Perspectives in Drug Discovery and Design, 2 (1994) 445-458 445 ESCOM

PD3 055

Host and Porphyromonas gingivalis proteinases in periodontitis: A biochemical model of infection and

tissue destruction

Jan P o t e m p a a, Robe r t Pike b and J im Travis c'*

aDepartment of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, 31-120 Krak6w, Poland

bBiochemistry Department, University of Natal, Private Bag X01, Scottsville 3209, South Africa CDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, US.A.

Received 4 April 1995 Accepted 10 May 1995

Key words: Proteolytic enzymes; Gingipain; Periodontal diseases; Bacterial infection; Inflammation

SUMMARY

Periodontitis is an excellent model of local tissue destruction due to the uncontrolled action of host and microbial proteinases. Although host enzymes are responsible for direct degradation of connective tissue, proteinases from the periodontopathogenic bacterium Porphyromonas gingivalis are able to activate the kallikrein/kinin cascade, disrupt coagulation, and activate complement-mediated chemotaxis. Since the hallmarks of adult onset of periodontitis include infection by this pathogen, bleeding, increased crevicular flow, and neutrophil accumulation, investigations of this disease at a biochemical level indicate a major role for bacterial proteinases in infections by P. gingivalis.

I N T R O D U C T I O N

During the development of infection, a number of biochemical reactions are known to occur which both initiate and prolong inflammation. These processes require enzymes which clearly arise from both the infecting organism and the host. Many of the hallmarks of such reactions involve proteolytic events leading to pain, edema, neutrophil infiltration, tissue destruction and bleeding. However, the role(s) of both foreign- and host-derived proteinases has, until recently, been somewhat vague. In this review we describe for the first time how members of this class of enzymes function in the development of a major inflammatory disease, periodontitis.

WHAT ARE PERIODONTAL DISEASES?

Dental plaque is an extremely complex bacterial ecosystem which has been shown to contain more than 400 distinct bacterial taxa in various samples [1]. Regardless of the degree of oral

*To whom correspondence should be addressed.

0928-2866/$ 10.00 © 1995 ESCOM Science Publishers B.V.

446

TABLE 1 HOST PROTEINS TARGETED BY PROTEOLYTIC ENZYMES ELABORATED BY P. gingivalis

Affected proteins Molecular consequence and/or relevance for plaque Relevance to clinical hall- bacteria marks of periodontitis

Inactivation of proteinase inhibitors cq-proteinase inhibitor, %- macroglobulin, Cl-inhibitor, c~2-antiplasmin, antithrombin III

117,181 Disturbance of the local balance between phagocyte proteinases and their inhibitors (~l-PI, c~2M) in- creases local proteolysis and provides free amino acids and peptides necessary for growth of asaccha- rolytic, anaerobic bacteria Dysregulation of the proteolytic cascades of blood coagulation (ATIII, Cl-inh) and fibrinolysis (c~2-AP) provides plaque bacteria with heme, an important growth factor for P gingivalis

Degradation of immunoglobulins !19-241 IgA, IgG, IgM Disabling antibody-dependent mechanisms of bac-

tericidal activity of phagocytes. Source of essential amino acids and small peptides for bacteria growth

Degradation of iron transporting/ sequestering proteins [25]

Haemopexin, transferrin, haptoglobulin

Coagulation cascade [17,26-28] Factor X, prothrombinase complex, HMW-kininogen, fibrinogen

Release of iron, an indispensable bacterial grow factor. Source of essential amino acids and small peptides for bacterial growth

Local disorder of blood coagulation, leading to flow of erythrocytes into the periodontal pocket; source of heine

Complement cascade [20,21,24,29-33] C3, C4, C5, factor B, factor D, Generation of C5a and degradation of other com- Cl-inh plement factors. Attenuation of complement-depen-

dent bactericidal activity

Kallikrein/kinin cascade I17,27,34-361 Prekallikrein, HMW-kininogen Release of bradykinin which causes long-lived vaso-

Degradation of extracellular matrix Collagen type I, type IV, fibronectin

Degradation of bactericidal pro- teins and peptides [43,44]

Lysozyme, cathepsin G, defen- sins, bacterial permeability in- creasing factor

Miscellaneous Activation of matrix metallo- proteinases [45]

Cell surface receptors [46,47]

dilatation and enhanced vascular permeability, as well as providing continuous supply of nutrients for bacteria in subgingival plaque

proteins [37-42] Direct destruction of basement membrane and periodontal ligament; damage to host cells; source of essential amino acids and small peptides for bacteria growth; exposure of cryptotopes for bac- terial adhesion to host tissue

Attenuation of the oxygen-independent bactericidal potency of neutrophils in periodontal pockets and surrounding tissues

Destruction of periodontal tissues by matrix metal- loproteinases. Source of essential amino acids and peptides for bacteria growth Platelet activation and interaction with neutrophils; Attenuation of neutrophil antimicrobial activity

Destruction of periodontal ligament - one of the fac- tors leading to loss of attachment and formation of periodontal pocket. Bleeding on probing, a phenomenon associated with periodontitis

Infestation of periodontal pocket with large numbers of periodontopathogenic bacteria

As directly above

Bleeding on probing, a phenomenon associated with periodontitis

Infiltration of neutrophils which are unable to kill bacteria due to a lack of opsonins

Increased volume of gingi- val crevicular fluid

Attachment loss and peri- odontal pocket formation

Periodontopathogen pro- liferation in subgingival plaque

Attachment loss and peri- odontal pocket formation

Maintenance of local chronic inflammation

447

hygiene regime, dental plaque is always present on teeth and can only be removed by pro- fessional cleaning. Immediately after the tooth surface becomes coated with a saliva-derived acquired pellicle, it is quickly colonized by streptococci, actinomyces and other gram-positive bacteria [2]. At this stage the plaque, which is generally easily controlled by daily oral hygiene, is beneficial and acts as a protective agent against colonization by more obnoxious bacterial species. However, if left undisturbed for more than 48 h, profound changes in the bacterial composition of plaque will occur which are manifested by the replacement of gram-positive aerobic bacteria with gram-negative, facultative anaerobes [3]. Thriving of these species in plaque will lead directly to gingivitis, an inflammation of the gingival tissue surrounding the tooth [4]. In this altered environment yet another series of bacterial species begin to flourish in plaque [5], and gingivitis can progress to periodontitis. Periodontitis, an inflammation of periodontal tissues, including those directly supporting the root of the tooth in alveolar bone sockets, is characterized by massive accumulation of neutrophils, bleeding on probing, increased flow of gingival crevicular fluid, bone resorption, loss of tooth attachment, and formation of periodontal pockets infested with specific bacteria [6]. Several distinct clinical categories of periodontitis can be distinguished, but the most common form is adult periodontitis [7], with Porphyromonas gingivalis being generally regarded as the major pathogen involved in the induc- tion and/or progression of this disease [8-11].

GETTING ACQUAINTED WITH THE MAJOR CULPRIT OF ADULT PERIODONTITIS

P. gingivalis (formerly Bacteroides gingivalis), a gram-negative, anaerobic, nonmotile, nonspor- ing short rod is very well equipped with a broad array of functional and structural features which enable this bacterium to colonize within its ecological niche of either the gingival salcus or the periodontal pocket. The organism prospers in this hostile environment by successfully evading host antimicrobial defenses, using such putative virulence factors as fimbriae and lectin-type adhesins, a polysaccharide capsule and lipopolysaccharide, hemagglutinating and hemolysing activity, release of toxic products of metabolism, outer membrane vesicles and numerous enzymes, all of which have been extensively discussed in several review articles [12-16].

PROTEOLYTIC ENZYMES OF P. gingivalis AS POTENT VIRULENT FACTORS IN PERI- ODONTITIS

Although several enzymes elaborated by P. gingivalis (e.g., hyaluronidase, keratinase and superoxide dismutase) have been implicated in invasion, tissue destruction and evasion of the oxygen-dependent bactericidal activity of neutrophils, the primary focus of research has been on the proteolytic enzymes which are produced in large quantities by this bacterium. In Table 1, a diverse variety of potentially harmful activities of P. gingivalis proteinases are summarized, and it is immediately apparent that these enzymes can significantly contribute to virulence and, at least in part, directly or indirectly, could be responsible for most of the clinical hallmarks of periodontitis. Therefore, P. gingivalis proteinases are perfect targets for the development of specific inhibitors which could be tested as alternative treatments to reduce adult periodontitis. For this purpose, however, rigorous purification and precise biochemical characterization of individual enzymes are necessary.

448

TABLE 2 SUMMARY OF PURIFICATION ATTEMPTS OF Arg-X- AND/OR Lys-X-SPECIFIC PROTEINASES FROM P gingivalis

Refi Source Purification Molecular Inhibitors b Synthetic Protein sub- Name ~'r mass substrate ~ strated (kDa) ~

57 Cell Ion-exchange, N,D, Leupeptin, BAPNA, but not Albumin Trypsin- envelope aminophenyl- EDTA, PCMB, Tos-Lys-Me like fraction mercury-sepharose antipain

58 Culture AmSO 4 precipita- 300* PCMB, TLCK, BAPNA, but not Azocasein, azo- Trypsin- media tion, ion-exchange, EDTA, NEM Bz-Lys-pNA coll, but not like

IEF collagen 59 Culture A m S O 4 precipita- 49** NEM, TLCK, BAPNA and Bz- N.D. Trypsin-

media tion, gel filtration, leupeptin, but Lys-pNA like ion-exchange not EDTA

43 Culture Acetone precipita- N.D. PCMB, NEM, BAPNA Lysozyme, Trypsin- media tion, gel filtration, leu-peptin, but casein like ~

Arg-sepharose not EDTA 61 Cell Benzamidine- 35* PMSF, EDTA, X-Y-Arg-pNA, Collagen type Trypsin-

extract sepharose, but PCMB and weak activity on IV, gelatin, but like gel filtration NEM are weak substrates with not collagen

inhibitors P~ Lys type I leupeptin, BAPNA but not gelatin, albu- TLCK Bz-Lys-pNA rain, but not

IgA

61 Cell AmSO4 precipita- 50* extract tion, ion-exchange,

hydroxyapatite, gel filtration

39 Culture AmSO 4 precipi- 58* media tation, gel filtra-

tion 62 Culture AmSO4 precipita- 43*

media tion, ion-exchange, gel filtration, IEF

63 Culture Acetone precipita- 72---80* media tion, gel filtration,

Arg-sepharose, ion-exchange

44*

64 Cell Gel filtration, 85* envelope ion-exchange fraction

65 Cell Gel filtration, 44* envelope ion-exchange, fraction Arg-sepharose

66 Culture Covalent chroma- media tography

67 Cell Gel filtration, extract ion-exchange

IEF

20 Vesicles Preparative SDS- PAGE

68 Culture AmSO 4 precipita- media tion, gel filtration,

ion-exchange

95*

43, 87, and 170"* or 43* g 80*

50*

Trypsin- lik&

TLCK, PCMB, BAPNA Azoalbumin, Trypsin- EDTA azo-casein, like e

azocoll NEM, TLCK, X-Y-Arg-pNA Casein, %M Arginine- leu-peptin, but but not Bz-Lys- specific not EDTA pNA protease ~

NEM, TLCK, X-Y-Arg-pNA Casein, (~aM Trypsin- leu-peptin, but and Bz-Lys-pNA like * not EDTA Histatin BAPNA and Z- N,D. Trypsin-

Lys-SBzl like

Leupeptin, X-Y-Arg-pNA, N.D. Trypsin- TLCK, NEM X-Y-Arg-MCA like ~

(no activity on substrates with P1 Lys)

2,2'-Dipyridyl BAPNA Azocasein Gingivain disulfide PCMB, TLCK, X-Y-Arg-pNA IgA, IgG, Fb, Trypsin- EDTA, NEM but not Bz-Lys- albumin like e

pNA

PCMB, IAA BAPNA IgG, IgM, C3 Trypsin- like

NEM, IAA, leu- X-Y-Arg-pNA Many different Gingi- peptin, TLCK, (no activity on proteins hut not pain "f EDTA substrates with collagen type I

P~ Lys)

N.D. BAPNA collagen type I, Trypsin- Fn like J

449

TABLE 2 (continued)

Ref. Source Purification Molecular lnhibitors b Synthetic Protein sub- Name °'f mass substrate c strate d (kDa) a

69 Culture AmSO4 precipita- 79 90 Antipain, TLCK X-Y-Arg-pNA Albumin Protease media tion, IEF and 47* I1, pro-

tease I 70 Culture AmSO 4 precipita- 48* TLCK but not X-Y-Lys-pNA IgG, IgA, Lysine-

media tion, ion-exchange, leu-peptin and (no activity on albumin specific gel filtration, EDTA substrates with protease e Lys-sepharose P~ Arg)

27 Cell Gel filtration, 68-70* IAA, FFRck X-Y-Lys-pNA HK, Fb Lys- extract hydroxyapatite (weak activity gingivain

chromatography on substrates with P~ Arg)

71 Culture Acetone precipita- 105" h IAA, TLCK, X-Y-Lys-pNA Fb and other Lys-gin- media tion, gel filtration, but not EDTA (no activity on proteins, but gipain ~'f

Arg-sepharose, or leupeptin substrates with not collagen ion-exchange Pl Arg) type I

95* ' IAA, TLCK, X-Y-Arg-pNA Arg-gin- leu-peptin, (no activity on PK, C5 and C3 gipain es EDTA substrates with and other pro-

P~ Lys) teins but not collagen type I

48 Culture Ion-exchange, 55* leupeptin, anti- X-Y-Arg-pNA Collagen type I, Trypsin- media gel filtration, pain, TPCK, (weak activity III, IV and V, like e

chromatofocusing NEM on substrates C3, Fb, Fn, with PI Lys) ~2M, oqPI, but

not IgA 72 Intact cells CHAPS extraction, 150" and TLCK, EDTA X-Y-Arg-pNA Azocoll, casein, Porphy-

ion-exchange, 120" (leu-peptin only and X-Y-Lys- Fn, but not pain e preparative SDS- inhibited Arg- pNA collagen PAGE specific activity)

49 Culture AmSO 4 precipita- 44* Leupeptin, X-Y-Arg-MCA, Collagen type I Argingi- media tion, ion-exchange, NEM, EDTA, GIy-Pro-MCA, and IV, IgG pain e

IEF, gel filtration TLCK Leu-MCA

Molecular mass was determined by SDS-PAGE (*) or gel electrophoresis (**). b Only some representative inhibitors used in a referenced study are reported in the table. TLCK= tosyl-L-Lys-chloro-

methyl keton; 1AA=iodoacetic acid or iodoacetamide; NEM=N-ethylamaleimide; PMSF=phenylmethylsulfonyl fluoride; PCMB =p-chloromethyl benzoate; FPRck = o-H-Phe-Pro-Arg-chloromethyl ketone.

c A single entry in this column means that no substrate with a P~ lysine residue had been used in the study, pNA =p-nitro- aniline; MCA = 4-methylcoumaryl-7-amide; SBzl = thiobenzyl ester; Bz- = benzoyl; X,Y = any amino acid or N-terminal blocking group; Me = methyl ester.

a Fn =fibronectin; Fb=fibrinogen; PK=prekallikrein; HK=HMW-kininogen; c~2M=a2macroglobulin. e Presented proof (SDS-PAGE) of homogeneity of purified proteinase. f Reported N-terminal sequence of purified proteinase. g Monomer (43 kDa) which can associate to form dimer (87 kDa) or trimer (170 kDa). h The proteinase is a complex of the catalytic domain (60 kDa) with hemagglutinins. ' The proteinase is a complex of the catalytic domain (50 kDa) with hemagglutinins. J In contrast to all other enzymes described in the table, activity of this proteinase was not stimulated by reducing agents.

C L A S S I F I C A T I O N OF PROTEINASES P R O D U C E D BY P. gingivalis

F o r a c o n s i d e r a b l e l e n g t h o f t i m e the p r o t e o l y t i c ac t iv i ty o f P. gingivalis has b e e n classif ied

in to two m a j o r g roups : e n z y m e s w h i c h specif ical ly d e g r a d e d co l lagen , a n d gene ra l ' t r yps in - l ike '

p ro t e ina se s w h i c h a p p e a r e d to be r e spons ib l e fo r o t h e r p r o t e o l y t i c activity. D e s p i t e s o m e c la ims

450

[40,48-51], no true collagenase has yet been purified to homogeneity and/or thoroughly character- ized, although a systematic analysis of the effect of six classes of proteinase inhibitors on P. gingivalis collagenolytic activity has strongly suggested that such an enzyme is produced by this organism, and it appears to be a cysteine proteinase requiring metal ions for activity [52]. The importance of this activity in periodontitis-associated tissue damage is not clear, because it was shown that degradation of collagen in vivo, at active periodontitis sites, is the result of the action of host-derived matrix metalloproteinases [53,54]. On the other hand, using electrophoretic zymography analysis, at least eight proteolytic enzymes with molecular masses in the range 29-110 kDa were identified in different fractions of P. gingivalis. Apart from two, which appeared to be serine proteinases with glycyl-prolyl peptidase activity, all other proteinases were shown to be activated by reducing agents and to hydrolyze the synthetic substrate Bz-Arg-pNA [55]. In this regard, the latter activity is the most prominent produced by P. gingivalis and could be detected in gingival crevicular fluid [56], indicating that such proteinases are produced in vivo and, more importantly, are not controlled by host proteinase inhibitors. Indeed, it would appear that host-derived collagenolytic activity is significantly more important than that secreted by P. gingivalis. Furthermore, assays of 'purified' collagenases [48,49] from this organism have, because of the methodologies utilized, actually been assays of gelatinase, so that no true P. gingivalis collagenase has yet been isolated and characterized.

Culture fluid I Acetone

precipitate

i SephadexG-150 I

90-110 kDa protein peak

['rg-,ep"arose I

Lysine

~ Arginine ( ~ gradient

105-kDa HRGP* KGP (95-kDa RGP-1)

50 kDa prote i peak

I DE'S2 cellul°se J

FIow through N.aCI J ~ Peak I

gradient ~ (BAPNA positive) Peak II l (BAPNA positive) [Mono S, FPLC ]

I Arg'sepharOse J

RG P-1 * RGP-2** (50 kDa) (50 kDa)

*Product of the same HRGP gene **Product of the second gene

Fig. 1. Scheme of gingipain purification from culture fluid of P. gingivalis.

451

Too many proteinases and too many names Since 1984, due to convenient assay systems with chromogenic or fluorogenic substrates, the

trypsin-like proteinases have become targets for purification, and at least 24 apparently different enzymes have been described. The purified proteins, despite having the common feature of being activated by reducing agents and cleaving synthetic substrates with arginine and/or lysine at the P~ position exclusively, differed widely in other characteristics, including molecular mass (in the range from 35 kDa to 300 kDa), association with hemagglutinin activity, susceptibility to syn- thetic inhibitors, and the ability to degrade a variety of proteins (Table 2). Unfortunately, because of the absence of structural data and/or the lack of convincing proof of homogeneity of the purified enzymes, it has been almost impossible to perform any reasonable comparative analysis of the isolated enzymes. An additional dimension to this chaos was added by the recent fashion of naming purified P gingivalis proteinases too quickly, and presently four different acronyms (gingivain, gingipain, porphypain and argingipain) are suggested in the literature. All these ambiguous data have created a confusing picture of P gingivalis as an organism producing literally a myriad of related, though nonidentical, proteinases.

Contrary to this seemingly extreme diversity, we were able to purify only three cysteine pro- teinases from culture media of P. gingivalis H66, two with Arg-X specificity and one with Lys-X specificity [68,71]. Since the first purified enzyme shared some properties with clostripain, includ- ing a similar molecular mass, requirements of calcium for stability and a narrow specificity limited to Arg-X peptide bonds, we proposed the acronym gingipain (P. gingivalis + clostripain = gingi-pain) [68]. Later, we have used this name with the prefix Arg- (Arg-gingipain, RGP) or Lys- (Lys-gingipain, KGP) to distinguish between Arg-X- and Lys-X-specific proteinases.

PURIFICATION AND CHARACTERIZATION OF GINGIPAINS

The simple and efficient purification procedure utilized here is outlined in Fig. 1 and has allowed simultaneous separation of a 105-kDa KGP, a 95-kDa RGP (high-molecular-mass RGR HRGP) and two very closely related, though different in some characteristics, 50-kDa RGPs (RGP-1 and RGP-2). Whereas RGP-1 was obtained in microgram quantities, the three other proteinases were purified with a yield of 2-6 mg of each homogeneous protein from 1000 ml of culture medium.

The availability of relatively large quantities of homogeneous proteins allowed us to perform detailed biochemical and enzymological characterization of purified proteinases. All four gingi- pain activities, either on synthetic substrates or on proteins, were totally dependent on the presence of reducing agents, and each could be totally and irreversibly inactivated by thiol group blocking reagents, including iodoacetamide, iodoacetic acid and N-ethylmaleimide, at rather high concentrations (10 mM) and after relatively long times of incubation (20-30 min). On the other hand, the active-site titrant E-64, which is exclusively specific for cysteine proteinases, inhibited only RGP-related enzymes, but not in the normal equimolar manner typical for enzymes from the papain family. In other words, in order to inhibit gingipain, E-64 must be applied in a several thousand-fold molar excess over the enzyme, while the interaction of papain is on a 1:1 molar basis. In comparison to E-64, all four enzymes are inhibited efficiently by the peptidyl chloro- methyl ketones TLCK and TPCK (potent inhibitors of serine and cysteine proteinases), although with different rate constants (Table 3). In contrast, bacterial-derived peptidyl aldehyde inhibitors,

452

TABLE 3 COMPARISON OF SOME CHARACTERISTIC FEATURES OF PURIFIED GINGIPAINS

Gingipain Polypeptide N-terminal Hemag- Gly-Gly (200 Inhibition composition sequence glutinin gM) stimula- (%) by leu-

activity a tion of ami- peptin or dolytic activ- antipain ity b (100 gM)

Rate constants [Kobsd/[I ] (M-Is-I)] for GP inactivation by chloromethyl ketones

TPCK TLCK

RGP-1 Single chain YTPVEEKQNGR None 18.8-fold 100 (50 kDa)

RGP-2 Single chain YTPVEEKENGR None 6.7-fold 100 (50 kDa)

95-kDa RGP 50 kDa YTPVEEKQNGR (HRGP) 44 kDa SGQAEIVLEAH

27 kDa ANEAKVVLAA 17 kDa PQSVWlERTVD 15 kDa ADFTETFESSTH

n.d. n.d.

257 27000

1.8 gg/ml 18.2-fold 100 1250 116400

105-kDa KGP 60 kDa DVYTDHGDLYN (KGP) 44, 30 and 27 ANEAKVVLAA 6 gg/ml Slight N.I.

kDa inhibition 17 kDa PQSVWIERTVD 15 kDa ADFTETFESSTH

252 79910

a Hemagglutinin titer, the lowest concentration of the protein at which human red cells agglutinated. b Assay performed at 10 mM concentration of cysteine as activating factor using BAPNA or Z-Lys-pNa as substrates

for RGPs and KGP, respectively. N.I. = no inhibition; n.d. = not determined.

leupeptin and antipain, as well as EDTA and ZnC12, exclusively inactivate Arg-gingipains, with EDTA exerting its inhibitory effect in a time-dependent, partially reversible manner.

The most profound difference between the two types of gingipains is their restricted specificity to cleavage after the carboxy-terminal side of arginine (RGPs) or lysine residues (KGPs), conveni- ently measured with synthetic substrates but also fully confirmed on a broad range of peptides and proteins. However, to avoid false results these experiments must be performed on rigorously purified enzyme, since both types of gingipains are very potent proteolytic enzymes. An interest-

ing feature of KGP is its ability to cleave Lys-Pro peptide bonds, a property rather unusual among mammal ian serine and cysteine proteinases.

In comparison to cross-group differences, the biochemical and enzymological characteristics of the 50-kDa RGPs are highly similar. The major differences are the fold of amidolytic activity stimulation by glycylglycine (Table 3), the kinetic constants of substrate turnover and inhibition by peptidyl chloromethyl ketones (J. Potempa et al., manuscript in preparation) and some minor variations at the pr imary structure level. In addition to a single amino acid substitution at the protein N-terminus (Table 3), a few other differences in the structure have been found during sequencing of tryptic and autodigestion peptides f rom RGP-2. Therefore, we believe that RGP-1 and RGP-2 are the products of two different, but closely related genes, which is in keeping with Southern blot analysis indicating the presence of two RGP genes in various strains of P. gingivalis (Pavloff, N. et al., unpublished data).

453

GINGIPAIN STRUCTURE AT THE GENE AND PROTEIN LEVEL

While the 50-kDa RGPs are single-chain proteins, the two high-molecular-mass proteinases are noncovalent, tight complexes of a catalytic domain with several polypeptide chains, believed to be involved in the hemagglutinin/adhesin activity of high-molecular-mass gingipains [71]. N- terminal amino acid sequence analysis of individual polypeptide bands of HRGP and KGP has revealed that, although the sequence of catalytic domains differed considerably (Table 3), the other components of the HRGP and 105-kDa KGP complexes are identical or, at the very least, closely related. This observation has allowed us to speculate that each complex might be as- sembled either from different gene products or, alternatively, is the result of posttranslational processing of a polyprotein. Molecular cloning and structural characterization of the HRGP gene [73] have shown that it codes for a 1704 amino acid residue polyprotein (185.4 kDa). Without the propeptide, the proteinase domain and adhesin/hemagglutinin fragments (HGP) would create a single-chain protein of 159.9 kDa, indicating that the secreted enzyme is most likely processed and assembled as a noncovalent complex of the proteinase with individual adhesin/hemagglutinin domains, as schematically depicted in Fig. 2.

HYPOTHETICAL MECHANISM OF HRGP SECRETION

In most P. gingivalis strains, the majority of the activity measured with BAPNA and Z-Lys- pNA is associated with cell surfaces and vesicles, which are the blebs of outer membranes [55,74-79]. This indicates that enzymes responsible for this activity are outer-membrane-associated proteins, and from data to be described later, it seems most likely that these proteinases are identical with RGPs and KGP [74]. This also indicates that, as part of the secretory pathway, they must traverse the cytoplasmic (inner membrane) and become inserted, at least transiently, into the outer membrane. In this regard, the gingipain journey to the cell surface and eventually to the extracellular milieu strikingly resembles the unusual secretory pathway utilized by the IgA proteases of Neisseria gonorrhoeae, Neisseria meningitidies, Haemophilus influenzae, as well as a serine protease of Serratia marcescens, all of which are secreted via an outer-membrane-anchored intermediate (reviewed in Refs. 80 and 81). Another appealing analogy between gingipains and these enzymes is the fact that all are derived from precursors which are substantially larger than the secreted products. Also, the structural organization of the HRGP polyprotein bears some similarity to the Neisseria IgA protease precursor, including an N-terminal signal peptide, fol- lowed by a domain corresponding to the mature secreted proteinase, and a C-terminal extension which, in the case of the IgA protease, harbors specific secretory functions for outer membrane transport. Therefore, it is tempting to propose the following events: (i) HRGP polyprotein first crosses the cytoplasmic membrane; (ii) the N-terminal signal peptide is cleaved when the gingipain precursor reaches the periplasmic space; (iii) the C-terminal part of the partially processed poly- protein integrates with the outer membrane and translocates the remaining part of the HRGP precursor to the cell surface; (iv) at the cell surface, the enzyme acquires its active conformation; and (v) the enzyme undergoes further proteolytic processing. In some strains of P. gingivalis the enzyme remains attached to the outer membrane, while in others it is shed into the media due to excessive proteolysis. Some experimental data strongly support this hypothetical scenario. For example, Ciborowski et al. [72] purified single-chain, membrane-associated, high-molecular-mass

454

Polyprotein encoded by HRGP gene (1704 amino acid residues, 185.4 kDa)

M-227 I:1-1 1:1492 1:1909 K1044 R1202 K1477

Propeptida HGP27 Catalytic domain HGP44 HGP15 HGP17

Proteolytic processing of polyprotein and assembly of putative secretory

forms of HRGP (95-kDa RGP)

V 94 kDa

92 kDa

94 kDa

Fig. 2. Structure of polyprotein encoded by the HRGP gene and its hypothetical processing towards secretory forms of proteinase.

forms (120 and 150 kDa) of Arg-X- and Lys-X-specific cysteine proteinases. In addition, our data from Western blot analysis, using anti-RGP antibodies and confirmed by the specific labeling of the active-site cysteine residue, revealed that membrane-associated enzymes contain an extra 15- kDa polypeptide [74], presumably involved in outer membrane anchoring.

UNITY AMONG DIVERSITY

Using the Western blot approach, combined with zymography and inhibition studies [74], it was shown that, regardless of the P. gingivalis strain and method of bacterium cultivation, RGPs occur as 110-, 95-, 70- to 90-, and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinins, while the other forms are single-chain enzymes. In contrast, the predominant form of KGP is a complex of the 6g-kDa catalytic domain with hemagglutinins. Moreover, lower molecular mass forms of both gingipains have also been observed, indicating that they could arise by excessive proteolysis from larger precursors. These data explain the seemingly extreme complexity of P. gingivalis proteinases and indicate that in most of the strains only two primary enzymes are present, one with arginine-X specificity and the other with lysine-X specificity, which occur in many different molecular forms and, working in concert, are respon- sible for the 'trypsin-like' activity associated with this bacterium. In this regard, it is obvious that multiple attempts to purify these proteinases have resulted in separation of one of several gingi-

455

pain forms, so that observed variations from different laboratories are due to different protocols. In one case, however, the lack of proper controls or certain precautions has led to gross misinter- pretations of the data [49], where an alleged novel proteinase with purported collagenolytic activity, named argingipain, turned out to be identical to RGP-1 after the gene coding for this protein was cloned and sequenced [82].

GINGIPAINS AND CLINICAL HALLMARKS OF PERIODONTITIS

The pathological potentials of the proteolytic activity elaborated by P. gingivalis are not in question, but most of the data (presented in Table 1) have been collected using whole bacterial cells, cell extracts, or other undefined mixtures of proteinases. To pinpoint the destructive func- tion of strictly defined, individual enzymes, we have investigated the effect of active-site titrated KGP and RGPs on blood coagulation (T. Imamura et al., Infect. Immunol., manuscript sub- mitted for publication), vascular permeability enhancement [34,35] and activation of the comple- ment cascade [33].

RGPs (RGP- 1, RGP-2 and HRGP) are very potent VPE factors of P. gingivalis, inducing this activity through plasma prekallikrein activation and subsequent bradykinin release. Since this activity was totally suppressed by anti-RGP antibodies in crude bacterial extracts, it is obvious that RGP is the only enzyme produced by P. gingivalis which can trigger bradykinin release through prekallikrein activation [35]. KGP by itself is not able to induce VPE in human plasma, but working in concert with RGP the pair can induce VPE by cleaving bradykinin directly from HMWK [34]. Thus, both gingipains are important VPE factors, responsible for gingival crevicular fluid production at periodontitis sites infected with P. gingivalis. In this way, they aid in the pro- vision of a continuous supply of nutrients that are important for bacterial growth and virulence.

RGP is also a very efficient enzyme in terms of the generation of a potent chemotactic factor, C5a, through direct cleavage of C5, which probably explains the significant leukocyte infiltration at periodontitis lesions. At the same time, the enzyme degrades C3 and in this way eliminates the creation of C3-derived opsonins, thus rendering P. gingivalis resistant to phagocytosis [24,33]. Importantly, the massive accumulation of neutrophils in the inflamed periodontal tissue reflects the very high levels of active granular proteinases (elastase, cathepsin G, gelatinase, collagenase) in gingival crevicular fluid [53,54,83-85] and, therefore, connective tissue destruction. In such a highly proteolytic environment, subgingival plaque bacteria would clearly thrive due to the presence of high concentrations of peptides and amino acids, thus further aggravating tissue destruction.

Among the plasma proteins, fibrinogen seems to be the major target for KGE In vitro, and already at nanomolar concentrations, this enzyme degrades the fibrinogen Aa chain within minutes [27], thus rendering it non-clottable. In normal plasma, fibrinogen degradation is mir- rored by a prolongation of plasma thrombin time, and KGP does exert a strong negative effect on plasma clottability at low nanomolar concentrations (T. Imamura et al., Infect. Immunol., manuscript submitted for publication). Indeed, KGP is the most potent fibrase characterized to date [27], and there is little doubt that its presence and nonrestricted activity in periodontal pockets would contribute to a bleeding tendency, especially since it also very efficiently destroys the procoagulant portion of high-molecular-weight kininogen [27]. The bleeding at periodontitis sites is of primary importance for P. gingivalis, because it delivers, through the hemoglobin in

456

erythrocytes, the richest source of heme and iron. The proteinase-hemagglutinin complexes may then be important in the uptake of these vital growth factors, via (i) hemagglutination [71]; (ii) hemolysis of erythrocytes [86]; and (iii) subsequent degradation of hemoglobin by bacterial and host proteinases.

GINGIPAIN AS AN ATTRACTIVE TARGET FOR T H E R A P E U T I C INHIBITOR DEVEL- OPMENT

There is thus no doubt that only two enzymes (RGP and KGP), with very restricted specifici- ties and produced in large quantities by P. gingivalis, may be directly responsible for the clinical features of periodontitis (GCF production, neutrophil accumulation, and bleeding), each of which is very beneficial for this periodontopathogen. In addition, gingipains seem to be the major players in disabling host antimicrobial mechanisms, either humoral or cellular. This hypothesis received a recent boost when it was shown that an allelic exchange mutant of P. gingivalis W83, in which the HRGP gene was inactivated, showed dramatically reduced virulence in a mice model in comparison with the wild-type W83 strain [87]. It is thus likely that attenuated virulence of P. gingivalis could also be induced by gingipain inactivation with specific inhibitors. Since these proteinases are not at all inactivated by host inhibitors and, in fact, degrade such proteins, only the development of either synthetic inhibitors or specific, neutralizing antibodies is a logical therapeutic consideration. These approaches would be appealing because (i) periodontitis is a very localized disease and there are already systems for local delivery (into a single periodontal pocket) of controlled-release antimicrobials [88,89]; and (ii) gingipains differ from other known protein- ases, so that highly specific inhibitors could be developed. In addition, inhibition of host protein- ases by locally administered inhibitors might also be beneficial for the patient, since the host proteinases appear to be primarily responsible for periodontal tissue destruction [90,91].

REFERENCES

1 Moore, W.E.C., Moore, L.V.H. and Cato, E.R, U.S. Fed. Culture Collect. Newslett., 18 (1988) 7. 2 Nyvad, B. and Kilian, M., Scand. J. Dent. Res., 95 (1987) 369. 3 L6e, H., Theilade, E. and Jensen, S.B., J. Periodontol., 36 (1965) 177. 4 Moore, L.V.H., Moore, W.E.C., Cato, E.R, Smibert, R.M., Burmeister, J.A., Best, A.M. and Ranney, R.R., J. Dent.

Res., 66 (1987) 989. 5 Moore, W.E.C., J. Periodontal Res., 22 (1987) 335. 6 Williams, D.M., Hughes, F.J., Odell, E.W. and Farthing, RM., Pathology of Periodontal Disease, Oxford University

Press, Oxford, 1992. 7 Lung, N.R, Arch. Oral Biol., 35 (1990) 9S. 8 Slots, J. and Listgarten, M.A., J. Clin. Periodontol., 13 (1988) 570. 9 Slots, J., Bragd, L., Wikstr6m, M. and Dahlen, G., J. Clin. Periodontol., 15 (1988) 85.

10 Holt, S.C., Ebersole, J., Felton, J., Brunsvold, M. and Kornmann, K.S., Science, 239 (1987) 55. 11 Lamster, I.B., Smith, Q.T., Celenti, R.S., Singer, R.E. and Grbic, J.T., J. Periodontol., 65 (1994) 511. 12 Mayrand, D. and Holt, S.C., Microbiol. Rev., 52 (1988) 134. 13 Holt, S.C. and Brumante, T.E., Crit. Rev. Oral Biol. Med., 2 (1991) 177. 14 Kolenbrander, RE., In Dworkin, M. (Ed.) Microbial Cell-Cell Interactions, American Society for Microbiology,

Washington, DC, 1991, pp. 303-329. 15 Sundqvist, G., FEMS Immunol. Med. Microbiol., 6 (1993) 125. 16 Cutler, C.W., Kalmar, J.R. and Genco, C.A., Trends Microbiol. Sci., 3 (1995) 45.

457

17 Nilsson, T., Carlsson, J. and Sundqvist, G., Infect. Immun., 50 (1985) 467. 18 Carlsson, J., Herrmann, B.E, Hofling, J.E and Sundqvist, G.K., Infect. Immun., 43 (1984) 644. 19 Sato, M., Otsuka, M., Maehara, R., Endo, J. and Nakamura, R., Arch. Oral Biol., 32 (1987) 235. 20 Grenier, D., Infect. Immun., 60 (1992) 1854. 21 Sundqvist, G., Carlsson, J., Herrmann, B. and Tarnvik, A., J. Med. Microbiol., 19 (1985) 85. 22 Grenier, D., Mayrand, D. and McBride, B.C., Oral Microbiol. Immunol., 4 (1989) 12. 23 Fishburn, C.S., Slaney, J.M., Carman, R.J. and Curtis, M.A., Oral Microbiol. Immunol., 6 (1991) 209. 24 Cutler, C.W, Arnold, R.R. and Schenkein, H.A., J. Immunol., 151 (1993) 7016. 25 Carlsson, J., Hofling, J.F. and Sundqvist, G.K., J. Med. Microbiol., 18 (1984) 39. 26 Wikstr6m, M.B., Dahlen, G. and Linde, A., J. Clin. Microbiol., 17 (1983) 759. 27 Scott, C.E, Whitaker, E.J., Hammond, B.E and Colman, R.W., J. Biol. Chem., 268 (1993) 7935. 28 Lantz, M.S., Rowland, R.W, Switalski, L.M. and Hook, M., Infect. Immun., 54 (1986) 654. 29 Sundqvist, G., Bengtson, A. and Carlsson, J., Oral Microbiol. Immunol., 3 (1988) 103. 30 Schenkein, H.A. and Berry, C.R., J. Periodontal Res., 23 (1988) 308. 31 Schenkein, H.A., Oral Microbiol. Immunol., 6 (1991) 216. 32 Schenkein, H.A., J. Periodontal Res., 23 (1988) 187. 33 Wingrove, J.A., DiScipio, R.G., Chen, Z., Potempa, J., Travis, J. and Hugli, T.E., J. Biol. Chem., 267 (1992) 18902. 34 Imamura, T., Potempa, J., Pike, R.N. and Travis, J., Infect. Immun., 63 (1995) 1999. 35 Imamura, I"., Pike, R.N., Potempa, J. and Travis, J., J. Clin. Invest., 94 (1994) 361. 36 Hinode, D., Nagata, A., Ichimiya, S., Hayashi, H., Morioka, M. and Nakamura, R., Arch. Oral Biol., 37 (1992) 859. 37 Wikstr6m, M. and Linde, A., Infect. Immun., 51 (1986) 707. 38 Uitto, V.-J., Haapasalo, M., Laakso, T. and Salo, T., Oral Microbiol. Immunol., 3 (1988) 97. 39 Smalley, J.W, Birss, A.J. and Shuttleworth, C.A., Arch. Oral Biol., 33 (1988) 323. 40 Lantz, M.S., Allen, R.D., Duck, L.W., Blume, J.L., Switalski, L.M. and Hook, M., J. Bacteriol., 173 (1991) 4263. 41 Uitto, V.-J., Larjava, H., Heino, J. and Sorsa, T., Infect. Immun., 57 (1989) 213. 42 Gibbons, R.J., Hay, D.I., Childs III, WC. and Davis, G., Arch. Oral Biol., 35 (1990) 107S. 43 Otsuka, M., Endo, J., Hinode, D., Nagata, A., Maehara, R., Sato, M. and Nakamura, R., J. Periodontal Res., 22

(1987) 491. 44 Odell, E.W and Wu, RJ., Arch. Oral Biol., 37 (1992) 597. 45 Sorsa, T., Ingman, T., Suomalainen, K., Haapasalo, M., Konttinen, Y.T., Lindy, O., Saari0 H. and Uitto, V.-J.,

Infect. Immun., 60 (1992) 4491. 46 Curtis, M.A., Macey, M., Slaney, J.M. and Howells, G.L., FEMS Microbiol. Lett., 110 (1993) 167. 47 Lala, A., Amano, A., Sojar, H.T., Radel, S.J. and De Nardin, E., Biochem. Biophys. Res. Commun., 199 (1994) 1489. 48 Bedi, G.S. and Williams, T., J. Biol. Chem., 269 (1994) 599. 49 Kadowaki, T., Yoneda, M., Okamoto, K,, Maeda, K. and Yamamoto, K., J. Biol. Chem., 269 (1994) 21371. 50 Lawson, D.A. and Meyer, T.F., Infect. Immun., 60 (1992) 1524. 51 Kato, T., Takahashi, N. and Kuramitsu, H.K., J. Bacteriol., 174 (1992) 3889. 52 Birkedal-Hansen, H., Taylor, R.E., Zambon, J.J., Barwa, EK. and Neiders, M.E., J. Periodontal Res., 23 (1988) 258. 53 Gangbar, S., Overall, C.M., McCulloch, C.A.G. and Sodek, J., J. Periodontal Res., 25 (1990) 257. 54 Overall, C.M., Sodek, J., McCuUoch, C.A.G. and Birek, R, Infect. Immun., 59 (1991) 4687. 55 Grenier, D., Chao, G. and McBride, B.C., Infect. Immun., 57 (1989) 95. 56 Wikstr6m, M., Potempa, J., Polanowski, A., Travis, J. and Renvert, S., J. Periodontol., 65 (1994) 47. 57 Yoshimura, F., Nishikata, M., Suzuki, T., Hoover, C.I. and Newbrun, E., Arch. Oral Biol., 29 (1984) 559. 58 Fujimura, S. and Nakamura, T., Infect. Immun., 55 (1987) 716. 59 Ono, M., Okuda, K. and Takazoe, I., Oral Microbiol. Immunol., 2 (1987) 77. 60 Sorsa, T., Uitto, V.-J., Suomalainen, K., Turto, H. and Lindy, S., J. Periodontal Res., 22 (1987) 375. 61 Tsutsui, H., Kinouchi, T., Wakano, Y. and Ohnishi, Y., Infect. Immun., 55 (1987) 420. 62 Fujimura, S. and Nakamura, T., Oral Microbiol. Immunol., 5 (1990) 360. 63 Hinode, D., Hayashi, H. and Nakamura, R., Infect. Immun., 59 (1991) 3060. 64 Nishikata, M., Kanehira, T., Oh, H., Tani, H., Tazaki, M. and Kuboki, Y., Biochem. Biophys. Res. Commun., 174

(1991) 625. 65 Nishikata, M. and Yoshimura, F., Biochem. Biophys. Res. Commun., 178 (1991) 336.

458

66 Shah, H.N., Gharbia, S.E., Kowlessur, D., Wilkie, E. and Brocklehurst, K., Microb. Ecol. Health Dis., 4 (1991) 319. 67 Fujimura, S., Shibata, Y. and Nakamura, T., FEMS Microbiol. Lett., 113 (1993) 133. 68 Chen, Z.X., Potempa, J., Polanowski, A., Wikstr6m, M. and Travis, J., J. Biol, Chem., 267 (1992) 18896. 69 Curtis, M.A., Ramakrishnan, M. and Slaney, J.M., J. Gen. Microbiol., 139 (1993) 949. 70 Fujimura, S., Shibata, Y. and Nakamura, T., Oral Microbiol. Immunol., 7 (1992) 212. 71 Pike, R., McGraw, W., Potempa, J. and Travis, J., J. Biol. Chem., 269 (1994) 406. 72 Ciborowski, R, Nishikata, M., Allen, R.D. and Lantz, M.S, J. Bacteriol., 176 (1994) 4549. 73 Pavloff, N., Potempa, J., Pike, R.N, Prochazka, V., Kiefer, M.C., Travis, J. and Barr, RJ., J. Biol. Chem., 270 (1995)

1007. 74 Potempa, J., Pike, R. and Travis, J., Infect. Immun., 63 (1995) 1176. 75 Grenier, D. and Mayrand, D., Infect. Immun., 55 (1987) 111. 76 Minhas, T. and Greenman, J., J. Gen. Microbiol., 137 (1989) 557. 77 Smalley, J.W. and Birss, J., J, Gen. Microbiol., 133 (1987) 2883. 78 Smalley, J.W. and Birss, J., Oral Microbiol. Immunol., 6 (199t) 202. 79 Smith, A.J., Minhas, T., Greenman, J. and Embery, G., Microbios, 73 (1993) 185. 80 Klauser, T., Pohlner, J. and Meyer, T.E, BioEssays, 15 (1993) 799. 81 Pugsley, A.R, Microbiol. Rev., 57 (1993) 50. 82 Okamoto, K., Misumi, Y., Kadowaki, T., Yoneda, M., Yamamoto, K. and Ikehara, Y., Arch. Biochem. Biophys.,

316 (1995) 917. 83 Cox, S.W. and Eley, B.M., J. Clin. Periodontol., t9 (1992) 333. 84 Gustafsson, A., Asman, B., Bergstr6m, K. and S6der, R-O., J. Clin. Periodontol., 19 (1992) 535. 85 Giannopoulou, C., Andersen, E., Demeurisse, C. and Cimasoni, G., J. Dent. Res., 71 (1992) 359. 86 Shah, H.N. and Gharbia, S.E., FEMS Microbiol. Lett., 61 (1989) 213. 87 Fletcher, H.M., Schenkein, H.A., Morgan, R.M., Bailey, K.A., Berry, C.R. and Macrina, EL., Infect. Immun., 63

(1995) 1521. 88 Medlicott, N.J., Rathbone, M.J., Tucker, I.G. and Holborow, D.W., Adv. Drug Deliv. Rev., 13 (1994) 181. 89 Kornman, K.S., J. Periodontol., 64 (1993) 782. 90 Genco, R.J., J. Periodontol., 63 (1992) 338. 91 Birkedal-Hansen, H., J. Periodontal Res., 28 (1993) 500.