©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Structural Characterization of the Arg-gingipain Proteinase of Porphyromonas gingivalis
BIOSYNTHESIS AS A PROTEINASE-ADHESIN POLYPROTEIN (*)

(Received for publication, September 27, 1994; and in revised form, November 18, 1994)

Nadine Pavloff (§) Jan Potempa (1) Robert N. Pike (2) Vaclav Prochazka Michael C. Kiefer James Travis (2) Philip J. Barr

From the  (1)From LXR Biotechnology Inc., Richmond, California 94804, the Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, 31-120 Cracow, Poland, and the (2)Department of Biochemistry, University of Georgia, Athens, Georgia 30602

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The identification of proteinases of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications in the study of host-pathogen interactions as well as in the discovery of potential therapeutic and immunoprophylactic agents. We have cloned and characterized a gene that encodes the 50-kDa cysteine proteinase gingipain or Arg-gingipain-1 (RGP-1) described previously (Chen, Z., Potempa, J., Polanowski, A., Wikstrom, M., and Travis, J. (1992) J. Biol. Chem. 267, 18896-18901). Analysis of the amino acid sequence of RGP-1 deduced from the cloned DNA sequence showed that the biosynthesis of this proteinase involves processing of a polyprotein that contains multiple adhesin molecules located at its carboxyl terminus. This finding corroborates previous evidence (Pike R., McGraw, W., Potempa, J., and Travis, J.(1994) J. Biol. Chem. 269, 406-411) that RGP-1 is closely associated with adhesin molecules, and that high molecular weight forms of the proteinase are involved in the binding of erythrocytes.


INTRODUCTION

Mammalian periodontal diseases result from complex interactions between the host and a variety of anaerobic microorganisms. A number of studies have suggested an important role for Porphyromonas gingivalis in human periodontal tissue destruction(1, 2, 3) . Several potential virulence factors, including the elaboration of proteinase activity, have been identified for this organism(4, 5, 6) . Proteinases have been proposed to play a major role in periodontal disease because of their capacity to degrade protective host immunoglobins and to hydrolyze host proteins that provide amino acids required for growth, and by their participation in the destruction of host connective tissue (7, 8, 9, 10, 11, 12, 13) . Furthermore, there are reports indicating a direct involvement of ``trypsin-like'' proteinases of P. gingivalis in its binding to erythrocytes and extracellular matrix components. This suggests that some of the P. gingivalis proteolytic enzymes associated with the cell surface function as adhesins that mediate bacterial adherence to host tissues (reviewed in (14) ). Several groups have reported the cloning of proteinase genes from P. gingivalis(15, 16, 17, 18, 19, 20) . We report here the molecular cloning of a gene that encodes gingipain-1 (RGP-1), (^1)a 50-kDa arginine-specific cysteine proteinase, described previously by Chen et al.(21) , and found predominantly in culture medium as 95- and 50-kDa proteins, or associated with bacterial membranous fractions as 110- and 70-90-kDa forms.

Although the role of the RGP-1 proteinase in the development of periodontal disease is not yet fully clear, recent results have indicated that this proteinase is the major vascular permeability enhancement factor of P. gingivalis, resulting in gingival crevicular fluid production at sites of periodontitis caused by infection with this organism(22) . It has been shown previously that 110- and 95-kDa RGP-1 protein complexes possess erythrocyte-binding properties(23) , suggesting an association between proteolytic and hemolytic activities. Here we show, from analysis of the amino acid sequence deduced from the rgp1 gene sequence, that this proteinase/adhesin association is derived from the biosynthesis of RGP-1 as a polyprotein that contains multiple adhesin domains at the carboxyl terminus of the previously identified proteinase. By comparison of the proteinase domain of the polyprotein with the sequences of other cysteine proteinases, RGP-1 appears to be a member of a new family of pathogenic proteinases.


EXPERIMENTAL PROCEDURES

Bacterial Strains

P. gingivalis strains H66 and W50 were obtained from Dr. Roland Arnold, Emory University, Atlanta, GA.

Nucleic Acids

Oligonucleotides were synthesized by the phosphoramidite method using an Applied Biosystems model 394 DNA synthesizer, purified by PAGE, and desalted on Sep-Pak cartridges (Millipore). DNA templates used for PCR were isolated from P. gingivalis strain H66 by standard procedures(24) .

Genomic Library Construction

A DASH P. gingivalis H66 DNA library was constructed using the DASH(TM) II/BamHI cloning kit (Stratagene). A library of 2 times 10^5 independent recombinant clones was obtained. ZAP P. gingivalis H66 and W50 DNA libraries were also constructed as described using the ZAP/BamHI cloning kit (Stratagene). Libraries of 3 times 10^5 and 1.5 times 10^5 independent recombinant clones were obtained from strains H66 and W50, respectively.

Southern Blot Analysis

BamHI, HindIII, or PstI digestions of P. gingivalis H66 DNA were blotted (24) and hybridized with P-labeled oligonucleotide probes. Membranes were washed as described below.

Screening of Genomic Libraries

Approximately 10^3 to 10^5 phage were grown on 5 times 150-mm plates, lifted in duplicate onto supported nitrocellulose transfer membranes (BAS-NC from Schleicher & Schuell). Hybridizations were performed overnight at 42 °C in 2 times Denhardt's solution(24) , 6 times SSC (SSC is 15 mM sodium citrate, 150 mM NaCl), 0.4% SDS (w/v), 500 mg/ml salmon sperm DNA. Filters were washed in 2 times SSC containing 0.05% SDS (w/v) at 48 °C. Positively hybridizing plaques were purified. Standard protocols for cDNA library screening, phage purification, agarose gel electrophoresis, and plasmid cloning were used(24) . For cloning of the 3`-region of the rgp1 gene, PstI/HindIII-digested DNA (50 µg) was size-selected on 1% agarose, and the region at 4.5 kbp was cloned into pBluescript SK(-). Positive clones were identified using a 20-mer oligonucleotide probe.

DNA Sequencing

Double-stranded DNA cloned into pBluescript SK(-) and single-stranded DNA cloned into M13mp18 and M13mp19 were sequenced by the dideoxy method(25) , using sequencing kits purchased from United States Biochemicals (Sequenase version 2.0). A 6327-base pair PstI/PvuII fragment encoding the full RGP-1 polyprotein gene was submitted to GenBank under the accession number U15282. Gene fragments obtained by PstI, PstI/Asp718, and BamHI/HindIII digestion of plasmid subclones were introduced into M13 vectors, and the sequence was obtained by single strand sequencing of M13 subclones in both directions.

Active Site Titration

RGP-1 from P. gingivalis H66 was purified and titrated as described previously(21, 22, 23) . Tosyl-L-lysine chloromethyl ketone (TLCK) and sequencing grade enzymes: Staphylococcus aureus V8 protease (Glu-C) and Asp-N endopeptidase were from Boehringer Mannheim. Bio-X-FPRck was purchased from Haematologic Technologies Inc. (Essex Jct., VT), and avidin (monomeric)-agarose was obtained from Sigma.

Active Site Labeling

RGP-1 (6.4 µM) was activated in 75 mM HEPES, 2 mM CaCl(2), 8 mM cysteine, pH 8.0, for 15 min at 37 °C, then treated with a 1.2 molar excess of Bio-X-FPRck and incubated for 15 min at room temperature. Residual RGP activity (less than 5%) was eliminated by treatment with TLCK (final concentration 2 mM), the sample was dialyzed extensively against 50 mM ammonium bicarbonate, pH 7.8 and lyophilized. Biotinylated RGP (50 nmol) was denatured in 6 M guanidine HCl, reduced with dithiothreitol, and S-carboxymethylated by the method of Waxdal et al.(26) . The sample was desalted on a PD-10 column (Pharmacia Biotech Inc.) equilibrated with 50 mM ammonium bicarbonate pH 7.8, and the protein concentration determined by the BCA method (Pierce).

Polypeptide Chain Fragmentation and Analysis

The derivatized protein (25 nmol) was digested in 25 mM ammonium bicarbonate buffer pH 7.8 with V8 protease at 25 °C for 12 h (1:100 enzyme to substrate weight ratio) or with Asp-N endopeptidase at 37 °C for 10 h (1:1000 enzyme to substrate weight ratio). Each digest (2 ml) was loaded on a 5-ml avidin-agarose column equilibrated with 0.1 M Tris, pH 8.0. The column was washed with 30 ml of the equilibration buffer, followed by 30 ml of 1.0 M NaCl/Tris, pH 8.0, then 50 ml of deionized H(2)O. Biotinylated peptides were eluted with HCl (pH 2.0), and 5-ml fractions were collected directly into 1 ml of 0.2 M ammonium bicarbonate. Biotinylated peptide-containing fractions were detected by dot blot analysis on nitrocellulose membranes. Blotted membranes were incubated with streptavidin-alkaline phosphatase conjugate and developed with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent kit from Bio-Rad. The fractions that contained biotinylated peptide were pooled, blotted onto polyvinylidine difluoride membranes, and subjected to amino-terminal amino acid sequence analysis using an Applied Biosystems 460A gas phase sequencer.


RESULTS AND DISCUSSION

We describe here that RGP-1, the major arginine-specific cysteine proteinase from P. gingivalis, is synthesized as a polyprotein that can function as an erythrocyte-binding protein through the presence of multiple adhesin domains at its carboxyl terminus. Chen et al.(21) have determined the primary structure of the amino terminus of RGP-1 by direct amino acid sequencing. This sequence information was used to prepare a mixture of synthetic oligonucleotides: primer GIN-1-32, a 32-mer coding for amino acids 2-8 of the mature protein (TPVEEKE); and primer GIN-2-30, a 30-mer coding for amino acids 25-32 of the mature protein (KDFVDWKN). These primers were used to amplify from genomic DNA the corresponding fragment of the rgp1 gene by PCR. The expected 105-base pair PCR product was cloned and sequenced. On the basis of this sequence, GIN-8S-48, a unique 48-mer oligonucleotide probe corresponding to the coding strand of rgp1, was synthesized and used to screen a DASH DNA library constructed from BamHI-digested P. gingivalis genomic DNA. DNA sequence analysis of positive clones indicated that the proteinase domain was encoded by these 3.5-kbp clones. However, since no transcriptional termination codon was evident within the large open reading frame encoding the proteinase, overlapping clones were isolated from a size-selected PstI/HindIII plasmid library, using a 20-mer oligonucleotide probe. Using this procedure, several 4.5-kbp-containing clones were obtained. In total, 7.8 kbp of genomic DNA from BamHI to HindIII sites (Fig. 1A) was isolated and characterized. The composite 6327-base pair PstI/PvuII fragment of this genomic DNA (Fig. 1A) was fully sequenced in both directions and is described here, with the first base of the 5`-PstI site (Fig. 1A) assigned as base 1. Within this composite sequence was found an open reading frame encoding a 1704 amino acid sequence (Fig. 1B), with the 5`-most ATG initiation codon at nucleotides 949-951. Between this ATG and the mature RGP-1 sequence are an additional 8 in-frame methionine codons. The exact ATG used for initiation of translation is currently unknown, although the presence of a consensus TATA box (TATAAT) at nucleotides 889-894 suggests the 5`-most ATG as the strongest candidate.


Figure 1: A, map of the cloned genomic DNA sequence encompassing the rgp1 gene. Only major restriction sites are indicated: B, BamHI; P, PstI; S, SmaI; A, Asp718; Pv, PvuII; H, HindIII. M13 subclones used for DNA sequencing are shown (arrows). The overlap at the 3`-PstI site was determined by sequencing a SmaI/BamHI plasmid clone. Also shown is a schematic representation of the RGP-1 polyprotein structure, including the proposed methionine used for translation initiation and experimentally determined basic residue cleavage sites. Also shown () is the experimentally determined active site cysteine residue of the previously identified 50-kDa mature RGP-1(21) . B, full deduced amino acid sequence of the RGP-1 polyprotein in single-letter code. Peptide sequences identified previously(23) , or as part of the present work, are underlined. The first amino acid of the mature RGP-1 proteinase (tyrosine) is assigned as amino acid 1.



The most striking feature of the deduced protein sequence is the presence of multiple homologous sequences immediately carboxyl-terminal to the proteinase coding domain (Fig. 1), leading to a calculated molecular mass of 185.4 kDa of the encoded polyprotein. Within these sequences can be found peptides identified by Pike et al.(23) as the components of high molecular mass gingipain (HGP) that confer adhesion activity on the high molecular mass RGP-1 complex. The polyprotein sequence deduced from the gene sequence now allows exact delineation of the primary structure of the mature RGP-1 proteinase. The amino terminus (23) is derived from proteolytic processing at an arginine residue (Fig. 1A). The carboxyl terminus is derived by processing at Arg-492 and also releases the amino terminus of the 44-kDa HGP (HGP44, Fig. 1, A and B, underlined) determined by Pike et al.(23) . Similar processing at Arg-1202 gives rise to the amino terminus of the 27-kDa HGP27 (Fig. 1, A and B, underlined) also found to be associated with RGP adhesion activity (23) .

More recently, we have used high resolution SDS-PAGE (27) to separate the 95-kDa form of RGP into 5 major bands of 50, 44, 27, 17, and 15 kDa. (^2)Amino-terminal sequence analysis confirmed the structures of the 50-, 44-, and 27-kDa fragments reported previously(23) . For the 17- and 15-kDa fragments, the following amino termini were determined: PQSVWIERTVDL and ADFTETFESSTHG, respectively. Thus, the 17-kDa polypeptide (HGP17, Fig. 1, A and B, underlined) is cleaved at lysine residue 1044, most likely catalyzed by Lys-gingipain, the other cysteine proteinase produced in large quantities by P. gingivalis(23) . The sequence of the 15-kDa fragment (HGP15, Fig. 1, A and B, underlined) reveals that processing occurs after Arg-909. The calculated molecular mass of 53.9 kDa for RGP-1 is in good agreement with its mobility of 50 kDa on SDS-PAGE. Similarly, the 417-, 275-, 158-, and 135-amino acid sequences of HGP44 (44.7 kDa), HGP27 (29.6 kDa), HGP17 (17.5 kDa), and HGP15 (14.3 kDa), respectively, correlate well with their SDS-PAGE mobilities. Together, the proteinase domain and adhesin/hemagglutinin fragments would create a polyprotein of 159.9 kDa, while the high molecular mass form of RGP (HGP) is only 95 kDa, indicating that the secreted enzyme is most likely processed and assembled as a non-covalent complex of the proteinase with different individual adhesin/hemagglutinin domains.

Three large repeats of homologous sequence are located within three of the cleavage products (Fig. 2A). The first is found in the middle of HGP44, the second in HGP17, and the third in the carboxyl-terminal region of HGP27. Amino acid sequence identities within these 49-amino acid stretches varies between 76 and 96%, indicating similar roles for each sequence, possibly in non-covalent interactions with the proteinase domain, or in the adhesion activity of the high molecular mass complexes.


Figure 2: A, alignment of conserved areas within the adhesin domains of the RGP-1 polyprotein sequence. B, identification of the active site cysteine residue as Cys-185 by active site labeling and separation of V8 proteinase- and Asp-N-derived peptides. X refers to unidentifiable amino acid residues at a given Edman degradation cycle. During such analyses, these positions are characteristically associated with cysteine or modified amino acid residues in the polypeptide chain.



The mature RGP-1 sequence exhibited no similarity with any cysteine proteinase reported previously except for the related enzyme from the same organism, referred to as Lys-gingipain (23) . (^3)Even in this case, the similarity is limited to the sequence around His-211 and Asn-442, suggesting that these residues, along with the active site cysteine residue (Cys-185), determined by active site labeling (Fig. 2B), encompass the catalytic triad. Interestingly, the lack of any similarity around these sequences with cysteine proteinases other than Lys-gingipain suggests that these bacterial proteinases represent a distinct branch of this family of proteolytic enzymes. That the catalytic apparatus of RGP-1 might be different from that of other known cysteinyl proteinases demands that residues other than Cys-185 involved directly in the hydrolysis of peptide bonds must be verified experimentally.

Molecular cloning of the rgp1 gene confirms previous findings that this arginine-specific proteinase is closely associated with adhesion activity. High trypsin-like activity has been shown previously to be an important virulence factor in P. gingivalis. Sequencing of the corresponding rgp1 gene of the virulent W50 strain of P. gingivalis revealed only two conservative amino acid changes in the mature enzyme sequence (data not shown). Thus, any involvement of RGP-1 in virulence would have to be due to its differential regulation, and enhanced expression in virulent strains. The availability of RGP-1 DNA sequences will now allow the further study of hemolysis of erythrocytes through the adhesin/hemagglutinin activities of this proteolytic polyprotein. Recombinant polypeptides can also be used for the development of potential immunoprophylactic and therapeutic agents against this human pathogen.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15282[GenBank].

§
To whom correspondence should be addressed: LXR Biotechnology Inc., 1401 Marina Way South, Richmond, CA 94804. Tel.: 510-412-9100; Fax: 510-412-9109.

(^1)
The abbreviations used are: RGP-1, Arg-gingipain; PAGE, polyacrylamide gel electrophoresis; Bio-X-FPRck, biotin--aminocaproyl-Phe-Pro-Arg-chloromethylketone; kbp, kilobase pair(s); HGP, high molecular mass gingipain; PCR, polymerase chain reaction; TLCK, tosyl-L-lysine chloromethyl ketone.

(^2)
J. Potempa, unpublished results.

(^3)
N. Pavloff, J. Potempa, R. N. Pike, V. Prochazka, M. C. Kiefer, J. Travis, and P. J. Barr, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical contributions of W.-C. A. Chen, T. Rigley, and K. Norris.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.