An Endo-Acting Proline-Specific Oligopeptidase from Treponema denticola ATCC 35405: Evidence of Hydrolysis of Human Bioactive Peptides
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INFECTION AND IMMUNITY, Nov. 1994, P. 4938-4947 Vol. 62, No. 11
0019-9567/94/$04.00+0
Copyright C 1994, American Society for Microbiology
An Endo-Acting Proline-Specific Oligopeptidase from
Treponema denticola ATCC 35405: Evidence of
Hydrolysis of Human Bioactive Peptides
PIRKKO-LIISA MAKINEN, KAUKO K. MAKINEN,* AND SALAM A. SYED
Department of Biologic and Materials Sciences, School of Dentistry,
The University of Michigan, Ann Arbor, Michigan 48109
Received 2 May 1994/Returned for modification 7 July 1994/Accepted 19 August 1994
An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified
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to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral
spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The
POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed
(optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at
the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin,
angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable
peptide size was tetrapeptide P3P2P,P',, while the maximum substrate size was ca. 3 kDa. An imino acid
residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the
following, with the Kj(8pp) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5),
neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical
modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends
on the catalytic triad of COOH Ser
... His but not on a metal. The amino acid sequence around the putative
...
active-site serine is Gly-Gly-Ser*-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue
near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71%
with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the
Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino
acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by
participating in the peptidolytic processing of those peptides.
Treponema denticola is one of the predominant members of 53, 62). A member of this enzyme family, the prolyl oligopep-
the human periodontal flora (15, 20-24, 34, 45). Previous tidase (POPase), may participate in the processing of brain
studies suggest that T. denticola is associated with periodontal angiotensin (Ang) (52) and in the degradation of oxytocin (59).
infections by adhering to epithelial cells (36), gingival fibro- After the N-terminal heptapeptide of Ang-I was also demon-
blasts (50), fibronectin (7), laminin, fibrinogen, gelatin, and strated to possess biological activity through a pathway that is
type I and type II collagens (12); by degrading basement not dependent on Ang-converting enzyme, POPase was de-
membrane collagen (48); by invading healthy tissue (9, 14, 21, fined as one of the putative Ang-I-processing enzymes and thus
32); by suppressing fibroblast proliferation (4); by causing part of the RAS cascade (51). It was further suggested that
microulceration of the sulcular epithelium (28, 30); by showing synthetic peptide inhibitors of POPase act as antiamnestic
keratinolytic activity (31); and by exhibiting mutual symbiotic agents (60). A POPase from human brain (16) and pig muscle
growth enhancement with another periodontal pathogen, Por- (38) has been characterized and may be involved in the
phyromonas gingivalis (10). Treponemal cells have been shown maturation and degradation of hormones and neuropeptides
to migrate through the basement membrane (11, 48), and T. (29, 49, 53).
denticola proteases activate host latent procollagenase (46). Mammalian POPases are sensitive to diisopropyl fluoro-
Because of the complexity of the overall process, the detailed phosphate, although they are also inhibited by p-hydroxymer-
chemical mechanism of human treponemal infections is not curibenzoic acid (pHMB) (49, 53). These and other character-
known, although the above studies suggest that proteases and istics have given the POPases an imprint of an "obscure" group
peptidases present in the outer cell envelope or in the periplas- of serine proteases (1). The substrate often used in the assay of
mic space of the treponemes may play a crucial role. Our the POPases is carbobenzoxyglycyl-L-prolyl-p-nitroanilide (Z-
research has focused on the cell-associated oligopeptidases of Gly-Pro-pNA), in which the enzyme hydrolyzes the Pro-pNA
treponemes and discovered in these cells a novel prolyl endo- bond. The same enzyme is responsible for all the activities
peptidase with a strict specificity profile. previously attributed to postproline endopeptidase, endooli-
Prolyl endopeptidases (EC 3.4.21.26; previously called post- gopeptidase B, TRH-deamidase, brain kinase B, and oxytocin-
proline endopeptidases) have received attention because of degrading enzyme (57). This enzyme was later termed POPase
their role in the metabolism of vasoactive peptides (29, 49, 52, and recently called prolyl oligopeptide hydrolase. This enzyme
family reflects a further distinct evolutionary line of serine
*
Corresponding author. Mailing address: Department of Biologic peptidases and differs in catalytic mechanism from the chymo-
and Materials Sciences, School of Dentistry, The University of Mich- trypsin and subtilisin families (40). In the family of POPases,
igan, Ann Arbor, MI 48109-1078. Phone: (313) 763-6166. Fax: (313) the order of catalytic residues (Asp ... Ser ...His) is different
747-3896. from that of chymotrypsin and subtilisin. POPase is an endo-
4938VOL. 62, 1994 PROLYL OLIGOPEPTIDASE FROM A SPIROCHETE 4939
peptidase with restricted specificity for substrate size, making it
an oligopeptidase. A m- -
I
Our studies on T. denticola ATCC 35405 showed that this 5
organism contains a POPase which hydrolyzes in human
bioactive peptides (HBPs) the bond involving the carboxyl
group of proline. It is possible that the chemical mechanism of 60 90 120 150 180 210
treponemal infection is associated with the degradation of
bradykinin (Bk), substance P (SP), Ang, vasopressin, or other
HBPs used in this study. A potential mechanism for regulating
the levels of HBPs in mammalian tissues could arise by
substrate competition for interaction with the enzyme, so that
one peptide may serve to regulate the in vivo level of another
peptide (13). It is possible that the POPases from pathogenic
organisms interfere with such reactions. The objective of this
study was to investigate the nature of the prolyl oligopeptidase 11
reaction catalyzed by cell extracts of T. denticola. The specific
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aims of this study included the development of a purification
procedure for the enzyme, the study of the specificity profile of
the enzyme and the enzyme's inhibition by peptide inhibitors,
and the study of the chemical modification of the enzyme for 0 3u 60u
its possible classification.
100
MATERLILS AND METHODS D
50
Source and cultivation of the organism and treatment of
cells. Cells of T. denticola ATCC 35405 were grown anaerobi-
.
cally for 48 h in a tryptone-yeast extract-heart infusion broth 0 20 40 60 RETENTION TIME (Minutes)
containing 10% heat-inactivated rabbit serum (35). For the
purpose of enzyme purification, 1.2-liter aliquots of the growth FIG. 1. Purification of the POPase by means of FPLC. (A) Sepa-
medium in 1.5-liter screw-capped flasks were inoculated with ration of the enzyme on a Calbiochem high-resolution hydroxyapatite
100-ml aliquots of cultures (27) and incubated anaerobically column (2.2 by 35 cm). The elution was carried out with phosphate
buffer (pH 6.8) 20 mmol per liter with EDTA (0.1 mmol/liter) (initial
for 4 days at 37°C. The optical density at 660 nm (OD660) was
used to determine the number of cells; an OD660 of 0.2
buffer), using a phosphate gradient from 0.02 to 1.0 mol/liter (0 to 35%
in 120 min and 35 to 100% in 40 min). The gradient was applied after
corresponded to 5 x 108 cells per ml (12). The cells were the application of the sample was completed. The fraction volume was
harvested by centrifugation for 10 min at 16,300 x g. This 3 ml (flow rate, 3 mUmin). (B) Separation of the enzyme from the
procedure and all subsequent steps of enzyme purification previous step on a phenyl-Sepharose CL-4B column (1 by 10 cm). The
were carried out at 0 to 4°C, except for fast protein liquid elution was carried out with the above buffer using a descending
chromatographic (FPLC) separations, which were carried out NH4C1 gradient from 3.5 to 0 mol/liter in 120 min. The column was
at 22°C. finally eluted with Milli-Q water. The fraction volume was 1 ml (flow
Chemicals. Unless specifically mentioned, the chemicals rate, 1 ml/min). (C) Separation of the enzyme from the previous step
on Fractogel EMD TMAE-650 anion exchanger (40 to 90 ,um; 1 by 40
used were obtained from Sigma. The water used in this study
was prepared with a Millipore Milli-Q system and had a
cm). The elution was performed using for the first 15 min 50 mmol of
Tris (pH 7.5) per liter at 2.0 ml/min and subsequently an NaCl gradient
resistance of 18 megaohms cm-'. from 0 to 1.0 mol/liter (0 to 50% from 15 to 80 min and 50 to 100%
Purification of the enzyme. The harvesting of the cells for from 80 to 100 min). (D) Final separation of the enzyme with two
enzyme purification was performed after 4 days of growth, successive runs on a Superose 12 column (10/30) using 0.25 mol of
because the cell mass reached a sufficiently high level by 4 days. NaCl per liter in 20 mmol of phosphate buffer (pH 6.8) per liter with
The cells were washed with 20 mmol of phosphate buffer (pH EDTA (0.1 mmolJliter) (the second separation is shown). The flow rate
was 0.5 ml/min. The elution of the POPase is shown in each panel with
6.8) per liter and subsequently suspended in the same buffer a bracket. The scale for the salt gradients (--- ) and the protein at 280
containing 0.1 mmol of EDTA (15 ml/8 g of cells, wet weight) nm (the latter shown as FPLC/Pharmacia printouts with values of 0.1
per liter. Small volumes of 10% Triton X-100 (Pierce) were to 2.0 for absorption units full scale [AUFS]) is shown on the left (from
added to a final concentration of 0.05%. Sixty minutes later, 0 to 100%).
the suspension was centrifuged for 15 min at 27,000 x g. A
large number of separate purifications were carried out by
subjecting suitable aliquots of the Triton X-100 extracts to
FPLC as described below. 50 volumes of 25 mmol of Tris (pH 7.5) plus 0.1 mmol of
(i) Hydroxyapatite-FPLC. The enzyme (normally in 50-ml EDTA per liter, changing the dialyzing solution once. The
aliquots) resulting from the detergent extraction was subjected resulting dialysate was concentrated as above.
to high-resolution hydroxyapatite-FPLC (Fig. 1A). The active (iii) Anion-exchange FPLC. The dialyzed enzyme was chro-
fractions were combined, and the enzyme was concentrated matographed through a strong (Tentacle type) anion ex-
using Amicon Centriprep-30 membrane filters. changer (Fractogel) (Fig. 1C). Normally, less than 10-ml
(ii) Phenyl-Sepharose FPLC. Solid NH4Cl was added to the volumes of the dialysate were applied to the column using a
enzyme from the previous step to a final concentration of 3.5 Superloop 10 sample applicator (Pharmacia). The resulting
mol/liter. The enzyme was then subjected to separation on a enzyme was concentrated as above.
phenyl-Sepharose gel involving a descending NH4Cl gradient (iv) Gel permeation chromatography. The enzyme was
(Fig. 1B). The active fractions were combined, and the enzyme finally subjected to two consecutive separations on a Superose
was dialyzed (Spectrapor; cutoff, 12 to 14 kDa) for 16 h against 12 column (Fig. 1D). The purified enzyme was stored in the4940 MAKINEN ET AL. INFECT. IMMUN.
TABLE 1. Purification of the POPase from T. denticola ATCC 35405
Step Vol (ml) Protein Total protein Sp acta Total activity
(mg/ml) (mg) (,umol/min/mg) (,umol/min)
1. Triton X-100 extract after centrifugation 226 5.86 1,324.4 0.089 117.9
2. After hydroxyapatite chromatography 96.7 2.58 249.5 0.38 94.5
3. After phenyl-Superose 11.0 2.02 22.2 2.63 58.4
4. After Fractogel/concentration 2.6 1.73 4.5 8.58 38.6
5. After Superose 12 3.0 n.d. n.d. n.d. n.d.
6. After Superose 12 6.0 0.102 0.61 24.02 14.8
a Determined with Z-Gly-Pro-pNA under conditions described in Materials and Methods with MES (50 mmol/liter, pH 6.5).
" n.d., not determined.
elution buffer at 4°C. The purification procedure is summa- enzyme) in no more than 2% of the total assay volume was
rized in Table 1. The actual enzyme yield was 0.61 mg from 80 added. The reaction was followed to establish the inhibited
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g (wet weight) of cells. This yield is about 12.5% of the rate (vi). Substrate concentration was kept constant by allowing
expected (theoretical) yield of about 5.0 mg. no more than 5% hydrolysis. Under these conditions, Ki(app)
Enzyme determinations. The POPase was discovered as a (i.e., Ki in the presence of substrate) was given by vGlvi = 1 +
contaminant in crude preparations of the FALGPA-peptidase [1]114(app) where [I] is the concentration of the inhibitor (43).
(an enzyme hydrolyzing 2-furylacryloyl-L-leucylglycyl-L-prolyl- The values of Ki proper was calculated according to Cornish-
L-alanine) (27), which hydrolyzed Bk at the Pro-7-Phe-8 bond Bowden (6).
in addition to the Phe-5-Ser-6 bond hydrolyzed by the purified Protein determination. The protein concentration was de-
enzyme proper (27). Subsequent studies showed that it was the termined spectrophotometrically at 220 nm (54).
POPase that hydrolyzed the Pro-7-Phe-8 bond and that this Chemical modification of POPase. Modification of seryl
enzyme could be conveniently assayed using Z-Gly-Pro-pNA residues was performed with diisopropyl fluorophosphate.
as the substrate. The activity of POPase was determined in Modification of histidyl residues by diethyl pyrocarbonate was
1.0-ml reaction mixtures containing 0.05 mol of MES (mor- studied according to Miles (33). Treatment of the POPase with
pholine ethanesulfonic acid) (pH 6.5), 0.2 mmol of Z-Gly-Pro- N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to
pNA (dissolved in methanol) (3), and 1 to 10 ,ul of enzyme per study the involvement of carboxyl groups in enzyme activity
liter at 30°C. After enzyme addition, the increase in absorption was performed at 25°C (39, 42). E-64 IL-trans-epoxysuccinyl-
at 405 nm was monitored for 5 min at 30°C using a Shimadzu leucylamide-(4-guanidino)-butane], Hg +, and pHMB were
UV-265 recording spectrophotometer and a thermostated used to study the modification of active sulfhydryl groups of
cuvette holder. The value of 8,800 M-1 cm-' was used for E405. the enzyme (2). Unless otherwise mentioned, all modifications
Determination of the cleavage site of peptides. The study of were carried out in an iced water bath.
the hydrolysis of peptides was based on the separation of the
products of hydrolysis by reversed-phase chromatography on a
Pharmacia PepRPC 5/5 column. The peptide substrates were RESULTS
first incubated for various periods of time (5 min to 2 h) in 50
mmol of MES (pH 6.5) per liter with 2 ,ug of enzyme and a Production and localization of POPase in the cells. The
suitable quantity of the peptide (0.1 mmol/liter) at 30°C. maximum yield of the enzyme was obtained after 2 to 4 days of
Aliquots of the mixtures were withdrawn at desired reaction growth. The maximum production thus took place during the
times, and the reactions were quenched by adding eluent A logarithmic growth phase, declining thereafter, although the
(see below) to the aliquot (1:1). The mixtures were immedi- growth of the cells had not reached a stationary phase by the
ately subjected to reversed-phase chromatography on a Pep- fourth day. No POPase activity was demonstrated in the
RPC R 5/5 column, using the Pharmacia FPLC system. Eluent growth medium. The enzyme activity was functional as a
A was 0.1% trifluoroacetic acid in water, and eluent B was component of intact cells. However, the cells need not lyse for
0.1% trifluoroacetic acid in acetonitrile or 0.05% trifluoroace- the POPase to be functional. Accordingly, thoroughly washed
tic acid in isopropanol. The increase in the percentage of B per whole cells of T. denticola ATCC 35405 were very active,
minute depended on the peptide used. The absorption of the suggesting that the enzyme may be located in the outer
peptide fragments was monitored at 214 nm. The fractions membrane or in the periplasmic space. Washing of the cells did
containing these fragments were combined, and the resulting not affect their microscopic morphology. Treatment of the cells
solution was evaporated to dryness using a SpeedVac evapo- with 0.05% Triton X-100 resulted in a virtually instantaneous
rator. The dry residues were hydrolyzed for 4 h at 145°C in 6 liberation of most POPase; even 0.01% detergent was effective.
mol of HCl per liter, and the resulting hydrolysates were Concentrations higher than 0.1% inhibited the enzyme.
evaporated to dryness. The final dry residues were dissolved in Purity of the enzyme. The purity of the POPase after step 6
a Beckman System 6300 dilution buffer for compositional (Table 1) was studied by means of sodium dodecyl sulfate-
amino acid analyses on a Beckman System 6300 High Perfor- polyacrylamide gel electrophoresis (SDS-PAGE) (using Phast-
mance Analyzer. The molar ratios of the individual amino Gel Gradient 8-25 and PhastGel SDS Buffer Strips) and FPLC
acids were used to determine the structure of the peptide on a protein reversed-phase ProRPC 5/10 column. The Phast-
fragments involved. System was from Pharmacia LKB Biotechnology Inc. The
Determination of Ki values. Determination of KI(app) values POPase was homogeneous in SDS-PAGE (Fig. 2) and in
in the POPase-catalyzed hydrolysis of Z-Gly-Pro-pNA was reversed-phase FPLC. The purity of the enzyme was indepen-
carried out by following the reaction in the absence of the dently reconfirmed by means of microbore-HPLC at the
inhibitor to establish the uninhibited linear rate of substrate University of Michigan Medical School Protein Structure and
hydrolysis (v0). Inhibitor (at least 20-fold molar excess over Sequencing Facility.VOL. 62, 1994 PROLYL OLIGOPEPTIDASE FROM A SPIROCHETE 4941
prolyl residue and the amino group of another amino acid
kDa residue that did not exhibit strict structural requirements.
Several peptides (human big endothelin, neuropeptide Y, and
40 "It insulin B-chain; see below) did not serve as substrates of the
29 _- - :~ ~4
POPase, although each one of these molecules contained one
2 or more suitable peptide bonds and although proline-contain-
45 _ .. ing fragments of big endothelin and insulin B-chain were
66 _. _o Om readily hydrolyzed. Accordingly, insulin B-chain with a molec-
97.4 _-
116w --
-
_W--75
-- kDa ular weight (mol. wt.) of 3495.9 and with a potentially scissile
205 _- "`
bond of Pro-28-Lys-29 was not hydrolyzed, but it was a potent
inhibitor. However, insulin B-chain fragment 22-30 (mol. wt.
!I 1086.3) was readily hydrolyzed at Pro-28-Lys-29. Similarly,
human big endothelin, with a mol. wt. of 4282.9 and with the
l 2 3 4 5 6 7 potentially scissile bonds of Pro-25-Glu-26, Pro-30-Tyr-31,
FIG. 2. SDS-PAGE of the enzyme after various purification steps. and Pro-36-Arg-37, was not hydrolyzed, but it acted as a strong
The PhastGel gradient 8-25 gels and PhastGel SDS Buffer Strips were inhibitor. Fragment 22-38 (mol. wt. 1,809) of the same mole-
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used in the PhastSystem (Pharmacia). Lanes: 1, size markers (Sigma cule was readily hydrolyzed at all above bonds. Neuropeptide
SDS 6H); 2, Triton X-100 extract of cells; 3, after hydroxyapatite Y (mol. wt. 4271.7) also contains bonds that in theory should
chromatography; 4, after phenyl-Sepharose; 5, after Fractogel; 6, after be hydrolyzed by the POPase (Pro-5-Asp-6, Pro-8-Gly-9, and
first gel permeation chromatography; 7, after second gel permeation
chromatography. Pro-13-Ala-14), but it is too large a molecule to act as a
substrate. These and other data (Table 3) showed that the
present POPase did not hydrolyze peptides with a mol. wt.
Amino acid composition, molecular weight, and isoelectric
higher than ca. 3,000. The hydrolysis of SP by POPase at
Pro-4-Gln-5 still left the fragment SP[5-11] biologically active
point. The amino acid composition of the POPase is shown in (the C-terminal heptapeptide of SP is biologically more potent
Table 2. These data gave for the molecular weight a range of than SP itself). The POPase hydrolyzed Bk at Pro-7-Phe-8 but
77,023 to 77,121 (mean, 77,072; SDS-PAGE gave a value of did not attack the Pro-3-Gly-4 bond. The minimum hydrolyz-
75,000; FPLC on Superose-12 and Superose-6 gave 75,000 and able peptide size was P3P2P1P'1.
77,000, respectively). The estimated minimum length of the Affinity and specificity constants. The values of Km and Vm.n
peptide was 689 amino acid residues. The isoelectric point of for the hydrolysis of Z-Gly-Pro-pNA, Bk, SP, and Ang-I were
the POPase was 6.5 (determined in free solution by means of determined in 0.02 mol of phosphate (pH 6.8) per liter at 30°C,
an LKB isoelectric focusing column and a pH gradient of 3.5 to using the Enzpack 3 program (Biosoft, Ferguson, Mo.). The
10). hydrolysis followed the normal Michaelis-Menten kinetics.
Substrate specificity. Substrate specificity studies are sum- Therefore, plots of Lineweaver-Burk, Hanes-Wolf, and Eadie-
marized in Table 3. In oligopeptides, the enzyme hydrolyzed Hofstee as well as the direct linear and the Wilkinson methods
internal peptide bonds which involved the carboxyl group of a gave essentially similar results, and the values of Ki,, as well as
those of kcat and the specificity constant (kcatlKm), are shown in
Table 4. The high affinity and specificity constants for SP and
TABLE 2. Amino acid composition of the POPase Ang-I deserve attention. Among the peptides studied, SP was
from T. denticola ATCC 35405 the best POPase substrate.
No. of Nearest Effect of pH on enzyme reaction. The enzyme hydrolyzed
residues integer Z-Gly-Pro-pNA most rapidly at pH 6.5 when tested in 50 mmol
Asparagine/aspartic acid 86.2 86 of MES per liter and Bis-Tris buffers (Fig. 3). Washed whole
Threonine 35.9 36 cells hydrolyzed Z-Gly-Pro-pNA, Bk, and SP rapidly near pH
Serine 44.4 44 6.5 as well.
Glutamine/glutamic acid 68.8 69 Elect of NaCl. The POPase was strongly activated by NaCl
Proline 28.1 28 at up to 1.5 mol/liter. The degree of activation increased with
Glycine 56.0 56 increasing pH (Fig. 4). The effect of NaCl was studied because
Alanine 46.4 46 this salt exerts a selective effect on proline-specific peptidases,
1/2Cystineb -3 3 i.e., low and high concentrations have quite different and
Valine 30.1 30 characteristic effects on the rate of hydrolysis of the favored
Methionine 10.6 11 substrates of three peptidases of T. denticola ATCC 35405
Leucine 60.7 61
Isoleucine 35.9 36 (Fig. 4). The activity of proline aminopeptidase (active on
Tyrosine 15.9 16 Not-L-prolyl-2-naphthylamine) (25) did not appreciably depend
Phenylalanine 42.6 43 on NaCl at the concentrations used, while the FALGPA-
Histidine 14.4 14 peptidase (27) was inhibited by all NaCl concentrations tested.
Lysine 83.2 83 This differentiation between treponemal peptidases by specific
Tryptophanc 9.0 9 NaCl effects may be important to the in vivo function of the
Arginine 17.6 18 enzymes, because their natural environment (human gingival
Unknown 0 0 crevice) may contain Cl- at relatively high levels, i.e., up to
a The hydrolysis of the enzyme (13.28 pmol) was carried out for 60 min at 0.01 mol/iter (8).
200°C. The values were not corrected for possible loss of amino acids during Effect of temperature. The stability of the isolated enzyme
hydrolysis. decreased rapidly above 37°C. The enzyme lost about 50% of
bDetermined after hydrolyzing the enzyme for 22 h at 1i10C in HCI (6 its activity after 35 min at 37°C in MES at 50 mmol/liter, pH 6.5.
mol/liter).
c Determined after hydrolyzing the enzyme for 22.5 h at 110°C in mercapto- Summary of chemical modification studies. The order of the
ethanesulfonic acid (3 mol/liter). catalytic triad residues in the members of the POPase family is4942 MAKINEN ET AL. INFEcr. IMMUN.
TABLE 3. Determination of the cleavage site of peptides hydrolyzed by the POPase from T denticola ATCC 35405
Peptides identified
Substrate Amino acid sequence after reaction with
POPasea
Z-Gly-Pro-pNA Z-GIP-pNA
Substance P R-P-K-PQ--Q-F-F-G-L-M-NH2 1-4; 5-11
Angiotensin I D-R-V-Y-I-H--P'F-H-L 1-7; 8-10
Angiotensin II D-R-V-Y-I-H-PIF 1-7;8
Neurotensin pE-L-Y-E-N-K-PAR-R-P4Y-I-L 1-7; 11-13
Neurotensin 8-13 R-R-P1Y-I-L 8-10
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Bradykinin R-P-P-G-F-S-P1F-R 1-7; 8-9
Oxytocin C-Y-I-Q-N-C-PLO-H2 1-7
[Arg8J -Vasopressin C-Y-F-QN-CN-P'R-G-NH2 1-7
[Lys8J-Vasopressin C-Y-F-Q-N-C-PK-G-NH2 1-7
FBI-Peptideb RSD-P- K-P 1-5; 6-10; 1-6
Hnge region pepddec P-T-P NS-N2 1-3
Endothdelin fragment 22-38d V-N-T-P 22-30; 31-36
22-25; 26-36
a The reactions were carried out for 60 min in 0.21-ml mixtures containing 2 ,ug of enzyme and 0.5 mg of the peptide in MES (50 mmol/liter, pH 6.5) at 30°C.
Z-Gly-Pro-pNA was tested at 0.2 mmol/liter. The peptide fragments were separated with a PePRP 5/5 column, and the compositional amino acid analysis was performed
after hydrolysis of the fragments. The arrow shows the site of cleavage. All substrates were from Sigma (unless otherwise shown), and all amino acids were in the L-form
(Z in Z-G-P-pNA stands for carbobenzoxy).
b Inhibits the binding of fibronectin to fibroblasts.
'Analog of the hinge region of human IgA2.
d Human big endothelin-1 fragment (Bachem, Philadelphia, Pa.).
suggested to be Asp ... Ser ... His (from N to C terminus) (1). and [I] is the concentration of the modifier (19), resulted in a
Therefore, the modification studies focused on these amino straight line with a slope of 1.1, indicating that an average of at
acid residues and also on the possible presence of a sulfhydryl least one modifier molecule binds to POPase when inactivation
group. occurs. Because hydroxylamine reactivated the enzyme, its
(i) Histidyl residues. When treated with 0.21 to 0.64 mmol inactivation most likely correlated with the carbethoxylation of
of diethyl pyrocarbonate per liter in 20-mmollliter phosphate one histidyl residue.
buffer, pH 6.8 (containing 0.1 mmol of EDTA and 0.25 mol of (ii) Seryl residues. Treatment of the enzyme with 43 to 540
NaCl per liter), the enzyme was inactivated in a time- and ,umol of diisopropyl fluorophosphate per liter in the above
dose-dependent manner. Addition of hydroxylamine (final buffer resulted in 80 to 100% irreversible inactivation of the
concentration, 0.2 mol/liter) to the inactivated enzyme not only enzyme in 10 min. The above plot gave a straight line with a
totally reversed the inactivation, but slightly (10 to 15%) slope of 0.9, suggesting that an average of at least one inhibitor
activated the enzyme. Plot of log(1/t05) versus [I], where tO5 molecule had reacted with POPase. Because dithiothreitol (1.0
stands for the time (in minutes) required for 50% inactivation mmol/liter) failed to reverse the inactivation (thus excluding
the possibility that diisopropyl fluorophosphate reacted with a
cysteinyl residue), it can be assumed that an active-site serine
TABLE 4. Kinetic constants of the hydrolysis of oligopeptides by residue is involved. Benzamidine and phenylmethyl sulfo-
the POPase from T. denticola ATCC 35405' nylfluoride (both at 1.0 mmol/liter) caused only 14 to 15%
inactivation, while aprotinin was without effect at 6 ,umol/liter.
Substrate Km (M) kcat klatIKm Diisopropyl fluorophosphate has been claimed to cause a rapid
(min-') (M-1 min-')
and irreversible inactivation of serine-dependent proteolytic
Z-Gly-Pro-pNA 8.3 x 10-4 6,767 8.15 x 106 enzymes, while phenylmethylsulfonyl fluoride is much less
Bk 9.1 x 10-5 1,078 1.18 x 107 reactive.
SP 1.1 x 10-5 1,286 1.17 x 108 (iii) Carboxyl groups. The enzyme was irreversibly inacti-
Ang-I 2.0 x 10-5 545 2.72 x 107
vated by 1.0 to 4.0 mmol of EEDQ (dissolved in methanol) per
a Each of the peptides was hydrolyzed at only one peptide bond (Table 3). The liter for 60 min at 25°C (tested in the above buffer). The above
enzyme reactions were performed in buffer containing 20 mmol of phosphate plot gave a straight line with a slope of 1.1, suggesting that at
buffer (pH 6.8) and 0.1 mmol of EDTA per liter at 30°C. The POPase least one carboxyl group had reacted with the EEDQ. A
concentration was 7.3 nmol/liter. The peptides were separated on a reversed-
phase column (PePRP 5/5) and identified and quantitated by means of amino protonated form of a carboxyl group is required for EEDQ
acid analysis. modification. The modification of POPase was therefore alsoVOL. 62, 1994 ~~~~~~PROLYL OLIGOPEPTIDASE FROM A SPIROCHETE 4943
0.1 io-A
~~~~MES
BIS-TRIS
0.4 25- ~~pH 6.8
~~~~~BIS-TRIS ~10O 0
PROPANE 0 0.5 1.0
(D
>
B
A40!5 150
_B
Downloaded from http://iai.asm.org/ on May 7, 2021 by guest
~~~~~~Z
(whole cells)
1004
2
0.4 25 -Z (sonicate) 50
1~~~~~SP(hlecls
2 r~
44
>2 (whole cells)
I
BKK(hlecls
0 1
3
23
NaCI Concentration (M)
6 7 8 pH
FIG. 4. Importance of NaCi to peptidase activity. (A) Effect of
5
NaCi at two pH values on the rate of the hydrolysis of 0.2 mmol of
FIG. 3. Effect of pH on POPase-catalyzed reactions. (A) Rate (in Z-Gly-Pro-pNA per liter in phosphate buffer (25 mmol/liter) at 300C.
A405) of the hydrolysis of 0.2 mmol of Z-Gly-Pro-pNA per liter by the (B) Effect of NaCi on the activity of three peptidases from T denticola
purified enzyme in different buffer systems (90 mmol/liter). (B) ATCC 35405. Curve 1, POPase; curve 2, proline iminopeptidase (25);
Hydrolysis of 0.1 mmol of Z-Gly-Pro-pNA (Z) per liter by washed curve 3, peptidase hydrolyzing FALGPA (27). The reactions were
whole cells (curve 1) and by the supernatant fluid of sonicated cells performed at 300C in 0.1 mol of bis-Tris-propane (pH 7.5) per liter
(curve 2) in MES (50 mmol/liter) and the hydrolysis of the Pro-4--Gln-5 with the iminopeptidase and in 0. 1 mol of MES (pH 6.5) per liter with
bond of 0.1 mmol of SP per liter (curve 3), and that of the Pro-7-Phe-8 other enzymes.
bond of 0.1 mmol of Bk per liter (curve 4), by washed whole cells in
MES (50 mmol/liter). The rate of the hydrolysis of SP and Bk was
determined from the size of PepRPC 5/5 peaks SP[1-4] and Bk[1-7],
respectively, and adjusted to the A45 axis. tions of 1 to 10 pLmol/liter (which are normally effective in the
study ofcysteinyl proteases). Also, because no thiols needed to
be added to the enzyme to maintain full activity, it is possible
performed pHs from 5 to 7 (the acid sensitivity of POPase
at that the above strongly inhibitory thiol reagents had reacted
did not allow experiments below pH 5). When the effect of with a cysteinyl residue that is not necessary for catalysis but is
EEDQ (0.75 mmollliter) was tested at 250C in MES (0.1 located close enough to the active site to affect the hydrolysis
mmol/liter) at pHs from 5 to 7, the rate of modification of Z-Gly-Pro-pNA. Such "reactive cysteines" have been re-
increased with decreasing pH, and the plot of kapp versus pH ported to be necessary for substrate binding.
gave a curve with a pK value below pH 5.5, suggesting that the Inhibition by peptides. Among the potentially inhibitory
reagent had reacted with the protonated form of the active peptides were Bk, Bk fragment 1-7, Bk potentiator B, SP and
(carboxyl) group. some of its fragments or modification products, peptides
(iv) Sulflhydryl reagents. The enzyme activity could be derived from TgA2, Ang, and vasopressin (Table 5). The most
effectively destroyed in the presence of typical thiol-s?ecific potent inhibitors were, however, human big endothelin, neu-
reagents. For example, pHMB and Hg2+ even at 10- molt rotensin, neuropeptide Y, and oxidized insulin B-chain. In
liter, caused a strong inactivation of the enzyme, which could general, larger peptides were stronger inhibitors than shorter
be reversed by 2-mercaptoethanol. The reactions were per- peptides. With peptides for which the value of was deter-
formed in 1.0-ml mixtures to which to 10 pxl of HgCl2 solution mnined, the inhibition was of the competitive type (except for
was added. The reaction time was 5 min. After each 5-mmn insulin B-chain). Because of the strong inhibition of the
reaction, resulting in the inhibition shown, addition of 2-mer- POPase by oxiddized insulin B-chain (in which the sulfhydryl
captoethanol to a final concentration of 1.0 mmol/liter imme- groups have been oxiddized), the effect of cysteic acid was also
diately returned the enzyme activity to approximately 90% of tested. The value of 2.14 mM for Kiap reflected low inhibi-
the original level. If left standing for several days at 40C, the tion.'(a
enzyme activity could not be returned. It can be seen in Fig. 5 Elfect of chelators, their analogs, and other inhibitors.
(inset) that pHMB inactivated the enzyme nearly stoichiomet- 1,10-Phenanthroline and its nonchelating homologs 1,7-phen-
rically. Titration with pHMB could thus be used for the anthroline and4,7-phenanthroline were equally inhibitory,
determination of the concentration of the purified POPase. indicating the involvement ofunspecific effects. EDTA had a
Iodoacetamide was much less effective. However, E-64, which stabilizing effect during purification of the enzyme and did not
has been claimed to be absolutely specific for active-site inhibit the purified enzyme. EDTA, 8-hydroxyquinoline sulfo-
(catalytic) sulfhydryl groups, was without effect at concentra- nic acid, and EGTA [ethylene glycol-bis(3-:aminoethyl ether)-4944 MAKINEN ET AL. INFECT. IMMUN.
TABLE 5. Inhibition of the T. den Iticola POPase by oligopeptidesa
Peptide Kiap Ki Action as a
(lfrM) (105 M) substrate'
I
- N.,
~~~~~~Bk 1.6 +
~~~~~~~~Bk[2-7] 16.8 -
50% Bk[1-7]
K
1.7 -
X
250
an I0>
Bk[1-6]
Bk[1-5]
7.4
19.0
-
-
cc [Lys']-Bk 2.3 +
0 2 4 6 Lys-Bk 1.7 +
Moles/L (108) pHMB Ile-Ser-Bk (T-kinin) 0.4 +
Met-Lys-Bk 2.0 +
co des-Pro2-Bk 2.5 +
des-Arg9-Bk 0.9 +
0
des-Arg9, [Leu8]-Bk 1.4 +
1 2 3 4 5 Bk potentiator 6.7 n.t.
Bk potentiator B 1.8 n.t.
Downloaded from http://iai.asm.org/ on May 7, 2021 by guest
Moles Hg2+/L (106) Bk potentiator C 10.1 n.t.
FIG. 5. Reactioj n between Hg2+ and pHMB with a "reactive" SP 3.2 4.0 (C) +
cysteinyl residue o If POPase. Residual activity plotted versus Hg2+ SP[1-9] 2.9 3.5 (C) +
concentration in t]he POPase-catalyzed hydrolysis of 0.2 mmol of SP[1-7] 5.5 10.0 (C) +
Z-Gly-Pro-pNA perr liter in 0.1 mol of MES (pH 6.5) per liter at 30°C. SP[2-11] 7.5 8.3 (C) n.t.
(Inset) Titration off 70 nmol of POPase per liter with pHMB in 20 SP[3-11] 4.1 3.9 (C) n.t.
mmol of phosphate buffer per liter (pH 6.8) with EDTA (0.1 mmoll SP[4-11] 2.9 2.2 (C) n.t.
liter) plus NaCl (0.225 mol/liter) at 30°C. Aliquots (1 ,ul) of 1.0 ,umol of [D-Pro2, D-Trp7'9]-SP 1.5 n.t.
pHMB per liter werre added at 10-min intervals to a 0.1-ml mixture (at IgA2 hinge regionc (Pro-Thr-Pro- 4.9 +
each step, 1.0 pmc)l of pHMB was added). The extrapolated curve Ser-NH2)
(dashed line) gave the value of 0.9 to [pHMB]/[E], where [E] is the FBI peptided 4.1 +
concentration of enizyme. IgGl Fc regione (Leu-Pro-Pro-Ser- 3.5
Arg)
Ang-I 2.7 3.4 (C) +
3.4 4.1 (C) +
N,N,N',N'-tetraa(cetic acid] de facto slightly activated the en- Ang-II 0.5 +
zyme. These findlings suggest that the POPase of T. denticola Neurotensin[8-13] 2.1 +
ATCC 35405 is not a metalloenzyme. Bacitracin inhibits ,-Casomorphin 3.0 nt.
mammalian POP;ases. Under the conditions used for sulfhydryl [Lys8]-vasopressin 1.7 +
reagents (see abowe), bacitracin gave a K( value of 14 uM, [Arg ]-vasopressin 1.3 +
while Zn2+ at 0.2 20 mmollliter inhibited by6P4%, with a Ki( ) Human big endothelin (38 residues; 0.36
of 130 pLM. mol. wt. 4282.9)
Effect of sulfhy,dryl compounds. Both tested sulfhydryl com- Human big endothelin fragment 0.06 +
pounds (dithioth ireitol and 2-mercaptoethanol) slightly acti- 19-38 (mol. wt. 2221.6)e
Human endothelin-1 (mol. wt. 0.05
vated (10 to 15% ) the enzyme. 2492)e
Amino acid secluence. The POPase showed an unblocked N Neuropeptide Y (36 residues; mol. 0.17
terminus. The N- terminal amino acid sequence for the first 24 wt. 4271.7)
residues is M-Q-'Y-K-K-S-D-V-S-D-N-Y-F-G-T-I-V-P-D-?-Y- Insulin A-chain (oxidized; 21 6.7
R-W-L. When tihis sequence was scanned for homology to residues; mol.-wt. 2531.6)
sequences of all Iproteins present in the database CDPROT26 Insulin B-chain (oxidized; 30 0.07 0.11 (NC)
(31,808 sequence s) (37), sequence 4 to 24 showed a relative residues; mol. wt. 3495.9)
1.2 +
score of 71% for Ihomology with sequence 32 to 52 of Flavobac- Insuli B-chain fragment 22-30
terium meningoseipticum POPase precursor (Fig. 6). Sequence
n f-L0L')A snowea
Ah1Z 0Insulin(51residu,
es;r Q Ltf
Insulin (51 residues1 mo. mol. wwt. 5700)
7 0) 0.93
IIlU U Iclose
o1
nomoIogy
h wiln sequenceil 1o 5 oi
a The reactions were carried out in MES (50 mmol/liter, pH 6.5) at 30°C using
Aeromonas hydrophila POPase. A 6.2-kDa fragment of the
Treponema POPase, obtained with CnBr treatment, was also 0.2 mmol of Z-Gly-Pro-pNA per liter as substrate (C, competitive; NC, noncom-
petitive). The molecular weight of some larger peptides is indicated in paren-
checked for homology. Sequence 2 to 41 of this fragment theses. Unless otherwise indicated, the peptides were from Sigma.
showed a 74% score with sequence 525 to 564 of the POPase b Suitability as a substrate of the POPase is indicated, if known (+, acts as a
precursor of F. meningosepticum and even greater homology substrate; -, is not hydrolyzed; n.t., not tested as substrate).
with the sequence 506 to 545 of the POPase of A. hydrophila. Analog of the hinge region of human IgA2; inhibits proteolysis of IgA by
'
Neisseria gonorrheae protease Type I.
A score of 68.9% with sequence 2 to 38 of pig brain POPase d Inhibits fibronectin binding to fibroblasts.
was obtained. Sequencing of the 6.2-kDa fragment of the From Novabiochem (La Jolla, Calif.)
Treponema POPase revealed the amino acid sequence around
the putative active seryl residue to be G-G-S*-N-P-G. A
putative active-site aspartic acid residue was located in this sp. (47), and A. hydrophila (17). The POPases isolated from
fragment (Fig. 6). organisms associated with human diseases display an interest-
ing substrate specificity profile: they hydrolyze certain proline-
DISCUSSION containing HBPs at a high rate. Although the role of POPases
in bacterial inflammations remains to be determined, the
The only bacterial POPases more thoroughly studied have possibility exists that microbial POPases have developed simul-
been obtained from F. meningosepticum (58), a Xanthomonas taneously with the human POPases to carry out functions thatVOL. 62, 1994 PROLYL OLIGOPEPTIDASE FROM A SPIROCHETE 4945
A
24 T-POP
29 52 F-POP
9 32 A-POP
I
B
2 KQtMF~45 T-POP 6.2 kDa
525 ITI M 568 F-POP W
506 ~~~~~~~~~~~~~~~~~~~~A
~~~~~~~~~
~ K E YF T D RL AR ~ ~ ~~ #~ M TO 549 A-POP
QiGt,V
523 Q AAY KEGCTPK RL NT1N CNG ,V A T C ANWQ 566 P-POP
Downloaded from http://iai.asm.org/ on May 7, 2021 by guest
FIG. 6. Homology between POPases from different sources. Alignment of the N-terminal segment (A) and the segments around the active-site
areas (B) of T. denticola POPase (T-POP) with segments of POPase from F. meningosepticum (F-POP) (61), A. hydrophila (A-POP) (17), and pig
brain (P-POP) (41). The putative active-site aspartic acid residues have been circled. The active seryl residues have been marked with an asterisk
(for the 6.2-kDa T-POP fragment, the involvement of the corresponding seryl residue in enzyme activity remains to be vindicated). The sequencing
studies of the 6.2-kDa fragment provided inconclusive results for residue 24 (which is either R or S), for residue 36 (which is most likely G), and
for residue 43 (which is most likely V). Residues identical to those of T-POP are shaded.
are necessary for the perpetuation of the pathogen in human amino acid sequence around the active-site serine residue
habitats. Such a functional adaptation to the host environment closely resembles that of POPase from F. meningosepticum, A.
may enable the enzyme to escape from being recognized by the hydrophila, and pig brain. Third, the mol. wt. and the pH
host's immune system as nonself (18) and consequently avoid optimum of the T. denticola peptidase were similar to those of
inactivation. Such bacterial functions may be pathogenic to the POPases. Furthermore, the sequence of N-terminal amino
host. This study demonstrated the presence in the cells of T. acid residues 4 to 24 showed >70% homology with residues 35
denticola ATCC 35405 of a POPase which is active on proline- to 52 of the F. meningosepticum POPase precursor (61), and
containing HBPs. On the criteria of purity presented above, residues 10 to 24 showed a close similarity with sequence 18 to
the T. denticola POPase was judged to be homogeneous, 32 of A. hydrophila POPase (17).
although the enzyme yield was regarded as somewhat unsatis- The T. denticola enzyme contains at least three Cys residues,
factory. The lower yield should be considered against the fact of which one reacts stoichiometrically with pHMB. It is
that in purification steps involving hydrophobic-interaction possible that this reactive Cys residue is necessary to confer
FPLC, the interactions that maintain protein conformation are conformational changes upon substrate binding. Previous lit-
also those that mediate retention. Therefore, the FPLC itself erature suggests that the Flavobacterium (58) and the Xantho-
can cause partial or even complete unfolding of a protein. On monas (47) POPases are not inhibited by sulfhydryl reagents.
the other hand, this FPLC step is sensitive enough to separate Consequently, the Treponema enzyme may be considered to
the pure native protein from other forms (Fig. 1B). The resemble in this respect more closely the mammalian POPases
POPase is an example of "proline-specific peptidases" (this which show susceptibility to pHMB (52). The POPase gene of
term implies that the peptidase requires an imino acid residue eukaryotes may have evolved further, with the introduction of
at or near the scissile bond). A typical proline-specific enzyme an additional Cys residue near the active site.
is proline aminopeptidase (EC 3.4.11.5), which is remarkably The biological role of the POPase must be closely associated
active in cells of T. denticola (25), suggesting that it plays an with the location of the enzyme in the cells. The T. denticola
important role in the propagation of this organism. A specific POPase may be regarded as a bacterial "ectopeptidase,"
function may also be ascribed to the FALGPA-peptidase, because quite low detergent levels liberated the enzyme virtu-
which acts on Bk and small collagen fragments (27) and ally entirely. Triton X-100 is known to remove the outermost
requires the presence of a proline residue at the P'2 position of layer of the cells of T. denticola (5), this structure being the
the substrate, which is hydrolyzed. The treponemal proline- source of several peptidases. The ability of intact, washed cells
specific peptidases, including the POPase, seem to be cell to bring about the rapid hydrolysis of Z-Gly-Pro-pNA, SP, and
associated, but so located (in the outer membrane?) that they Bk with enzymatic characteristics similar to those of the
can readily inactivate HBPs present at the site of inflammation. purified POPase (to be reported) must be emphasized. The
It is thus possible that the proline-specific peptidases are enzyme indeed displayed high affinity for Bk, SP, Ang-I, and
important in the chemical aggressiveness of the treponemes. others. It is possible that the POPase interferes with the
Based on the evidence presented in this paper, the T. normal balance of such proline-containing peptides, thereby
denticola peptidase can be considered to belong to the POPase contributing to the maintenance of the inflammatory condition
family of serine proteases (40). This proposition is primarily in periodontal infections. For example, fragment SP[5-11],
based on the strict specificity of the peptidase for a proline which is produced by the POPase from intact SP, has been
residue in the P1 position of peptides ranging from tetrapep- found to be biologically more active than SP itself (56).
tides to about 3-kDa oligopeptides. Second, the chemical Furthermore, the POPase can also serve as an ancillary
modification studies suggested the involvement of an active Ang-I-converting system, because the T. denticola peptidase
seryl residue, an active histidyl residue, and an active carboxyl also liberated fragment 1-7 from the intact Ang-I. This frag-
group, which form the catalytic triad in the POPase family; the ment is biologically active (52). Therefore, although it is4946 MAKINEN ET AL. INFECT. IMMUN.
possible that the hydrolysis of proline-containing HBPs by the 8. Ferris, G., T. E. Grow, S. B. Low, and R. T. Ferris. 1987.
POPase is incidental, with no pathological meaning, the Measurement of gingival crevicular fluid conductivity, in vivo. J.
POPase is expressly an oligopeptidase with a strict specificity, Periodontol. 58:46-50.
not a protease. The enzyme showed too low affinity constant 9. Goldhaber, P., and D. B. Giddon. 1984. Present concepts concern-
values for several proline-containing HBPs to be categorically ing the etiology and treatment of acute necrotizing ulcerative
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10. Grenier, D. 1992. Nutritional interactions between two suspected
substrate binding. The cells of T. denticola possess the POPase periodontopathogens, Treponema denticola and Porphyromonas
for a purpose, and it is conceivable that the natural substrates gingivalis. Infect. Immun. 60:5298-5301.
of this enzyme include natural, proline-containing HBPs. Fu- 11. Grenier, D., V.-J. Uitto, and B. C. McBride. 1990. Cellular location
ture research must elucidate the possibility of collagen frag- of a Treponema denticola chymotrypsinlike protease and impor-
ments and salivary proline-rich peptides acting as potential tance of the protease in migration through the basement mem-
substrates of the POPase. It is noteworthy that peptides can brane. Infect. Immun. 58:347-351.
replace free amino acids as preferential growth substrates of T 12. Haapasalo, M., V. Singh, B. C. McBride, and V.-J. Uitto. 1991.
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The reason for larger peptides being inhibitors and not nin and other proteins. Infect. Immun. 59:4230-4237.
13. Hersch, L. B. 1981. Immunological, physical, and chemical evi-
substrates may be the strict spacial geometry of the active site dence for the identity of brain and kidney post-proline cleaving
of the POPase. The active site may have to accommodate the
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entire scissile peptide (VOL. 62, 1994 PROLYL OLIGOPEPTIDASE FROM A SPIROCHETE 4947
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