(Received for publication, April 9, 1996, and in revised form, August 27, 1996)
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 ¶ Department of Biochemistry, University of Georgia, Athens, Georgia 30602
The proteinases of Porphyromonas gingivalis are key virulence factors in the etiology and progression of periodontal disease. Previous work in our laboratories resulted in the purification of arginine- and lysine-specific cysteine proteinases, designated gingipains, that consist of several tightly associated protein subunits. Recent characterization of arginine-specific gingipain-1 (gingipain R1; RGP-1) revealed that the sequence is unique and that the protein subunits are initially translated as a polyprotein encoding a proteinase domain and multiple adhesin domains (Pavloff, N., Potempa, J., Pike, R. N., Prochazka, V., Kiefer, M. C., Travis, J., and Barr, P. J. (1995) J. Biol. Chem. 270, 1007-1010).
We now show that the lysine-specific gingipain (gingipain K; KGP) is also biosynthesized as a polyprotein precursor that contains a proteinase domain that is 22% homologous to the proteinase domain of RGP-1 and multiple adhesin domains. This precursor is similarly processed at distinct sites to yield active KGP. The key catalytic residues in the proteinase domain of KGP are identical to those found in RGP-1, but there are significant differences elsewhere within this domain that likely contribute to the altered substrate specificity of KGP. Independent expression of the proteinase domain in insect cells has shown that KGP does not require the presence of the adhesin domains for correct folding to confer proteolytic activity.
The anaerobic Gram-negative rod bacterium Porphyromonas gingivalis is implicated in the initiation and progression of certain forms of periodontitis, including juvenile and adult periodontal disease (1-3). Mechanisms by which P. gingivalis evades the host defense response and elicits hard and soft tissue damage in the periodontal pocket are under investigation, with many studies being focused on the proteinases produced by the organism. To date, the combined proteolytic activities of P. gingivalis have been shown to be capable of degrading and inactivating host defense proteins (iron-binding proteins, immunoglobulins, and complement components), structural proteins (collagen, fibronectin, and fibrinogen), and plasma proteinase inhibitors (4-10). In addition, proteinase activity is associated with the ability of the organism to adhere to collagenous substrata and to hemagglutinate and lyse red cells, thus allowing the organism to remain in the periodontal pocket while meeting its nutritional requirements for both heme and peptides (11, 12).
Recent work in our laboratories has resulted in the identification and purification of multiple cysteine proteinases, designated gingipains, some of which comprise several tightly associated protein subunits. The recent complete gene sequence of arginine-specific gingipain-1 (gingipain R1; RGP-1)1 revealed a unique proteinase that is synthesized initially as a polyprotein encoding a proteinase domain and four adhesin domains (13). The protein subunits are generated by subsequent proteolytic processing to mature RGP-1.
We have also purified a lysine-specific gingipain (gingipain K; (KGP) from P. gingivalis. This enzyme is a 60-kDa cysteine proteinase that is also found associated with adhesin molecules (14, 15). Amino-terminal protein sequencing of the 60-kDa proteinase subunit of KGP showed that it also is a unique proteinase. Here we present the complete DNA sequence of the kgp gene and its encoded protein. This represents the second fully characterized proteinase from an emerging family of cysteine proteinases that have structural features distinct from those of all other known families.
The sequence shows that KGP is similar to RGP-1 in its structural organization, biosynthesis, and maturational processing. We also show by expression of the KGP proteinase domain in insect cells that KGP proteinase activity is not dependent on the presence of the adhesin domains for correct folding. It is likely that the tightly regulated coexpression of proteinase and adhesion functions is present in other soluble or membrane-bound forms of gingipains that have been described and shown to be key factors in contributing to the pathogenicity of the organism.
P. gingivalis strains HG66 and W50 were obtained from Dr. Roland Arnold (Emory University, Atlanta).
Oligonucleotide SynthesisOligonucleotide primers for PCR
probes and DNA sequencing were synthesized by the phosphoramidite
method on an automated DNA synthesizer (Applied Biosystems Model 394),
purified by polyacrylamide gel electrophoresis, and desalted on Sep-Pak
C18 cartridges (Millipore Corp.) using standard protocols.
Primer MK-9-29 was designed to complement the noncoding strand of
kgp DNA corresponding to the six amino-terminal residues of
the mature protein (DVYTDH) (14). The sequence of this 29-base primer
consisted of 17 kgp-specific bases, a 6-base
EcoRI restriction site, and 6 extra bases at the 5-end
(underlined) as follows:
5
-
GAATTCGA(C/T)GT(A/C/G/T)TA(C/T)AC(A/C/G/T)GA(C/T)CA-3
. Primer MK-10-29 was designed to complement the coding strand of kgp DNA corresponding to residues 16-21 of the mature
protein (MLVVAC) (14). The sequence of this 29-base primer comprised 17 kgp-specific bases, a 6-base HindIII restriction
site, and 6 extra bases at the 5
-end (underlined) as follows:
5
-
AAGCTTCC(A/C/G/T)GC(A/C/G/T)AC(A/C/G/T)AC(A/C/G/T)A(A/G)CAT-3
. Primer Lys-1-33 (5
-CATACGAACCGGCGTATTATACAAGTCGCCATG-3
) was designed
to complement the noncoding strand of kgp DNA corresponding to amino-terminal residues 6-16 of the mature protein (HGDLYNTPVRM) and was designed on the basis of partial sequence information on
kgp (nucleotides 1351-1383; see Fig. 1A). This
primer was used as a probe to screen a
DASH P. gingivalis
genomic DNA library (see below). An additional oligonucleotide primer,
Lys-1-35, was used as a probe to identify and clone the
PstI/Asp718 3
-end fragment (see Fig.
1B) of the kgp gene from genomic DNA. Primer
Lys-1-35 was designed to complement the noncoding strand of
kgp DNA corresponding to 27 bases specific for the 3
-end of
the kgp gene (5
-TTCTACCGTAACGTCTTTACATACCTT-3
, nucleotides
3445-3471). Finally, primer HGP27-1 was used as a probe to identify
and clone the HindIII/HindIII 3
-end fragment (see Fig. 1B) of the kgp gene from genomic DNA.
Primer HGP27-1 was designed to complement the noncoding strand of
kgp DNA corresponding to 30 bases specific for the 3
-end of
the kgp gene (5
- GTAACCCGTATTGTCTCCCCATACGTTGTC-3
, nucleotides 2893-2922).
PCR
PCR amplification was performed on P. gingivalis strain HG66 DNA using primers MK-9-29 and MK-10-29 and
yielded consistently a single 76-base pair product (P76) representing a
kgp DNA fragment. After Klenow treatment and digestion with
EcoRI/HindIII, P76 was subcloned into M13mp18 and
M13mp19 vectors (New England Biolabs Inc.) and sequenced. Based on
these results, the specific primer (Lys-1-33) was synthesized,
32P-labeled, and used to screen the DASH library.
Incorporated radiolabeled nucleotides were separated from
unincorporated nucleotides on a Sephadex G-25 column (Boehringer
Mannheim).
The P. gingivalis
strain HG66 DASH and P. gingivalis strain W50
ZAP DNA
libraries used here have been described previously (13).
Approximately 2 × 105 phage were grown on 5 × 150-mm plates, lifted in
duplicate onto supported nitrocellulose transfer membranes (BAS-NC,
Schleicher & Schuell), and hybridized with the 32P-labeled
Lys-1-33 probe. Hybridizations were performed overnight at 42 °C in
2 × Denhardt's solution, 6 × SSC (15 mM sodium
citrate and 150 mM NaCl), 0.4% (w/v) SDS, and 500 mg/ml
fish sperm DNA. Filters were washed in 2 × SSC containing 0.05%
(w/v) SDS at 48 °C. The DNA from positive plaques was purified and
subjected to Southern analysis (see below). A 3.8-kb BamHI
fragment and 3.4-kb PstI fragment were identified, excised,
and cloned into pBluescript SK(). The 3.4-kb PstI fragment
and a 0.9-kb PstI/BamHI-generated 3
-end fragment
of the 3.8-kb BamHI fragment were cloned into M13mp18 and
M13mp19 vectors and sequenced. Standard protocols for cDNA library
screening,
phage purification, agarose gel electrophoresis, and
plasmid cloning were employed (16). To clone the 3
-end of the
kgp gene, PstI/Asp718- and
HindIII-digested DNAs were size-selected on 1% agarose, and
the regions at ~0.2 and 4.5 kilobase pairs, respectively, were cloned
into pBluescript SK(
). Positive clones were identified by probe
hybridization, and smaller fragments were subcloned into M13 for DNA
sequencing (see Fig. 1B).
Membranes were washed as described above. BamHI-, HindIII-, or PstI-digested P. gingivalis genomic DNAs were hybridized with 32P-labeled Lys-1-33.
DNA SequencingDouble-stranded DNA cloned into pBluescript
SK() and single-stranded DNA cloned into M13mp18 and M13mp19 vectors
were sequenced by the dideoxy terminator method (17) using sequencing
kits purchased from U. S. Biochemical Corp. (Sequenase Version 2.0). DNA was sequenced using the M13 universal primer, reverse sequencing primer, and internal primers (see Fig. 1B).
KGP from P. gingivalis HG66 was purified and titrated, and the active site was labeled as described previously (13). Biotinylated KGP (20 nmol) was denatured in 6 M guanidine HCl, reduced with 2-mercaptoethanol, and S-pyridylethylated by the method of Howke and Yuan (18). The sample was desalted by dialysis against 25 mM ammonium bicarbonate, pH 7.8.
Polypeptide Chain Fragmentation and AnalysisThe derivatized protein (25 nmol) was digested in 25 mM ammonium bicarbonate buffer, pH 7.8, with trypsin at 37 °C for 16 h (1:50 enzyme/substrate weight ratio). Each digest was analyzed as described earlier (13). Biotinylated peptides were analyzed for amino-terminal sequence, amino acid composition, and molecular mass.
Construction of Recombinant BaculovirusThe KGP proteinase
domain was cloned into the pBlueBac III vector (Invitrogen). A PCR
primer corresponding to DNA encoding the 5-end of the proteinase
domain was used in conjunction with a second primer complementary to
DNA encoding the 3
-end of the proteinase domain. The
5
-oligonucleotide contained six extra nucleotides, a BamHI
site, the Kozak consensus sequence GCC (19), an initiation codon, and
the first six amino acids of the KGP proteinase domain. The
3
-oligonucleotide contained six extra nucleotides, a PstI
site, a termination codon, and the last six amino acids of the
proteinase domain. PCR fragments were cloned into pBlueBac III, and
recombinant plasmids were isolated. Two of them were sequenced as
described above and used to generate recombinant viruses by in
vivo homologous recombination. Recombinant viruses were used to
infect Spodoptera frugiperda clone 9 (Sf9) cells. After
48 h, recombinant viruses were collected, identified by PCR, and
further purified. Standard protocols for plasmid cloning were used
(16). Standard procedures for selection, screening, and propagation of
recombinant baculovirus were performed as described by the supplier
(Invitrogen).
Sf9 cells were infected at a multiplicity of infection of 5 with either wild-type baculovirus or baculovirus engineered to encode the KGP proteinase domain. At 24, 48, and 72 h, cells and media were collected and centrifuged (5000 rpm, 3 min). Supernatants (2 ml) and pellets (resuspended in KGP assay buffer) were assayed for lysine-specific amidolytic activity as follows. Aliquots (200 µl) of the supernatant or resuspended pellet were added to 800 µl of assay buffer (0.2 M Tris, 0.1 M NaCl, 10 mM L-cysteine, and 5 mM CaCl2, pH 7.6) and mixed for 1 min. Substrate (S-2251, Chromogenix) was added to a final concentration of 100 µM, and amidolytic activity was measured at 405 nm. The amount of active recombinant KGP proteinase domain expressed was quantitated by comparison with the activity of a fixed amount of KGP purified from P. gingivalis.
Pike et al.
(14) have previously determined the primary structure of the
NH2 terminus of Lys-specific gingipain by direct amino acid
sequencing. This information was used to prepare a mixture of synthetic
oligonucleotides complementary to amino acids 1-6 and 16-21 of the
mature protein. These primers were used to amplify, by PCR,
kgp gene sequence from P. gingivalis DNA. A
single 76-base pair product (P76) was identified, cloned, and sequenced to determine codon usage for the amino-terminal residues of KGP. On the
basis of this sequence, a 32P-labeled oligonucleotide probe
(Lys-1-33) corresponding to the coding strand of this partial
kgp DNA was synthesized and used to screen the DASH
P. gingivalis DNA library.
DNA from positive clones was extracted, purified, and subjected to
restriction enzyme analysis. All clones gave an ~3.8-kb BamHI fragment and an ~3.4-kb PstI fragment.
Similar results were obtained by Southern analysis of P. gingivalis total genomic DNA (data not shown). We therefore
concentrated on one clone designated A2. The 3.8-kb BamHI
and 3.4-kb PstI fragments from clone A2 were cloned into
pBluescript SK(). The 3.4-kb PstI fragment and a 0.9-kb
PstI/BamHI-generated 3
-end fragment of the
3.8-kb BamHI fragment were subcloned into M13mp18 and
M13mp19 vectors and sequenced. To clone the region of the
kgp gene containing the termination codon, overlapping
clones were isolated from size-selected
PstI/Asp718 and HindIII plasmid
libraries using oligonucleotide probes (see "Experimental
Procedures"). Using this procedure, several ~0.7- and 5.7-kb
fragment containing clones were obtained, respectively. In total,
~8.1 kb of genomic DNA, from PstI to HindIII
sites (Fig. 1B), was isolated and
characterized. The composite 7.304-kb PstI/NcoI fragment of this genomic DNA (Fig. 1B) was fully sequenced
in both directions and is described here.
Within the composite
kgp gene sequence was found an open reading frame encoding a
1723-amino acid sequence, with the 5-most ATG initiation codon at
nucleotides 652-654 (Fig. 1). Between this ATG codon and the published
amino-terminal sequence of KGP are an additional four in-frame
methionine codons. The exact ATG codon used for initiation of
translation is currently unknown, but the presence of a consensus TATA
box (ATAAATT) at nucleotides 635-641 and a 15-amino acid signal
peptide sequence immediately following the 5
-most ATG codon suggests
it to be the strongest candidate (amino acids
227 to
213; Fig.
1A).
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. 1B), leading to a
calculated molecular mass of 186.8 kDa for the encoded polyprotein. As
described for RGP-1 (13), within these sequences can be found peptides identified by Pike et al. (14) as the components of 95-kDa
gingipain R (high molecular mass gingipain (HGP)) that likely confer
adhesion activity on the high molecular mass KGP complex. The
polyprotein sequence deduced from the gene sequence now allows exact
delineation of the primary sequence of the mature KGP proteinase. The
amino terminus is derived from proteolytic processing at an arginine residue (Fig. 1B). The carboxyl terminus of the proteinase
domain is derived by processing at Arg-509, which also releases the
amino terminus of a novel subunit that is a hybrid of the 27-kDa
(HGP27) and 44-kDa (HGP44) polypeptide chains described recently in the rgp-1 gene structure (Fig. 1B) (13). Previously,
we identified three large repeats of homologous sequence located within
three of the HGP cleavage products (13). It is interesting to note that
the hybrid HGP27/HGP44 appears to result from fusion within this
repeated region. The molecular mass of this hybrid protein is 44.7 kDa.
The first 147 amino acids are identical to HGP27. The remaining 271 amino acids show 99% identity to HGP44, including one amino acid
change and two amino acid deletions (98% identity at the nucleotide
level). Okamoto et al. (20) have cloned and sequenced RGP-1
from P. gingivalis strain 381. The encoded protein also
contains a hybrid of the HGP44/HGP27 molecules that results from fusion
within this homologous sequence. It is, however, in the reverse
combination to that observed for KGP. The remainder of the processing
events are identical to those described for RGP-1. The alignment of
each structure is shown in Fig. 1B. Processing at Arg-927
gives rise to HGP15; processing at Lys-1062 gives rise to HGP17 (with
two amino acid changes compared with HGP17/RGP-1 (13)); and processing
at Arg-1220 gives rise to HGP27. The homology to the rgp-1
gene is also evident in the first 120 nucleotides of the 3-noncoding
sequences.
The proteinase domain of KGP comprises 509 amino acids with a
calculated molecular mass of 55.9 kDa. This is lower than the 70-kDa
molecular mass of Lys-gingivain first described by Scott et
al. (21), but corresponds more closely to the 60-kDa molecular mass determined by Pike et al. (14). A comparison of the
deduced amino acid sequences of the proteinase domains of RGP-1 and KGP indicates 22% identity using the Macaw alignment program (22). This
includes a long stretch of homologous sequence in the carboxyl terminus
that incorporates the motif LTATT present in all HGP repeat sequences
(Fig. 2, A and B) (13). This
sequence includes Asn-463, which aligns with Asn-442 in RGP and is the
putative asparagine in the catalytic triad. Similarly, His-216 in KGP
and His-211 in RGP-1 are other putative residues in the catalytic triads (Fig. 2, B and C). Previously, Cys-185 in
RGP-1 was thought to be the active-site residue using active-site
labeling and subsequent reduction and S-carboxymethylation
(13). For KGP, an alternative methodology (described under
"Experimental Procedures") was used to determine that Cys-249 is
the active-site residue of this proteinase. This residue aligns with
Cys-244 in RGP-1. This prompted us to re-examine the active site of
RGP-1. This methodology, which utilizes a more sensitive reduction and
pyridylethylation step, identified Cys-244 as the actual active-site
cysteine of RGP-1, and not Cys-185 as reported previously (13).
A, comparison of the deduced amino acid sequences of the proteinase domains of RGP-1 and KGP, and of HGP44, and HGP17 in the putative fibrinogen-binding sites using the Macaw program (22). Residues identical in at least two sequences are shown in white. Residues conserved in all sequences are boxed. Residues identical in all three sequences of proteins with experimentally determined fibrinogen binding activity are underlined. B, alignment of the deduced amino acid sequences of the proteinase domains of KGP and RGP-1 using the Macaw program (22). Identical residues are shaded. C, conservation of sequences around the potential catalytic cysteine, histidine, and asparagine residues of some cysteine proteinase families with those in the new gingipain cysteine proteinase family. Residues identical to those in either RGP-1 or KGP are underlined. The catalytic triad Cys, His, and Asn are in boldface. The cysteine proteinase alignment was based on that of Rawlings and Barrett (28).
KGP Has No Homology to Known Cysteine Proteinase Families
To date, some 20 families of cysteine proteinases are recognized. In Fig. 2C is shown the alignment of active sites of several members of these families and the putative active-site sequences in RGP-1 and KGP. As observed previously, RGP-1 exhibits no similarity to any known cysteine proteinase. This is also true for KGP. Thus, both P. gingivalis proteinases appear to represent an emerging distinct branch of this family of proteolytic enzymes in which the catalytic apparatus is likely different from that of other known cysteine proteinases. We also have evidence of the existence of a third member of this family. Restriction endonuclease-digested P. gingivalis DNA hybridized with an oligonucleotide corresponding to the amino terminus of mature RGP-1 (13) and showed two BamHI fragments of ~9.4 and ~3.5 kb and two PstI fragments of ~10 and ~3 kb (data not shown). Isolation of positive recombinant clones from the library revealed, upon analysis, one clone with a 3.5-kb BamHI fragment and a 3-kb PstI fragment. This corresponded to the rgp-1 gene described previously (13). We also identified independent clones containing a 9.4-kb BamHI fragment and an ~10-kb PstI fragment. Preliminary sequence analysis confirms that these clones encode a protein that is different from RGP-1 and that the translated sequence corresponds to peptide sequences of high molecular mass protein sequenced earlier in our laboratories and designated RGP-2 (data not shown).
Recombinant KGP Proteinase Domain Is Functionally ActiveBecause of the lack of a specific antibody against KGP, we
tested the expression of the KGP proteinase domain in the baculovirus system by measuring its activity. Lysine-specific amidolytic activity was detectable after 48 h and was maximal at 72 h (Fig.
3) only in the supernatant of KGP recombinant
baculovirus-infected cells. No amidolytic activity was observed in
resuspended pellets or in the supernatant of wild-type infected cells.
Calculations based on the activity of full-length KGP purified from
P. gingivalis cultures indicate that at least 2 mg/liter KGP
proteinase domain is being expressed. These results show the expression
of the KGP proteinase domain in the baculovirus system and support the
notion that the membrane-bound forms of gingipains likely associate
with the membrane via the adhesin domains and imply that this domain does not require the remainder of the precursor protein for correct folding into a catalytically active conformation.
Implications of KGP Structure for P. gingivalis Pathogenicity
Molecular cloning of the kgp gene confirms previous findings that, as shown for RGP-1, KGP is also closely associated with adhesion activity. Sequencing of the corresponding kgp gene of the virulent W50 strain of P. gingivalis revealed 94% identity in the proteinase domain sequence (data not shown). Thus, as described for RGP-1, any involvement of KGP in virulence is likely due to its differential regulation and enhanced expression in virulent strains. Microorganism adhesion to host tissues is the initial critical event in the pathogenesis of most infections and an essential step for colonization of surfaces exposed to continuous fluid flow (23, 24). The specific binding between the microbial cell-surface components (adhesins) and host cell, or other microorganism, surfaces is mediated by either protein-protein (non-lectin adhesin) or protein-carbohydrate (lectin adhesin) interactions. Besides mediating bacterial cell attachment to host tissues, the non-lectin adhesins of P. gingivalis have been implicated in hemagglutination with both activities associated with bacterial proteinases. A clearer understanding of the association between hemagglutinins/adhesins and proteinases occurred recently when we elucidated the structure of the gene encoding RGP-1 (13). We have now shown that both RGP-1 and KGP are synthesized as proteinase-adhesin polyproteins. It remains to be determined which polypeptide chain of gingipain complexes possesses affinity for microbial adhesion substrates such as fibrinogen. Fibrinogen is found in high concentrations in blood plasma, where it plays roles in blood coagulation and wound-healing processes. Its deposition on foreign bodies and at sites of trauma allow it to serve as a substrate for microbial adhesion. Sequence alignments of the proteinase domains of RGP-1 and KGP and of HGP17 and HGP44 reveal a region of high homology that might function as an adhesin domain (Fig. 2A). The ability of RGP-1, KGP, and other membrane-associated P. gingivalis gingipains to adhere to matrix molecules indicates similarity to the fibrinogen-binding microbial surface components recognizing adhesive matrix molecules (25, 26). Recently, two P. gingivalis cell-surface cysteine proteases, named porphypains 1 and 2, were purified and shown to bind and degrade fibrinogen (27). These likely correspond to two forms of high molecular mass complexes of gingipains.
The availability of RGP-1 and KGP DNA sequences will allow the further study of erythrocyte hemolysis mediated by the hemagglutinin/adhesin activities of this family of proteolytic polyproteins. In addition, recombinant polypeptides derived from these sequences may also prove useful in the development of potential immunoprophylactic and therapeutic agents against this human pathogen.
The technical contributions of D. Wong, T. Rigley, and I. Beletskaya are gratefully acknowledged.
During the review of this manuscript, two papers were published describing the cloning and sequencing of kgp-like-gene from P. gingivalis strains 381 and W12 (Barkocy-Gallagher, G. A., Han, N., Patti, J. M., Whitlock, J., Progulske-Fox, A., and Lantz, M. S. (1996) J. Bacteriol. 178, 2734-2741; Okamoto, K., Kadowaki, T., Nakayama, K., and Yamamoto, K. (1996) J. Biochem. (Tokyo) 120, 398-406).