Comparative Properties of Two Cysteine Proteinases (Gingipains R), the Products of Two Related but Individual Genes ofPorphyromonas gingivalis (1998)

Proteolytic enzymes produced byPorphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of α-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, α-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function asP. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components. Proteolytic enzymes produced byPorphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of α-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, α-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function asP. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components. Periodontal disease, the major cause of tooth loss in the general population of industrial nations (1Shaw J.H. N. Engl. J. Med. 1987; 317: 996-1004Crossref PubMed Scopus (50) Google Scholar, 2Williams R.C. N. Engl. J. Med. 1990; 422: 373-382Crossref Scopus (441) Google Scholar), is a chronic inflammatory disorder of tissues directly supporting the root of the tooth in alveolar bone sockets. The disease is characterized by bone resorption, loss of tooth attachment, and formation of periodontal pockets infested with specific bacteria (3Williams D.M. Huges F.J. Odell E.W. Farthing P.M. Pathology of Periodontal Disease. Oxford University Press, Oxford1992Google Scholar). Although more than 400 distinct bacterial taxa have been identified in dental plaque samples, very few can be correlated with the development and/or progression of the specific clinical variants of periodontitis (2Williams R.C. N. Engl. J. Med. 1990; 422: 373-382Crossref Scopus (441) Google Scholar). Adult onset disease, the most common form of periodontitis, is strongly associated with infections byPorphyromonas gingivalis (4Socransky S.S. Haffajee A.D. J. Periodontol. 1992; 63: 322-331Crossref PubMed Scopus (849) Google Scholar), and this is further substantiated by the finding that infection by this bacterium initiates periodontitis in primates (5Holt S.C. Ebersole J. Felton J. Brunsvold M. Kornmann K.S. Science. 1987; 239: 55-57Crossref Scopus (375) Google Scholar) and can be avoided by animal immunization with P. gingivalis antigens (6Persson G.R. Engel D. Whitney C. et al.Infect. Immun. 1994; 62: 1026-1031Crossref PubMed Google Scholar). P. gingivalis, a Gram-negative, anaerobic, nonmotile, nonsporing short rod elaborates a multiplicity of virulence factors involved in invasion, tissue destruction, and evasion of host defenses, all of which enable this bacterium to colonize within its ecological niche of either the gingival sulcus or periodontal pocket (reviewed in Ref. 7Cutler C.W. Kalmar J.R. Genco C.A. Trends Microbiol. 1995; 3: 35-51Abstract Full Text PDF Google Scholar). Among these factors, the primary focus of research has been on proteolytic enzymes that are produced in large quantities by this bacterium and could add to its pathogenicity. Indeed, it was shown that these proteinases can directly or indirectly degrade constituents of the periodontal tissues, destroy host defense elements, dysregulate coagulation, complement, and kallikrein-kinin cascades. Thus, they could be responsible for most of the clinical hallmarks of periodontitis (reviewed in Ref. 8Potempa J. Pike R. Travis J. Prospect. Drug Discovery Design. 1995; 2: 445-458Crossref Scopus (33) Google Scholar). Until recently, proteinases belonging to two catalytic classes and produced by P. gingivalis have been identified. A serine proteinase with prolyl peptidase activity was found to be associated with the bacterial cell surface (9Grenier D. McBride B.C. Infect. Immun. 1989; 57: 3265-3269Crossref PubMed Google Scholar), whereas large amounts of cysteine proteinases with trypsin-like activity were detected in either a soluble form in culture media or combined with whole bacteria or their membranous fragments, including vesicles (10Potempa J. Pike R. Travis J. Infect. Immun. 1996; 63: 1176-1182Crossref Google Scholar). Because of convenient assay systems with chromogenic substrates, trypsin-like enzymes were targets for numerous attempts at purification, but only recently has it become apparent that two distinct proteinases are responsible for this activity. One enzyme, described as an Arg-X specific proteinase, was purified from the culture media of P. gingivalis HG66, either as a single chain 50-kDa proteinase (RGP-1) 1The abbreviations used are: RGP-150-kDa gingipain R1 or arginine-specific gingipain 1HRGP95-kDa gingipain R1 or high molecular mass arginine-specific gingipain 1KGPlysine-specific gingipain or gingipain KBAPNAbenzoyl-l-arginine-p-nitroanilideFFRckPhe-Phe-Arg-chloromethyl ketonePAGEpolyacrylamide gel electrophoresisα1-Achyα1-antichymotrypsin.1The abbreviations used are: RGP-150-kDa gingipain R1 or arginine-specific gingipain 1HRGP95-kDa gingipain R1 or high molecular mass arginine-specific gingipain 1KGPlysine-specific gingipain or gingipain KBAPNAbenzoyl-l-arginine-p-nitroanilideFFRckPhe-Phe-Arg-chloromethyl ketonePAGEpolyacrylamide gel electrophoresisα1-Achyα1-antichymotrypsin. (11Chen Z.X. Potempa J. Polanowski A. Wikström M. Travis J. J. Biol. Chem. 1992; 267: 18896-18901Abstract Full Text PDF PubMed Google Scholar) or a high molecular mass (95 kDa) complex of a 50-kDa catalytic domain with hemagglutinins/adhesins (12Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar). Recently, molecular cloning and characterization of its gene revealed that all components of the 95-kDa gingipain-R (HRGP) are created by proteolytic processing of a single polyprotein (13Pavloff N. Potempa J. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1995; 270: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Similarly, a Lys-X specific proteinase (gingipain K, KGP) was separated as a 105-kDa protein complex composed of a unique catalytic domain (60 kDa) associated with hemagglutinins/adhesins (12Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar). Since the first purified enzyme shared some properties with clostripain, it was named as a gingipain (P. gingivalis clostripain) so that, following recommendations by the IUB, these proteinases are referred to as gingipain-R and gingipain-K to account for their unique specificity. 50-kDa gingipain R1 or arginine-specific gingipain 1 95-kDa gingipain R1 or high molecular mass arginine-specific gingipain 1 lysine-specific gingipain or gingipain K benzoyl-l-arginine-p-nitroanilide Phe-Phe-Arg-chloromethyl ketone polyacrylamide gel electrophoresis α1-antichymotrypsin. 50-kDa gingipain R1 or arginine-specific gingipain 1 95-kDa gingipain R1 or high molecular mass arginine-specific gingipain 1 lysine-specific gingipain or gingipain K benzoyl-l-arginine-p-nitroanilide Phe-Phe-Arg-chloromethyl ketone polyacrylamide gel electrophoresis α1-antichymotrypsin. The construction of isogenic P. gingivalis mutants deficient in the gingipain-R gene has shown unequivocally that these enzymes are pivotal virulence factors (14Nakayama K. Kadowaki T. Okamoto K. Yamamoto K. J. Biol. Chem. 1995; 270: 23619-23626Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) and, therefore, a perfect target for the development of specific potentially therapeutic inhibitors. However, before such compounds can be designed and tested as alternative treatments to reduce adult periodontitis, the biochemical characterization of rigorously purified gingipain-R forms is absolutely necessary. In this report, we examine distinct forms of this enzyme that have been found to be products of closely related but discrete genes. In addition, we clarify a controversy regarding: (i) putative collagenolytic activity, (ii) synthetic substrate specificity, (iii) inhibition by plasma proteinase inhibitors, and iv)stimulation of activity by glycyl-glycine. Bz-l-Arg-pNA and tosyl-l-lysine chloromethyl ketone were purchased from Sigma. S-2238 (d-Phe-Pip-Arg-pNA), S-2288 (d-Ile-Pro-Arg-pNA), S-2366 (pyroGlu-Pro-Arg-pNA), and S-2444 (pyroGlu-Gly-Arg-pNA) were obtained from Pharmacia-Harper. Z-Arg-pNA, Gly-Arg-pNA, Z-Arg-Arg-pNA, Z-Lys-Arg-pNA, Z-Phe-Arg-pNA, Z-Tyr-Lys-Arg-pNA, Boc-Val-Leu-Gly-Arg-pNA, and Leu-Thr-Arg-pNA were the product of Bachem. Chromozym TRY (Z-Val-Gly-Arg-pNA), Chromozym U (Bz-Ala-Gly-Arg-pNA), Chromozym t-PA (MeS-Phe-Gly-Arg-pNA), Chromozym PK (Bz-Pro-Phe-Arg-pNA), and Chromozym TH (Tos-Gly-Pro-Arg-pNA) were purchased from Boehringer, whereas Bz-Phe-Val-Arg-pNA, Boc-Leu-Gly-Arg-pNA, Bz-Ile-Glu-Gly-Arg-pNA, Z-Lys-Phe-Arg-pNA, and Sar-Pro-Arg-pNA were obtained from Calbiochem. Acid-soluble collagens type I from calf skin, rat tail, human placenta, and kangaroo tail were purchased from Sigma, whereas guinea pig collagen type I was a gift from Dr. Hideaki Nagase (University of Kansas, Kansas City, KS). In all cases, collagen integrity was checked by its resistance to degradation by porcine trypsin. The strain of P. gingivalis (HG66) was a gift of Dr. Roland Arnold (University of North Carolina, Chapel Hill). The cells were grown in 200 ml of broth containing 6.0 g of Trypticase Soy broth (Difco), 2.0 g of yeast extract, 1 mg of hemin, 200 mg of cysteine, 20 mg of dithiothreitol, and 0.5 mg of menadione (all from Sigma) anaerobically, at 37 °C for 48 h in an atmosphere of 85% N2, 10% CO2, 5% H2. The culture was used to inoculate 5 liters of the same broth, which was then incubated anaerobically, at 37 °C for about 48–60 h until the late stationary phase of bacteria growth (final absorbance at 660 nm > 2). The initial steps of gingipain R2 (RGP-2) purification were performed according to the methods designed for HRGP and KGP isolation (12Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar). Briefly, the cell-free culture fluid was precipitated with acetone, and the protein pellet was redissolved in 20 mm Bis-Tris, 150 mm NaCl, 0.02% NaN3, pH 6.8, containing 1.5 mm4,4′-dithiopyridine disulfide. The solution was then dialyzed, first against the above buffer (one change), followed by two changes with Bis-Tris/NaCl buffer supplemented with 5 mmCaCl2 but without 4,4′-dithiopyridine disulfide. The dialyzed fraction was clarified by centrifugation (40,000 ×g, 2 h), concentrated by ultrafiltration (Amicon PM-10 membrane), and applied to a Sephadex G-150 column equilibrated with Bis-Tris/NaCl buffer. The column was developed at a flow rate of 30 ml/h with three peaks of activity on BAPNA being found. The highest molecular mass activity peak was used for the purification of HRGP, exactly as described by Pike et al. (12Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar), whereas the low molecular mass peak (50 kDa), having the majority of the activity on BAPNA, was pooled, concentrated, extensively dialyzed against 50 mm Bis-Tris, 1 mm CaCl2, pH 6.5, and loaded onto a DE-52 cellulose (Whatman) column (1.5 × 20 cm), equilibrated with Bis-Tris/CaCl2 buffer at a flow rate of 20 ml/h. The column was washed until the A280 nmbase line fell to zero, followed by application of a gradient of 0–200 mm NaCl in a total volume of 200 ml. Fractions (5 ml) were assayed for activity on BAPNA. Some activity was found in the void volume of the column, but the major peak was eluted at about 100 mm NaCl. The latter activity was pooled, dialyzed, and applied to an arginine-Sepharose column (1.5 × 30 cm, 50 ml) equilibrated with 50 mm Tris, 1 mmCaCl2, pH 7.4, with 0.02% NaN3 at a flow rate of 20 ml/h. The column was washed with buffer until activity on BAPNA fell below 20 mOD/min/μl, and the remaining enzyme then eluted with 0.5 m NaCl. Five pools of activity obtained in this step (Fig. 1), nonadsorbed (A), retarded (B–D), and eluted with NaCl (E) were collected, concentrated, and dialyzed against 25 mm Bis-Tris, pH 6.3. Different isoforms of gingipain R2 were obtained from these pools by chromatofocusing on a mono-P column (Pharmacia, fast protein liquid chromatography system) equilibrated with 25 mm Bis-Tris, pH 6.3, using a pH gradient developed with 50 ml of 10× diluted Polybuffer 74 (Pharmacia) adjusted to a pH of 4.0. Routinely, amidolytic activities of gingipains R were measured with l-BAPNA (1 mm) in 1.0 ml of 0.2 m Tris-HCl, 0.1 m NaCl, 5 mm CaCl2, 10 mml-cysteine, pH 7.6, at 37 °C. After a specific time of incubation, the reaction was stopped by addition of 0.05 ml of glacial acetic acid, and the O.D. at 405 nm then measured against a blank sample containing no proteinase. To determine the effect of glycyl-glycine on RGP activity, buffer (0.1 m Tris-HCl, 5 mm CaCl2, 10 mm cysteine, pH 7.6) was supplemented to a desired dipeptide concentration by mixing with 1.0 m glycyl-glycine, 5 mm CaCl2, 10 mm cysteine, pH 7.6. To compensate for any change of the ionic strength to the control without glycyl-glycine, appropriate volumes of 0.1 m Tris, 0.2 m NaCl, 5 mm CaCl2, 10 mm cysteine, pH 7.6, were added. General proteolytic activity was measured with 1.0% (w/v) azocasein (15Barrett A.J. Kirschke H. Methods Enzymol. 1981; 80: 535-561Crossref PubMed Scopus (1727) Google Scholar), whereas the amount of active enzyme in each batch of gingipain was determined by active site titration using FFRck (16Potempa J. Pike R. Travis J. Biochemistry. 1997; 378: 223-230Google Scholar). Gingipain purification and protein degradation was monitored by Tricine SDS-PAGE using the Tris-HCl/Tricine buffer system (17Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10460) Google Scholar). To avoid protein degradation during boiling, all samples were treated with 0.05 mmFFRck, boiled in nonreducing SDS-treatment buffer, and then reboiled under reducing conditions. For amino-terminal sequence analysis, proteins resolved in SDS-PAGE were electrotransferred onto polyvinylidene difluoride (18Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Zymography analysis was performed on samples solubilized in SDS buffer (4% SDS, 20% glycerol, 0.125m Tris-HCl, pH 6.8) for 30 min at 37 °C and electrophoresed on 10% SDS-PAGE with gelatin (0.1 mg/ml, Difco Laboratories, Detroit, MI) incorporated into the gel (19Heussen C. Dowdle E.B. Anal. Biochem. 1980; 102: 196-202Crossref PubMed Scopus (1845) Google Scholar). The two serpins were incubated with gingipains at molar ratios from 10:1 to 1000:1 in 0.02m Tris-HCl, 0.15 m NaCl, 5 mmCaCl2, 10 mm cysteine, pH 7.6, at 37 °C. At specific time intervals, aliquots were removed and treated with FFRck to stop the reaction, with residual inhibitory activity being measured against the target proteinases, human cathepsin G (α1-Achy) and neutrophil elastase (α1-PI). Inhibitor-treated samples were also analyzed by SDS-PAGE (17Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10460) Google Scholar). The kcat andKm values were measured at 21 °C using substrates at concentrations ranging from 0.005 to 2 mm, with a final concentration of active site titrated enzyme of 3.2 nm in 0.1 m Tris-HCl, 5 mm CaCl2, 10 mm cysteine, 200 mm glycyl-glycine, pH 7.6. In the absence of glycyl-glycine, 12.5 nm gingipain was used in 0.2 m Tris-HCl, 5 mm CaCl2, 10 mm cysteine, 0.1 m NaCl, pH 7.6. The assay was performed in a total volume of 0.2 ml on microplates coated with albumin (20Wikström M. Potempa J. Polanowski A. Travis J. Renvert S. J. Periodontol. 1994; 65: 47-55Crossref PubMed Scopus (22) Google Scholar). To 0.05 ml of substrate, 0.15 ml of enzyme solution was added with a multichannel pipette, and the initial turnover rate at 12 different substrate concentrations was recorded at 405 nm using a micro plate reader (Molecular Devices, Vmax). TheKm and kcat values were calculated using Hyperbolic Regression Analysis, a program written by J. S. Easterby (University of Liverpool, UK) and obtained through shareware. Stimulation of gingipain amidolytic activity on different substrates by glycyl-glycine (stimulation factor) was determined at substrate concentrations at least five times higher than the Km value. RGP-2 was denatured in 6m-guanidine HCl, reduced with dithiothreitol, andS-carboxymethylated or S-pyridylethylated using protocols provided by Applied Biosystems. After rapid desalting on a PD-10 column (Pharmacia) equilibrated with 50 mm ammonium bicarbonate, pH 7.8, the modified protein solution was lyophilized. The derivatized protein was subjected to digestion with cyanogen bromide (in 70% w/v formic acid) at room temperature in the dark for 18 h at a 1:1000 molar ratio with respect to methionine. Enzymatic fragmentation was performed withl-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin and Glu-C endopeptidase. Generated peptides were separated by reverse phase high pressure liquid chromatography using a Vydac protein C18-10 column (4 × 30 mm). The peptides were eluted with 0.1% trifluoroacetic acid and acetonitrile containing 0.08% trifluoroacetic acid using a gradient from 0 to 80% acetonitrile over 60 min. Peptides were monitored at 220 nm and collected manually. For active site cysteine residue determination, RGP-2 was first labeled with biotin-ε-aminocaproyl-Phe-Pro-Arg-chloromethylketone (Hematologie Technologies, Inc., Essex Junction, VT) and thenS-pyridylethylated and subjected to proteolytic fragmentation with trypsin. Biotinylated peptide was purified on avidin-agarose (Sigma) and analyzed for both amino acid composition and sequence as described previously (13Pavloff N. Potempa J. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1995; 270: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). analysis was performed with an Applied liquid phase with analysis using an Applied high pressure liquid chromatography system according to the For amino acid purified RGP-2 isoforms and peptides were for h at °C in containing 0.1% The were and with several times before were applied to a high amino acid with a The molecular of the purified isoforms of RGP-2 were by SDS-PAGE using the Tris-HCl/Tricine buffer system (17Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10460) Google Scholar) and the of PubMed Scopus Google Scholar) on 10 or molecular mass were performed on all major isoforms of RGP-2 with mass using a mass chromatography of the obtained from culture a protein and activity BAPNA and to that previously (12Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar). the low molecular mass active fraction containing the majority of the activity against BAPNA was used for further chromatography of this fraction in the of two peaks of activity, that not to DE-52 cellulose and a major activity that eluted at 100 mm NaCl concentration and of the total activity. The activity in the fraction was not to the column during its all activity through the The of this fraction that of the 50-kDa gingipain R1 (RGP-1) purified by et al. (11Chen Z.X. Potempa J. Polanowski A. Wikström M. Travis J. J. Biol. Chem. 1992; 267: 18896-18901Abstract Full Text PDF PubMed Google Scholar). The major peak of activity was subjected to affinity chromatography on an arginine-Sepharose column, with three active being obtained (Fig. (A), retarded (B–D), and the of each fraction was checked by and it was shown that of proteinases were with reducing or at the site with tosyl-l-lysine chloromethyl each fraction was eluted from the column in a specific of differing for residues. gingipains in the collected the same molecular mass containing the of the native proteinase, also a protein of about (Fig. The amino acid sequence was the same for both that the a and or apparent molecular all gingipain in specific activity the most active being in and of gingipain at 37 at 37 °C. in a gingipain was subjected to with enzyme isoforms being purified A an of as as an protein In was and in isoforms and (Fig. 2). In the of gingipain isoforms was whereas in with an form from the column at a pH lower than any form not isoforms as as the same mobility in SDS-PAGE (Fig. to a molecular mass of as determined using of several The of and were determined by and these are in with from In addition to in molecular mass, the various isoforms also in that were determined to be and for isoforms from I to (Fig. In contrast, the of HRGP was found to be not The in of RGP-2 isoforms correlated with their mobility in gelatin zymography (Fig. in that forms with the higher were more retarded in the comparison of in and purified RGP-2 it is apparent that is the major form of this gingipain gingivalis The gene RGP-2 has been and from two different of P. and a chain with a acid followed by a catalytic domain of residues. The calculated molecular mass of for the catalytic domain was than its mass determined by mass and a at the The in the molecular mass different RGP-2 isoforms were that the chain of RGP-2 was residue mass (Fig. The amino acid analysis of the isoforms to any and that the various were a few amino acid than from molecular mass analysis not To clarify this the primary structure of RGP-2 was determined by sequence analysis of and proteolytic enzyme and of for a acid residue peptide all of RGP-2 could be purified and that RGP-2 is composed of 422 amino acid with the sequence being through of the gingipain chain by a proteinase (Fig. acid analysis such a structure (Fig. although its calculated molecular mass is about lower than determined from the mass is most of some amino acid chain that to formation of the RGP-2 these no effect on enzyme as in it was found that the isoforms were with to pH and proteolytic activity. in described below properties of RGP-2 and HRGP, either fraction from the arginine-Sepharose chromatography step or purified was has been that and were created by the two in P. gingivalis, through K. Microbiol. 1997; PubMed Scopus Google Scholar). Indeed, the primary structure of RGP-2 is generally to the catalytic domain of HRGP, although both within the (Fig. also to the containing the active site cysteine in HRGP has been a of controversy (13Pavloff N. Potempa J. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1995; 270: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, M. Biochem. Biol. 1995; Google Scholar). to determine the of this residue in RGP-2 by specific of the active site residue using biotin-ε-aminocaproyl-Phe-Pro-Arg-chloromethylketone N. Potempa J. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1997;