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Infection and Immunity, August 2005, p. 4864-4878, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4864-4878.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Expression of Arg-Gingipain RgpB Is Required for Correct Glycosylation and Stability of Monomeric Arg-Gingipain RgpA from Porphyromonas gingivalis W50

MRC Molecular Pathogenesis Group, Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts & The London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom,¹ School of Biological and Chemical Sciences, Birkbeck College, University of London, Gordon Square, London WC1 H0PP, United Kingdom²

Received 23 February 2005/ Accepted 18 March 2005

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the -catalytic chain and the ß-adhesin chain, the monomeric soluble Arg-gingipain comprising only the -catalytic chain (RgpA_cat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the -catalytic chain (mt-RgPA_cat), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpA_cat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpA_cat and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpA_cat from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpA_cat from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is considered an important etiological agent in periodontal disease. The organism produces several extracellular proteolytic enzymes of which the Arg-X-specific and Lys-X-specific enzymes are predominant. These cysteine proteases cause the degradation of several physiologically important proteins, including collagens (6), fibrin and fibrinogen (28, 44), and fibronectin (47, 52). They also account for deregulatory effects on several host systems (21, 34, 43, 49, 55) and are therefore considered important virulence determinants. The adhesin domains of Arg-gingipains mediate the adherence of P. gingivalis to epithelial cells (8).

The extracellular Arg-X-specific proteases of P. gingivalis W50 are encoded by two genes, rgpA and rgpB (9). Three enzymes, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the -catalytic chain and the ß-adhesin chain, the monomeric soluble Arg-gingipain comprising only the -catalytic chain (RgpA_cat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the -catalytic chain (mt-RgpA_cat), are derived from rgpA (41) and two enzymes, RgpB and mt-RgpB, from rgpB (40). HRgpA occurs as a dimer (41) or multimer (39) composed of a catalytic chain (55 kDa) noncovalently associated with a polypeptide(s) derived from the long C-terminal extension of the initial full-length translation product. RgpA_cat (55 kDa) and mt-RgpA_cat (70 to 80 kDa) are monomers of the catalytic chain with different amounts of posttranslational modifications (10). The rgpB protease gene lacks the long C-terminal extension coding region of rgpA but is otherwise almost identical (40), and the two protease isoforms derived from this gene, RgpB and mt-RgpB, are structurally and kinetically similar to the monomeric RgpA isoforms. Mikolajczyk et al. (30) expressed the full-length precursor of RgpB and showed that three sequential autolytic processing steps at the N and C termini are required for full activity and that the N-terminal propeptide may serve as an intramolecular chaperone rather than as an inhibitory peptide.

Biochemical analysis of the Rgps has established that the monomeric enzymes are glycoproteins containing between 14% (RgpA_cat and RgpB) and 30% (mt-RgpA_cat and mt-RgpB) carbohydrate by weight (10). The monosaccharide composition of RgpA_cat has been examined in some detail and contains at least nine different sugars, including high levels of GalNAc and Neu5(Ac). Furthermore, the glycan additions to this isoform are immunologically related to a polysaccharide preparation of this organism, suggesting a common link between the maturation pathway of the Rgps and the synthesis of this macromolecule. The functional significance of the glycosylation of these proteases has not been determined, although the recognition of these glycan additions by periodontal serum immunoglobulin G antibody may indicate a role in immune subversion (46). Takii et al. (50) have recently isolated a cell-associated gingipain complex of a 660-kDa mass existing as a homodimer of two catalytically active monomers which comprise their catalytic and adhesin domains. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide with the catalytic domains and a hemagglutinin domain of Rgp and Kgp in the complex. Takii et al. (50) also showed that the functional domains of lipopolysaccharide were structurally masked by the complex proteins, which suggested the importance of the complex in the evasion of host defense mechanisms as well as in host tissue breakdown.

The molecular mechanism of glycosylation of these enzymes is not known. However, results obtained previously in this laboratory (2) have implicated the products of rgpB in the posttranslational events leading to the generation of the different enzyme isoforms derived from rgpA. Insertional inactivation of rgpB led not only to significantly reduced levels of total Arg-X protease activity but also to alterations to the chromatographic characteristics of the monomeric isoforms derived from rgpA (2). Most strikingly, the vesicle-associated monomer, mt-RgpA_cat, was shown by active-site labeling and migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to lack the extensive posttranslational modifications present on the wild-type enzyme. In addition, RgpA_cat from the mutant appeared to be chemically different from the parent enzyme since the procedures routinely used for the purification of wild-type RgpA_cat were unsuccessful in isolating this isoform from the D7 mutant. Conversely, HRgpA, which appears to show very low levels of glycosylation in the parent strain, was unaffected with respect to the amount of enzyme present, its subunit composition, and the ease of purification in this mutant (2). These data led us to suggest that the correct maturation/modification of the monomeric isoforms of rgpA was dependent on a functional rgpB.

The possibility that the RgpA monomeric isoforms in the rgpB mutant are aberrantly posttranslationally modified with respect to carbohydrate additions provided us an opportunity to examine the influence of glycosylation on the enzymatic properties and immune recognition of these enzymes. In this paper, we first provide confirmation of the participation of RgpB in the maturation of rgpA-derived enzymes by showing that the loss of rgpB has a specific effect on rgpA protein maturation via proteomic analysis of the wild type and mutant strain. We also show that this effect is not due to a polar effect on transcription at the rgpB locus. Second, we describe the purification, properties, monosaccharide analysis, and identification of sugars linking oligosaccharides to Ser/Thr residues in RgpA_cat from strain D7 and how these are different from RgpA_cat from the parent strain. These data demonstrate the importance of posttranslational modifications to the stability of the monomeric RgpA_cat from this organism.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials. DEAE-Sephacel, Sephacryl S-200HR, and phenyl-Sepharose (high performance) were obtained from Pharmacia-Biotech (St. Albans, Hertfordshire, United Kingdom). L-Arg conjugated to 4% beaded agarose and N-benzoyl-DL-Arg-p-nitroanilide (DL-BRpNA) were purchased from Sigma Chemical Co. (Poole, Dorset, United Kingdom). N-acetyl-Lys-p-nitroanilide and leupeptin were obtained from Bachem Feinchemikalein AG (Bubendorf, Switzerland). Zwittergent 3-14 detergent and Dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DNS-EGR-CK) were purchased from Calbiochem Novabiochem UK Ltd. (Nottingham, United Kingdom). All other chemicals and reagents were the purest grades available and were from BDH Chemicals (Poole, Dorset, United Kingdom) or Sigma Chemical Co. Restriction and DNA-modifying enzymes were purchased from either Amersham Biosciences (St. Albans, Hertfordshire, United Kingdom) or Roche (East Sussex, United Kingdom). Reagents for PCR (Reddy Load) were obtained from ABgene (Epsom, Surrey, United Kingdom). DNA isolation reagents were from either Flowgen (Lichfield, United Kingdom) (chromosomal) or QIAGEN (Crawley, United Kingdom) (plasmids, DNA fragments, and amplicons). The monoclonal antibody (MAb) 1B5 used in Western blotting has been described previously (10). Rabbit antiserum to the RgpA catalytic domain was prepared by immunizing rabbits with recombinant His₆-tagged RgpA (R²²⁷-R⁷¹⁹) which was prepared in Escherichia coli XL-1 Blue transformed with a derivative of pQE3010 described previously (2). Horseradish peroxidase-labeled anti-species antibodies were supplied by DAKO A/S, Denmark.

Bacteria and growth conditions. Porphyromonas gingivalis W50 and isogenic mutants D7 (rgpB), DE1 (dppIV, PG0503), L1 (lipA, PG0504), H7 (ubiA, PG0509), and C7 (cicA, PG0508) were cultured anaerobically on blood agar or in brain heart infusion medium (Oxoid, Basingstoke, United Kingdom) supplemented with hemin (5 µg ml^–1) as described previously (2). E. coli XL1 Blue and SCS110 (Stratagene) were propagated in Luria-Bertani medium at 37°C. Ampicillin was added to 100 µg ml^–1 for plasmid selection. P. gingivalis allelic exchange mutants were selected on blood agar plates containing clindamycin hydrochloride at 5 µg ml^–1.

DNA manipulations. Manipulation of DNA, transformation of E. coli, and agarose gel electrophoresis were done as described by Sambrook et al. (42).

PCRs. Three separate amplicons corresponding to the loci shown in Fig. 1 were used to clone DNA fragments for the inactivation of dipeptidyl peptidase (dppIV), lipoic acid synthetase (lipA), and ClpXP protease (cicA) or hydroxybenzoate octaprenyl transferase (ubiA). Primer pairs used were 5'-TTATGCTCGCAGTGCAGG-3' (DppIVF1) and 5'-TGCCTCTGTAAAAAGCATCG-3' (DppIVR1), 5'-GGAATACCCGCTATCATCTC-3' (LipAF1) and 5'-CTGCTCCATTATGCTTTTCC-3' (LipAR1), and 5'-TGCGGATTGAAAGACAAGAG-3' (UbiF1) and 5'-GAATCGACAATCTCCAAACG-3' (UbiR1), respectively.

View larger version (5K): [in this window] [in a new window]   FIG. 1. Organization of genes around the rgpB locus of Porphyromonas gingivalis. The genes rgpB and dppIV (dipeptidyl peptidase) have been characterized in P. gingivalis. lipA (lipoic acid synthetase), cicA (clpXP protease), and ubiA (hydroxybenzoate octaprenyl transferase) are genes based on similarity searches of nucleotide and protein databases. The direction of transcription is shown as bold arrows above the line. Regions below the line were amplified by PCR, and vertical bars and letters above the amplicons represent restriction enzyme sites. B, BclI; C, ClaI; E, EcoRV; H, HincII; N, NcoI. The erm cassette was inserted at restriction sites (::) or following the deletion of sites () in the cloned amplicon. Plasmids containing PCR products were propagated in E. coli SCS110 (Stratagene) in order to render BclI and ClaI sites cleavable by restriction enzymes.

Allelic exchange. Suitable restriction enzyme sites were identified in the cloned amplicons (Fig. 1), and a blunted BamHI-KpnI fragment of pVA2198 containing an Erm cassette (14) was cloned into these sites to inactivate the genes in vitro. The cassette replaced a 900-bp ClaI-BclI fragment in the amplicon for dppIV, a 330-bp BclI-BclI fragment in ubiA, and a 131-bp EcoRV-HincII fragment in lipA. In the case of cicA, the Erm cassette was inserted at an NcoI site. These constructs were then retrieved from their pUC18not derivatives by NotI digestion and electroporated into P. gingivalis W50 as described previously (40).

Enzyme assays. Arg-X protease activity was measured at 30°C with DL-BRpNA (500 µM) as the substrate. One unit of protease catalyzes the formation of 1 µmol of p-nitroaniline min^–1 in this assay system (41). L-BRpNA (250 µM) was used as substrate for K_m measurements. Lys-X protease activity was measured with N-acetyl-L-lysine-p-nitroanilide (Ac-LyspNA) (250 µM) as substrate in the same reaction buffer and under the same conditions as described above.

Enzyme purification. Enzymes from the P. gingivalis D7 mutant were purified as follows. All steps were performed at 4°C. Cells from 4 liters of a 6-day culture were removed by centrifugation at 10,000 x g (60 min; Sorvall RC 5C Plus, SLA-3000), and solid ammonium sulfate (enzyme grade) was added to the supernatant to 85% saturation as previously described (41). Solubilization of enzyme activity from the ammonium sulfate precipitate, gel filtration, and affinity chromatography on Arg-agarose columns were performed essentially as described previously (41). Elution of HRgpA bound to the Arg-agarose column was performed as described previously (41) except that 0.1 M L-arginine was used. Enzyme fractions judged to be pure by constant specific activity and SDS-PAGE were stored at 4°C. This enzyme could be purified by rechromatography on Arg-agarose (41). This Arg-X protease is referred to herein as HRgpA/D7 or HRgpA(rgpB).

Purification of RgpA_cat(rgpB). Purification of enzymes present in the effluent from affinity column fractions (above) was achieved by dialysis against 20 mM Tris-acetate buffer, pH 7.5, followed by ion exchange chromatography on DEAE-Sephacel equilibrated in the same buffer. Enzyme was eluted by a linear gradient of NaCl to a final concentration of 0.15 M in equilibration buffer. Enzyme fractions were combined, and solid ammonium sulfate was added to make the solution 2 M in ammonium sulfate and applied to a column of phenyl-Sepharose equilibrated in 20 mM Tris-acetate buffer, pH 7.5, plus 0.1 M NaCl-2 M ammonium sulfate. Pure enzyme was eluted with linear gradients of ammonium sulfate from (i) 2 M to 0.6 M and (ii) 0.6 M to 0 M. Enzyme fractions judged to be pure by high constant specific activity and SDS-PAGE were stored at 4°C. This Arg-X protease is referred to herein as RgpA_cat/D7 or RgpA_cat(rgpB). The protein was essentially pure at this stage.

Determination of protein concentration. Protein concentrations were determined by measurement of absorbance at 280 nm and 260 nm (11). Protein concentration (mg ml^–1) was calculated according to the formula 1.55 A₂₈₀ – 0.76 A₂₆₀.

Gel electrophoresis. SDS-PAGE was carried out at 5°C in 12.5% polyacrylamide slab gels (10 by 7 by 0.15 cm) (26). Samples of protease (10 to 20 µg) were first treated with 50 µl of leupeptin (1 mg ml^–1) at 22°C for 20 min, heated at 100°C for 5 min, and dried in vacuo.

Immunoblotting. Western blotting of purified enzymes (2 µg) was performed following inhibition of the proteases with leupeptin (1 mg ml^–1). The samples were electroblotted from 12.5% polyacrylamide gels onto nitrocellulose membranes (Schleicher & Schuell, Germany) and probed using monoclonal antibody 1B5 (1:100) or rabbit antiserum to the recombinant catalytic domain of RgpA (1:250). Horseradish peroxidase-labeled anti-species antibodies were used at a 1:500 dilution with diamino-benzidine as the substrate.

N-terminal sequencing of proteins. Arg-X proteases were subjected to N-terminal sequencing after either 8 M urea-PAGE (17) for HRgpA/D7 or SDS-PAGE for RgpA_cat/D7 following transfer to Immobilon membranes (Millipore) took place. Sequencing was performed by the Haemostasis Research Group, Royal Postgraduate Medical School, London, United Kingdom, using an ABI 477A gas phase sequencer for 16 to 20 cycles.

Fluorescent labeling of proteases with DNS-EGR-CK. Six-day culture supernatants (500 µl) of P. gingivalis W50 and isogenic mutants D7, DE1, L7, H7, and C7 were treated with 750 µl of ice-cold acetone in a bath of freezing mixture (–10°C) for 30 min. The solution was centrifuged at 13,000 x g (Heraeus Biofuge) for 15 min, and the pellet was subjected to reduction and labeling (2). Pure RgpA_cat enzymes from W50 and D7 were labeled as described previously (17) and reprecipitated by the addition of 225 µl of ice-cold acetone in a bath of freezing mixture (–10°C) for 30 min. Labeled protein was centrifuged at 13,000 x g for 15 min, and the pellet was treated with SDS-PAGE sample buffer for gel electrophoresis.

Two-dimensional electrophoresis. Cells from 50 ml of P. gingivalis cultures (24 h) were harvested by centrifugation and washed three times with equal volumes of 10 mM Tris-HCl buffer, pH 7.5, with the final wash containing 1 mM N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and a cocktail of protease inhibitors (Roche, Germany). Cell pellets were resuspended in 3 ml of lysis solution (0.5 to 1.0% SDS, 1 mM TLCK, cocktail of protease inhibitors) and sonicated five times (10-s bursts) with cooling on ice for 1 min between bursts. DNase I (10 U/ml) and RNase A (1 U/ml) were added and incubated at 37°C for 1 h. The lysate was collected by centrifugation and precipitated with 5 volumes of ice-cold 10% trichloroacetic acid in acetone containing 0.2% dithiothreitol, and the pellet was washed twice with the same volume of ice-cold acetone. The precipitated protein pellets were solubilized in 1.5 ml of solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 50 mM dithiothreitol, 0.002% bromophenol blue, 2% immobilized pH gradient [IPG] buffer, pH 3 to 10; Amersham Biosciences) at room temperature for 2 h with occasional mixing. The suspension was centrifuged, and the clear supernatant was stored in 100-µl aliquots at –70°C. Soluble and outer membrane proteins were separated according to the method of Murakami et al. (31).

First dimension: isoelectric focusing. Isoelectric focusing with immobilized pH gradients (IPG strips) in the IPGphor Isoelectric Focusing System (Amersham Biosciences UK Ltd., Bucks, United Kingdom) was carried out according to the manufacturer's instructions using 7-cm or 13-cm IPG strips in the pH range 4 to 7 (linear) or 13-cm IPG strips in pH 3 to 5.6 (nonlinear).

Second dimension: gel electrophoresis. IPG strips were incubated in 10 ml of equilibration buffer (18) with gentle shaking for 15 min to denature the proteins and reduce disulfide bridges, followed by alkylation of cysteine residues (18). SDS-PAGE was performed in either a SE260 mini-vertical system or a Hoefer SE600 standard vertical system without the use of stacking gel. Gels were stained with colloidal Coomassie brilliant blue according to the method of Neuhoff et al. (33).

In-gel digestion and MALDI-TOF MS analysis. Protein spots were excised, destained, and digested with sequencing-grade trypsin (13 µg/ml) overnight (45). Peptide extracts were desalted and concentrated using C₁₈ resin Zip-Tip. Peptides (0.5 µl) were mixed with an equal volume of saturated matrix solution (10 mg/ml -cyano-4-hydroxy cinnamic acid in 50% acetonitrile, 0.05% trifluoroacetic acid), applied to a matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS sample plate, and allowed to dry in air.

Peptide mass fingerprinting was performed using MALDI-TOF MS. Identification of peptides was carried out using the RgpA sequence of P. gingivalis W50 (1). In some cases, quadrupole time of flight tandem mass spectrometry (Q-TOF MS-MS) was performed on selected high-intensity peptides to confirm the identity of the protein (MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London, United Kingdom).

Measurement of stability of RgpA_cat/D7. The stability of RgpA_cat/D7 was measured as a function of pH in the presence or absence of 2-mercaptoethanol (10 mM) and in the presence or absence of CaCl ₂ (10 mM) at 30°C. Buffers used were acetate (pH 4.5 to 6.0) and Tris-HCl (pH 7.3 to 8.3) buffers. Buffer (0.1 M) containing the desired additives was incubated in screw-cap Sarstedt tubes (Sarstedt Ltd., Leicester, United Kingdom) at 30°C in a water bath for a minimum of 1 h. A small volume of concentrated enzyme solution was added to the buffer to give a concentration of 12.5 to 15 µg ml^–1. After thorough mixing, 2x 20-µl aliquots were withdrawn at various times and assayed immediately at pH 8.1, 30°C.

Monosaccharide analysis of HRgpA/D7 and RgpA_cat/D7. HRgpA/D7 (2 mg) and RgpA_cat/D7 (0.2 mg) proteases were dialyzed against 5% (vol/vol) aqueous acetic acid to remove salts and detergents and freeze-dried. Monosaccharide analysis was essentially performed as described previously (10).

Release of O-linked oligosaccharides by alkaline ß-elimination. RgpA_cat (1.5 mg) and RgpA_cat/D7 (3 mg) were dialyzed against 5% (vol/vol) aqueous acetic acid to remove salts and detergents and freeze-dried. ß-Elimination (28) and separation of released oligosaccharides was performed as described previously (10). Peak fractions were dried under a vacuum or by freeze-drying and the monosaccharide composition of oligosaccharides determined by methanolysis as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
P. gingivalis W50 produces five extracellular Arg-X-specific proteases, HRgpA, RgpA_cat and mt-RgpA_cat, encoded by rgpA, and RgpB and mt-RgpB, encoded by rgpB (Table 1). HRgpA is a dimer of the catalytic chain and a ß adhesin chain, whereas the other four enzymes are monomers containing the catalytic chain with different degrees of posttranslational modifications.

Analysis of mutants in the rgpB locus. P. gingivalis W50, D7(rgpB), and mutants DE1 (dppIV), L7 (lipA), H7 (ubiA), and C7 (cicA) were grown in brain heart infusion supplemented with hemin as described in the Materials and Methods section. Whole cultures and supernatants were assayed for Arg-X and Lys-X enzyme activities after 24 h and 6 days. Mutants DE1, L7, H7, and C7 had enzyme activities comparable to those of the parent W50 strain (data not shown), whereas mutant D7 had approximately 50% of Arg-X activity and unaltered Lys-X activity in both whole culture and culture supernatant compared to the W50 strain. The 6-day culture supernatants of all six strains were treated with DNS-EGR-CK, the fluorescently labeled irreversible inhibitor of Arg-X activity. This method permits the separation and detection of mt-RgpA_cat and mt-RgpB (a), Kgp (b), and HRgpA, RgpA_cat, and RgpB (c) (Fig. 2).

View larger version (39K): [in this window] [in a new window]   FIG. 2. Gel electrophoresis of DNS-EGR-CK- labeled culture supernatants. Six-day culture supernatants from P. gingivalis strains W50, D7 (rgpB), DE1 (dppIV), L7 (lipA), H7 (ubiA), and C7 (cicA) were labeled with the fluorescent irreversible inhibitor DNS-EGR-CK as described in the text and subjected to SDS-PAGE in 12.5% acrylamide gels. Gels were viewed under UV light to visualize labeled proteins. The strains are indicated above the corresponding lanes. Arrows show the positions of mt-RgpAcat (a), Kgp (b), and HRgpA and RgpAcat (c) in descending order of molecular size.

Two-dimensional electrophoresis of soluble cellular proteins. Two-dimensional gel electrophoresis of cellular proteins, soluble cytoplasmic proteins, and outer membrane preparations of P. gingivalis strains W50 and D7 were performed using Immobiline Dry IPG strips in the pH range 4 to 7 (linear) or 3 to 5.6 (nonlinear) essentially according to the instructions of the manufacturer (Amersham). Reproducible two-dimensional gel patterns were obtained in all cases (Fig. 3). Comparisons showed that the main difference in total cell proteins between W50 and D7 was the presence of mt-RgpA_cat in W50, which was absent in D7 (Fig. 3A). The absence of mt-RgpA_cat in D7 was also shown by fluorescent labeling of culture supernatants (Fig. 2).

View larger version (199K): [in this window] [in a new window]   FIG. 3. Two-dimensional electrophoresis of proteins of P. gingivalis W50 and rgpB. Samples were prepared as described in the text. Isoelectric focusing was performed in immobilized pH gradients in 13-cm strips in the linear pH range 4 to 7 or 3 to 5.6 (nonlinear) followed by SDS-PAGE in 12.5% acrylamide gels and stained with colloidal Coomassie brilliant blue. Protein spots were cut out from the gel, digested with trypsin, and subjected to peptide mass fingerprinting by MALDI-TOF MS and Q-TOF MS-MS in certain cases as described in the text. (A) Total cellular proteins, pH 4 to 7. Black circles show the presence of mt-RgpAcat in W50 and absence in the rgpB strain. (B) Cytoplasmic proteins, pH 3 to 5.6. Arrows and numbers are described in the text. (C) Outer membrane proteins, pH 3 to 5.6. Arrows and numbers are described in the text. RgpAcat is enclosed in boxes to highlight differences in pIs. Molecular masses of marker proteins in kDa are given alongside one gel in each set.

Purification of Arg-X proteases from P. gingivalis/D7 (rgpB). Arg-X activity was solubilized in aqueous buffer from the ammonium sulfate precipitation and contained both HRgpA and RgpA_cat. Gel filtration chromatography of this fraction resulted in partial separation of HRgpA and RgpA_cat. Selective purification of these two forms was achieved by affinity chromatography on Arg-agarose columns, to which only HRgpA bound under the conditions used. This behavior was identical to that of HRgpA from P. gingivalis W50 (41), which will henceforth be referred to as HRgpA/W50. Elution from the affinity column was performed first with 0.3 M L-Lys, which eluted predominantly Lys-X protease, followed by 0.1 M L-Arg, which eluted HRgpA activity. Further purification of HRgpA could be achieved by rechromatography on Arg-agarose columns after dialysis against affinity column equilibration buffer. Use of high-detergent (0.05%) buffers resulted in the solubilization of the bulk of the remaining Arg-X activity from the ammonium sulfate precipitate. HRgpA from this fraction was purified as described above but at 22°C and will henceforth be referred to as HRgpA/D7 or HRgpA(rgpB).

Ion exchange chromatography of Arg-X activity not bound to the affinity column was performed at pH 7.5 in Tris-HCl buffer using DEAE-Sephacel columns (Fig. 4A). This yielded RgpA_cat which appeared to contain trace amounts of Lys-X activity and other contaminants. However, hydrophobic chromatography of the DEAE-Sephacel fractions on phenyl-Sepharose columns yielded essentially pure RgpA_cat of very high specific activity (Fig. 4B). The remaining Arg-X activity in ammonium sulfate precipitates was solubilized with buffer containing 0.05% detergent and was subjected to ion-exchange chromatography at 22°C, but the recovery and specific activity of RgpA_cat were very low. This enzyme is referred to hereafter as RgpA_cat/D7 or RgpA_cat(rgpB). The following recovery of enzymes was obtained: 22.2% for HRgpA/D7 and 2.6% for RgpA_cat/D7. The corresponding yields in the parent strain were 16% for HRgpA and 18% for RgpA_cat. The specific activities of mutant enzymes were 6.1 U/mg for HRgpA/D7 and 20.6 U/mg for RgpA_cat/D7, with DL-BRpNA as substrate at 30°C based on protein concentrations measured by absorbance at 280 nm and 260 nm. These values are comparable to the specific activities of the parent enzymes (41).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Purification of RgpAcat/D7. (A) Ion exchange chromatography of fraction S-I (unbound) on DEAE-Sephacel. Fraction I (unbound) from the Arg-agarose affinity column was applied to a column of DEAE-Sephacel (1.6 cm [inner diameter] by 9 cm) in 20 mM Tris-HCl buffer, pH 7.5. Enzyme was eluted with a gradient of NaCl (0 to 0.15 M; total volume, 180 ml) in 20 mM Tris-HCl buffer, pH 7.5. , A280 nm; , DL-BRpNA activity (units/ml); ---, Arg-X-specific activity (units/mg). (B) Hydrophobic chromatography of DEAE-Sephacel fractions on phenyl-Sepharose. DEAE-Sephacel fractions containing RgpAcat activity were combined, and solid ammonium sulfate was added to make it 2 M with respect to ammonium sulfate. The sample was applied to a column of phenyl-Sepharose (1.6 cm [inner diameter] by 7 cm) equilibrated in 20 mM Tris-HCl buffer (pH 7.5)-0.1 M NaCl-2 M ammonium sulfate. Enzyme was eluted with two consecutively applied gradients of ammonium sulfate: (i) from 2 M to 0.6 M (total volume, 160 ml) and (ii) from 0.6 M to 0 M (total volume, 160 ml) in 20 mM Tris-HCl buffer (pH 7.5)-0.1 M NaCl. The arrow shows the start of the gradient (ii). , A280 nm; , DL-BRpNA activity (units/ml); , Ac-LyspNA activity (units/ml); ---, Arg-X-specific activity (units/mg). RgpAcat/D7 fractions (40 to 51) were used in all experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Western blot analysis of rgpA-derived enzymes from P. gingivalis W50 and P. gingivalis/D7. Enzymes HRgpA, RgpAcat, and mt-RgpAcat from P. gingivalis W50 and HRgpA and RgpAcat from P. gingivalis/D7 were subjected to SDS-PAGE on 12.5% acrylamide gels, Western blotted, and reacted with rabbit antiserum to recombinant RgpAcat (A) or MAb 1B5, which recognizes a glycan addition to RgpAcat (B). Lanes 1, 2, and 3 contain W50, and lanes 4 and 5 contain D7. HRgpA (lanes 1 and 4), RgpAcat (lanes 2 and 5), and mt-RgpAcat (lane 3) are shown.

  chain, YTPVEEKQNGRMIVIVAKKY..., and

 ß chain, SGQAEIVLEAHDVWND...

in RgpA_cat/D7,

 YTPVEEKQNGRMIVIVAKKY... .

Attempts were made to determine the C-terminal sequences of at least 4 residues using the PE-Applied Biosystems Procise-C C-terminal protein sequencing system at the School of Biochemistry and Molecular Biology, University of Leeds, Leeds, United Kingdom. These experiments were not successful.

Physical properties of enzymes. Maximum enzyme activity for both HRgpA/D7 and RgpA_cat/D7 proteases was obtained only in the presence of 10 mM L-cysteine and 0.02 to 10 mM CaCl₂ at pH 7.9 to 8.1. No enzyme activity was detected in the absence of reducing agents. Ca²⁺ was not required for enzyme activity but improved enzyme stability, especially at higher temperatures and pHs (discussed later), as in the case of the parent enzymes.

The K_m for the chromogenic substrate L-BRpNA at pH 8.1 and 30°C for RgpA_cat/D7 was 5.6 µM and compares favorably with the values obtained for RgpA_cat/W50 (5 µM) and RgpB (4 µM) (40).

The thermostability of RgpA_cat/D7 was measured at 30°C under a variety of conditions, as shown in Table 2. First-order rate constants for the loss of enzyme activity at 30°C as a function of pH in the absence or presence of 10 mM 2-mercaptoethanol and in the absence or presence of 10 mM CaCl₂ for RgpA_cat/D7 showed that this enzyme is far less stable than wild-type RgpA_cat, especially at higher pH values. The latter enzyme was stable at all pHs tested in the presence of 10 mM CaCl_2, whereas RgpA_cat/D7 lost activity at pH 8.3 even in the presence of 10 mM CaCl₂ (half-life = 21.7 h). The (in)stability of RgpA_cat/D7 at pH values >7.5 in the absence of 10 mM CaCl₂ was even more dramatic, where the half-lives were between 40 and 80 times lower than the half-lives of the RgpA_cat wild-type enzyme either in the absence or in the presence of 10 mM 2-mercaptoethanol (Table 2).

Monosaccharide analysis. The monosaccharide composition of proteases HRgpA/D7 and RgpA_cat/D7 was determined after consecutive methanolysis, N-acetylation, and conversion of methyl glycosides to O-trimethylsilyl (O-TMS) ethers followed by gas chromatography-mass spectrometry (GC-MS). Table 3 shows the percentages and molar ratios of monosaccharides in the oligosaccharides of HRgpA, RgpA_cat, HRgpA/D7, and RgpA_cat/D7. These data are expressed both as a percentage and as an empirical formula of the monosaccharide constituents and help to predict the types of oligosaccharides present in the glycoprotein. Whereas HRgpA contained Rha, Man, Gal, Glc, GalNAc, and GlcNAc totaling 2.1% protein weight (10), HRgpA/D7 contained only small amounts of Man and Glc totaling <0.5% protein weight. RgpA_cat/D7 contained Ara, Rha, Fuc, Gal, Glc, and GlcNAc totaling 14% protein weight. Although the total sugar content was similar to that present in RgpA_cat (14.4%) (10), there were significant differences in composition (Table 3). GalNAc and Neu5(Ac), the most abundant sugars in RgpA_cat/W50, were not present, and the level of GlcNAc was greatly reduced. Ara, Fuc, and Glc levels were significantly raised, whereas Rha and Gal were comparable in the two enzymes.

View larger version (12K): [in this window] [in a new window]   FIG. 6. HPLC separation of oligosaccharides released by alkaline-borohydride treatment of RgpAcat/W50 and RgpAcat/D7. RgpAcat (1.5 mg) (A) and RgpAcat/D7 (3 mg) (B) were subjected to ß-elimination as described in the text. The oligosaccharides released were separated on a Hypersil porous graphitized column using a gradient of 0 to 40% acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid over 45 min. Absorbance of the column effluent was monitored at 210 nm, and 1-ml fractions were collected. Forty millivolts corresponds to a full-scale deflection of 0.04 absorbance units. Peak fractions were either freeze-dried or dried in vacuo, and their monosaccharide composition was determined by methanolysis as described in the text.

RgpA_cat/D7 was also treated with alkaline borohydride to release O-linked oligosaccharides and analyzed as described for RgpA_cat (Fig. 6B). Table 5, shows the monosaccharide composition of O-linked oligosaccharides obtained from RgpA_cat/D7 which were purified by HPLC. Eight fractions labeled A through H contained carbohydrate, of which only B and C appeared to be mixtures. Fractions A, B, C, and E contained Ara as the linking sugar, whereas B, C, and D contained Gal in glycosidic linkage to Ser/Thr in RgpA_cat/D7. C also contained an oligosaccharide linked via Fuc, whereas F, G, and H contained oligosaccharides with apparently increasing chain lengths linked via Glc. Although fraction B appeared to be a mixture, it contained only two reduced sugars, arabitol and galactitol, suggesting that only a single pentose and a single hexose residue were attached to two sites in RgpA_cat/D7. Fraction A contained a disaccharide Ara-Ara in glycosidic linkage, whereas D, E, F, G, and H contained longer oligosaccharide chains. Hence, there were significant differences in the composition and nature of the linking sugars in the oligosaccharides present in RgpA_cat/W50 and RgpA_cat/D7.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Two-dimensional electrophoresis of fluorescently labeled RgpAcat and RgpAcat/D7. Fluorescently labeled RgpAcat and RgpAcat/D7 were subjected to isoelectric focusing with immobilized pH gradients in 7-cm-long strips in the linear pH range 4 to 7 followed by SDS-PAGE in 12.5% acrylamide gels and viewed under UV light. The positions of marker proteins are indicated alongside the gels.

View larger version (57K): [in this window] [in a new window]   FIG. 8. Amino acid sequence of propeptide and -catalytic regions of Porphyromonas gingivalis W50 RgpA derived from the DNA sequence. Amino acid sequence in the propeptide region is indicated in italics. The arrow indicates the site of cleavage of propeptide from the -catalytic region. Amino acid sequences which are underlined were found in peptides from RgpAcat from both W50 and rgpB strains. Plus signs above the line indicate amino acid sequences found only in RgpAcat(rgpB). Boxes enclosing amino acid sequences indicate those sequences not detected in either RgpAcat (W50) or RgpAcat(rgpB).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Now that there is a growing acceptance that bacteria have developed the necessary cellular machinery to perform glycosylation reactions, the location, mechanism, enzymology, and physiological consequences of these processes are coming under increasing scrutiny (51). We have previously shown that the monomeric RgpA proteases of P. gingivalis W50 are variably glycosylated (10). In these studies, we demonstrated that insertional inactivation of rgpB in P. gingivalis W50 influences the biochemical behavior of the monomeric RgpA protease isoforms (2), suggesting that RgpB may have a role in the maturation pathway of rgpA-derived enzymes. In the absence of a suitable autonomously replicating plasmid for genetic complementation studies in the Porphyromonads, in our earlier study, we were unable to exclude the possibility that polar effects on transcription at an adjacent locus could also have accounted for these findings. Hence, in the present investigation, we performed targeted insertional inactivation of the genes upstream and downstream of rgpB on the P. gingivalis chromosome in order to provide additional evidence for a specific involvement of RgpB in the maturation of rgpA-derived monomeric enzymes. In the present work, we used proteomic analysis of whole-cell proteins of wild-type W50 and mutant rgpB to confirm that the inactivation of rgpB leads to a complete loss of mt-RgpA_cat, that this is a highly specific effect, and that there are no other major alterations to the cellular protein profile of this mutant.

Four major open reading frames surround rgpB (Fig. 1). The gene immediately upstream encodes a 278-amino acid protein with 69% similarity over 275 amino acids with Rickettsia prowazekii lipoic acid synthase (LipA) (3) and also to LipA from a variety of organisms including bacteria, yeast, and plants. A dipeptidyl peptidase (DppIV) which shows extensive similarity to both prokaryotic and eukaryotic DppIV enzymes is encoded 73 bp upstream of lipA (24, 25). An orf 553 bp downstream of rgpB encodes a 200-amino acid protein which has 47% similarity to CicA from Caulobacter crescentus involved in ClpXP protease function (37) and is transcribed in the opposite direction to dppIV, lipA, and rgpB. Downstream of cicA and overlapping by 4 bp is ubiA, which encodes a 321-amino acid protein with strong similarity to 4-hydroxy benzoate octaprenyl transferases (4) of Mycobacterium tuberculosis (50% similarity over 281 amino acids). However, insertional inactivation of each of these flanking genes had no effect on either Arg-X enzyme activity or the appearance of fluorescently labeled extracellular proteases on SDS-PAGE compared to the parent strain. Moreover, a screen of eight independently isolated P. gingivalis rgpB mutants showed exactly the same phenotype as the original D7 mutant described previously: a 50% reduction in total Arg-X activity and loss of the extensively modified mt-RgpA_cat isoform (not shown). Hence, it appears unlikely that the altered protease phenotype following the insertional inactivation of rgpB is a consequence of a polar effect on transcription at an adjacent locus and suggests a specific involvement of RgpB in the maturation of rgpA-derived enzymes.

Analysis of purified protease revealed that one of the main differences between RgpA_cat and RgpA_cat/D7 was in their isoelectric points. The latter is far less acidic at pH 5.3, at which anion exchange chromatography is routinely performed during purification of RgpA_cat and mt-RgpA_cat. Consequently, RgpA_cat/D7 was purified at pH 7.5 in order to accommodate the increase in its isoelectric point. Another important difference is that, unlike RgpA_cat from the parent strain, the mutant enzyme also appears to have hydrophobic regions or patches which enable it to bind to phenyl-Sepharose in the presence of 2 M ammonium sulfate. Despite these differences, purified HRgpA and RgpA_cat from D7 had almost identical specific activities and enzymatic properties compared to the enzymes from strain W50. RgpA_cat/W50 and RgpA_cat/D7 had identical N-terminal amino acid sequences and the same apparent molecular sizes as observed on gel filtration and on SDS-PAGE gels. In order to establish that both RgpA_cat enzymes had similar C-terminal sequences, attempts were made to determine the C-terminal sequences of at least 4 residues using the PE-Applied Biosystems Procise-C C-terminal protein sequencing system. However neither protease yielded a sequence either due to the limitations of C-terminal sequencing or because of posttranslational modification. Hence, pure RgpA_cat/W50 and RgpA_cat/D7 were subjected to two-dimensional electrophoresis and peptide mass fingerprinting. Both enzymes showed differences in their isoelectric points but had identical mobilities in the second dimension (Fig. 7). Both proteins gave very similar peptide mass fingerprints suggesting that they had almost identical polypeptide chain lengths. However, peptides in the C-terminal region beyond residue 574 could not be detected for both RgpA_cat enzymes. Thus, either both proteins had extensive posttranslational modifications in their C-terminal regions or both had residue 574 as their C terminus. The differences in isoelectric points of the two enzymes were also apparent in the two-dimensional electrophoresis gels (Fig. 3C) of outer membranes of 24-h cells, suggesting that charge differences are generated during the initial maturation of the enzyme prior to secretion into the supernatant.

A striking difference in the enzymatic properties of RgpA_cat and RgpA_cat/D7 is the decrease in stability at 30°C at pHs >7.5 of the mutant enzyme. In the absence of 10 mM Ca²⁺, the half-lives of RgpA_cat/D7 are between 40 and 80 times lower than those of RgpA_cat at pHs 7.8 and 8.3 (Table 2). In the presence of 10 mM Ca²⁺, RgpA_cat/D7 has a half-life of 21.7 h at pH 8.3 whereas RgpA_cat is stable at all pHs tested under these conditions. Thus, aberrant glycosylation of RgpA_cat appears to have a direct and dramatic influence on enzyme stability. The decreased stability does not appear to be caused by increased susceptibility to autolysis since the half-life of RgpA_cat/D7 was unaffected in the presence of reducing agent, which is essential for enzyme activity. It is possible that the loss of certain types of oligosaccharide chains could cause minor changes in the local conformation of several regions of the RgpA_cat/D7 molecule leading to instability. As indicated above, RgpA_cat/D7 appeared to contain hydrophobic patches which enabled this protein to bind to phenyl-Sepharose columns, which could again be due to changes in local conformation. The prevalence of shorter oligosaccharide chains in this protease could result in differences in protein surfaces exposed to solvent compared to RgpA_cat/W50.

These properties were partially explained by the different sugar composition of RgpA_cat/D7 compared to RgpA_cat from strain W50. Monosaccharide analysis of RgpA_cat/D7 showed that although it contained a similar amount of carbohydrate as the parent enzyme (14% versus 14.4% carbohydrate weight), the sugar composition was significantly different (Table 3). The most striking differences were that RgpA_cat/D7 lacked Neu5(Ac) and GalNAc and the level of GlcNAc was greatly reduced whereas Ara, Fuc, and Glc were present in significantly larger amounts. The lack of Neu5(Ac) could account for the increase in the isoelectric point of RgpA_cat/D7 relative to that of RgpA_cat. Several O-linked oligosaccharides are present in RgpA_cat. Two of these are linked through Glc, one through Gal, and at least five through GalNAc, analogous to mucin-type oligosaccharides (15). The absence of GalNAc in RgpA_cat/D7 may indicate that the glycan chains normally linked through this sugar are absent. Furthermore, since Neu5(Ac) is often linked to nonreducing terminal GalNAc, the absence of this sugar may also explain the absence of sialylation of the mutant enzyme. Analysis of the O-linked oligosaccharides of RgpA_cat/D7 showed dramatic differences compared to the parent enzyme. The oligosaccharides of RgpA_cat (W50) were composed of between 7 and 35 monosaccharide residues (Table 4), whereas RgpA_cat/D7 contained shorter chain oligosaccharides ranging from mono-, di-, and tetrasaccharides to a few longer (10- to 18-residue) chains. Oligosaccharide linkages were predominantly through Ara, Gal, Glc, and Fuc, unlike the parent strain enzyme, in which GalNAc and Gal were the main linking sugars. S-layer glycoproteins of Halobacterium halobium and Halobacterium salinarum have also been shown to contain oligosaccharides linked via Glc and Gal to Ser/Thr residues (29). In addition, the cellulase complexes of Bacteroides cellulosolvens and Clostridium thermocellum contain oligosaccharide moieties linked by Gal to Ser/Thr residues (16, 17). However, it is clear that inactivation of rgpB causes changes in both the composition and the linkage of oligosaccharides to RgpA_cat/D7.

Further confirmation that the glycan composition of the mutant enzyme is different from that of the parent came from immunochemical analysis with MAb 1B5. This monoclonal antibody was originally raised to RgpA_cat but also recognizes a carbohydrate epitope(s) in mt-RgpA_cat, mt-RgpB, and a polysaccharide of this organism (10). The polysaccharide preparations from P. gingivalis D7 retained immunoreactivity with MAb 1B5, indicating that while the epitope for this antibody is no longer present in RgpA_cat/D7, the necessary machinery required to synthesize the epitope is still functioning in this mutant.

Although it is well established that glycosylation of eukaryotic proteins is important for the maintenance of protein conformation and stability (38) and protection against proteolytic degradation (35), there are few examples of these functions in the bacterial glycoprotein literature. A ß-1,4-xylanase/exo-ß-1,4-glucanase from Cellulomonas fimi was more resistant to proteolysis and had an increased affinity for microcrystalline cellulose (27, 36) when expressed in a glycosylated form. Finally, it has been suggested that glycosylation of a mycobacterial surface antigen acts to regulate the proteolysis of a linker region close to the N terminus of this molecule (20). Given the nature of the ecological niche occupied by P. gingivalis, it is possible that glycosylation of the RgpA proteases is necessary to ensure their stability and protection against proteolytic degradation in the hostile inflammatory conditions at periodontal sites undergoing destructive disease. There are other roles described for glycosylated bacterial proteins. Fischer and Haas (13) have shown that site-directed mutagenesis of a putative glycosylation site in Helicobacter pylori RecA results in the production of an unmodified RecA protein. This posttranslational modification is not involved in membrane targeting or cell division functions but is necessary for the full function of RecA in DNA repair. Karlyshev et al. (23) have described mutants of Campylobacter jejuni deficient in their ability to glycosylate a number of proteins, which reduces the ability of C. jejuni to adhere to and invade human epithelial cells and to colonize chicks.

The precise role of rgpB in the correct glycosylation of the RgpA monomers is under continuing investigation. However, there are some interesting parallels involving the role of extracellular proteases of other pathogenic species in macromolecule biosynthesis. For example, it has recently been established that elastase, the product of lasB, in Pseudomonas aeruginosa plays an important role in the synthesis of alginate, the extracellular polysaccharide which is responsible for the mucoid phenotype of isolates of this organism in the cystic fibrosis lung (7, 22). In this case, cell-associated elastase is responsible for the proteolytic modification of the nucleoside diphosphate kinase (Ndk) of P. aeruginosa from a 16-kDa form to a 12-kDa moiety. Normally, Ndk is responsible for the synthesis of all nucleoside triphosphates or their deoxy derivatives. However, following elastase proteolysis, the truncated Ndk generates predominantly GTP, which is an important substrate in alginate biosynthesis. Thus, the inactivation of lasB in a mucoid strain leads to the abolition of alginate biosynthesis and the overexpression of elastase in a nonmucoid strain causes an increased synthesis of this polysaccharide. Similarly, it has recently been reported that inactivation of the extracellular cysteine protease, SpeB or streptopain, of Streptococcus pyogenes causes a decrease in the polysaccharide capsule expression in this organism although the molecular mechanism underlying this phenomenon has not been established (56). Hence, these two examples emphasize that proteolytic enzymes considered classical extracellular virulence factors may have additional cellular functions related to pathogenicity involving complex carbohydrate synthesis.

In the case of the involvement of rgpB in the glycosylation of rgpA-derived enzymes, it is plausible that RgpB may be required for the activation of an enzyme(s) involved in the transfer of sugar residues to amino acid residues in proteins or in the extension of oligosaccharide structures. Loss of these activities in the rgpB mutant may disable this process. The immunoglobulin superfamily-like domain at the C terminus of RgpB with a presumptive role in protein recognition/binding (12) could conceivably be involved in such a targeted proteolytic action. Nakayama et al. (32) have shown that Rgps play an important role in the proteolytic maturation of precursors of the outer membrane and structural proteins on the surfaces of P. gingivalis cells, which supports an activation hypothesis. In the present work, two-dimensional electrophoresis of soluble cytoplasmic proteins of P. gingivalis strains W50 and rgpB in the pH range 3 to 5.6 showed several differences. The most striking was the absence of enolase and a slightly lower level of phosphoribosylamine glycine ligase in D7 and the absence of thiol protease tpr in W50. However, at present, it is uncertain whether these differences are related to the aberrant maturation of rgpA-derived enzymes in rgpB. An alternative hypothesis is that loss of the RgpB template for glycosylation could influence the nature of glycan additions to RgpA-derived enzymes. While the precise mechanism remains to be determined, the data in the present report emphasize that RgpB is an important accessory protein in the maturation pathway of the RgpA protease monomers leading to the enhanced stability of RgpA_cat. Thus, the Rgps have multiple and overlapping functions in the pathogenicity of P. gingivalis.

	ACKNOWLEDGMENTS

 
This research was funded by the Medical Research Council (United Kingdom) by grant no. PG9318173.

	FOOTNOTES

 
* Corresponding author. Mailing address: MRC Molecular Pathogenesis Group, Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts & The London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom. Phone: 44-0207-882-2300. Fax: 44-0207-882-2181. E-mail: m.a.curtis{at}qmul.ac.uk.

Editor: J. D. Clements

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