is not clear. The active enzyme could comprise three subunits, or some association with membranous material might have altered the gel permeation properties of the native enzyme. SDS-PAGE of the purified extract indicated that the preparation was homogeneous and the purified enzyme had a molecular mass of approximately 80 kDa, which is in keeping with the theoretical mass obtained from the sequence analysis.
The dpp-IV gene of Pre. albensis is similar to the majority of peptidase genes, which do not exist as an operon, are monocistronic and are generally transcribed from a 70 promoter (Gonzales & Robert-Baudouy, 1996
). Putative promoter regions were identified within the upstream region of dpp-IV (CAGAT and TATCTTTG), similar to those previously identified in Por. gingivalis (CAGAT and TATAAT; Jackson et al., 2000
), as was the suggested promoter sequence for the Bacteroidaceae, including B. fragilis (TANNTTTG; Bayley & Smith, 1998
). These show distinct differences from the consensus
70 -35 and -10 promoter sequence of E. coli (TTGACA and TATAAT; Hawley & McClure, 1983
), not only in terms of sequence but also in terms of nucleotide spacing between the transcription start point and the individual promoter regions. There is not the optimal 17 nt spacing observed between the -10 and -35 promoter regions, and in many instances transcription initiation occurs well upstream from the translation start site.
DPP-IV activity was increased by the presence of peptides or even free amino acids in the growth medium. Thus, peptides are apparently not required to induce DPP-IV activity. This is consistent with the suggestion that peptidase activity in Pre. albensis is co-ordinately regulated along with other enzymes involved in nitrogen metabolism (Walker, 2002). The identification of a regulatory element consensus motif would further substantiate these findings. It may also lead to the identification of other genes involved in nitrogen metabolism.
Like the mammalian DPP-IV, the Pre. albensis enzyme had a preference for a proline residue at the penultimate position and hydrolysed the bond on the carboxyl side of the proline (Misumi & Ikehara, 1998). The group beyond the penultimate amino acid affected the rate of hydrolysis, in that GlyPro-pNA was more rapidly hydrolysed by DPP-IV than GlyPro-MNA. This may be due to differences in the steric or hydrophobic nature of the chromogenic/fluorogenic group. The proline as penultimate N-terminal amino acid could also be substituted by alanine, although the rate of hydrolysis was significantly decreased. This activity was also observed in the other DPP-IVs and in PepX of the lactic acid bacteria (Chich, 1998
; Misumi & Ikehara, 1998
). The ability to recognize and hydrolyse more than one type of substrate indicates a degree of overlapping specificity in the DPP-IV of Pre. albensis. Overlapping specificity is also found in the peptidases of other bacteria (Lazdunski, 1989
; Mierau et al., 1996
; Yen et al., 1980
). Studies in which peptidase-deficient mutants of Prevotella bryantii were isolated (Madeira et al., 1997
) demonstrated that there must be a degree of overlapping specificity between different peptidases in this organism because peptidase activity could not be completely abolished in mutants that were deficient for DPP-I.
The DPP-IV from Pre. albensis was inhibited by dichloroisocoumarin and PMSF, recognized serine protease inhibitors, and also by diprotin A (Ile-Pro-Ile), which is a substrate-analogue inhibitor of DPP-IVs. These inhibitors have already been shown to inhibit DPP-IV activity in crude cell extracts of Pre. albensis (Wallace et al., 1997), once again indicating the close similarity between the cloned enzyme and DPP-IV of Pre. albensis. The optimum pH (7·8) and broad pH range over which the enzyme was active was also similar to mammalian DPP-IVs (Misumi & Ikehara, 1998
), as was the optimum temperature (39 °C in comparison to 37 °C). Here, the pH and temperature optima were determined using crude extract from sonicated clone DPP-IV-2B; as the control clone with a 16S rRNA gene insert had no detectable DPP-IV activity, the results can be expected to be valid for the recombinant DPP-IV. Thus, the biochemical properties, in conjunction with the sequence data, of the first peptidase to be cloned from a ruminal bacterium indicate that it has properties very similar to DPP-IVs from closely related Gram-negative bacteria, such as Porphyromonas, and to DPP-IVs in distantly related mammalian species, despite very different biochemical conditions, environmental circumstances and, possibly, metabolic functions. It remains to be seen how the other main DPPs of Pre. albensis fit into the wider scheme of peptidase enzymes.
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ACKNOWLEDGEMENTS |
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Received 11 November 2002;
accepted 26 March 2003.
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