Vol. 178, No. 1, 1991 July 15, 1991

BIOCHEMICAL

CHARACTERIZATION

OF PORPHY

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 336-342

ROMONAS

HEMAGGLUTININ

Makoto ICentral Sapporo

Received

May

Nishikatal

(~ACTEROIDES)

GINGIVALIS

AS A PROTEASE

and Fuminobu

Yoshimura2

Research Division, School of Dentistry, Hokkaido University, 060 and *Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya 464, Japan

23,

1991

A hemagglutinin (HA) was purified to homogeneity from the membrane fraction of the oral bacterium Porphyromonas gingivalis. The HA possessed protease activity hydrolyzing proteins and arginine-containing synthetic subby thiol-blocking reagents, and The protease activity was inhibited strates. The HA functions as hence the HA can be characterized as a cystein protease. an attachment factor and its substrate-binding site is responsible for the attachment to an erythrocyte. B 1991 Academic Press, Inc.

Porphyromonas unique but

hemagglutinin is

valis

by

antipain

and

inhibited HA

fraction

by and

participates have

and

leupeptin. protease

a purified

to

which,

is

inhibited

that

the

direct

lectins,

that by

binding therefore

binding

periodontal is

pathogen not

arginine-containing

demonstrated

activities

obtain

putative unlike

and

We

in erythrocyte

attempted

by using

(4)

TLCK,

a

arginine

investigation membrane

both

(HA)

inhibited

previous

is

gingivalis,

and

peptides

of

inhibitors

the

bacterial

speculated

that and

evidence

inhibited

hemagglutination

protease

the

that

that

same

substrate p.

(l),

site

by

sugars

(2,3).

Our

by

the

such

as

cells

to

the

1.

gingi-

leupeptin,

erythrocytes

E, gingivalis of

has

HA

HA

has

molecule

binding.

In this

study,

gingivalis

HA

a protease

is

we

HA preparation.

Abbreviations: HA, hemagglutinin; 2-ME, 2-mercaptoethanol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate; TLCK, Tos-Lys chloromethyl ketone; ALCK; AC-Lys chloromethyl ketone: Bz, benzoyl; Z, benzyloxycarbonyl; Boc, tbutyloxycarbonyl; Tos, tosyl; AC, acetyl; pNA, e-nitroanilide; MCA, 4-methylcoumaryl-7-amide. OOCS291W91

Copyright All rights

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

336

Vol.

BIOCHEMICAL

178, No. 1, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Assay of HA: Serially twofold-diluted samples in trisodium citrate and 0.15 M NaCI, pH 7.5) containing with an equal volume of 0.5% (v/v) chicken erythrocyte a titer plate with U-bottom wells. The mixture was at room temperature. The titer was expressed as the dilution showing hemagglutination.

0.5 ml of SSC (15 mM 4 mM 2-ME were mixed suspension in SSC using left standing for 90 min reciprocal of the highest

Assay of Protease: Unless otherwise stated, protease activity was assayed with Bz-Arg-pNA as the substrate. A sample was mixed with 1 ml of 50 mM Tris-HCI (pH 8.5) containing 50 mM 2-ME and 0.2 mM Bz-Arg-pNA. After an appropriate incubation time at 25OC, 0.2 ml of 50% acetic acid was added and the increase in absorbance at 410 nm was measured. Purification of HA: The membrane fraction (wet) of E. gingivalis 381 prepared as described previously (5) was homogenized with a Teflon-pestle homogenizer in 50 volumes of 20 mM phosphate buffer (pH 7.3) containing 1% CHAPS, 25 mM 2-ME and 5 mM methylguanidine. The homogenate was incubated for 2 h at 40°C to effect solubilization and centrifuged at 110,000 x g for lh. After the supernatant had been incubated for 30 h at 40°C to dissociate HA aggregates, HA was purified from this supernatant (10 ml) by sequential steps of chromatography consisting of Bio-Gel A-0.5m (Bio-Rad) gel filtration (Fig. lA), DE-52 (Whatman) ion-exchange chromatography (Fig. 1B) and arginine-cellulose affinity chromatography (Fig. 1 C). The affinity adsorbent was prepared by coupling of arginine methyl ester with CM-52 (Whatman) using a water-soluble carbodiimide followed by trypsrn treatment for the removal of the methoxy group.

RESULTS Purity cellulose ular

of HA:

affinity

mass

SDS-PAGE

column

of 44 kDa

of the

(Fig.

under

HA

1C)

gave

reduced

and

preparation

a single

obtained

band

non-reduced

from

with (not

an

an arginine-

apparent

shown)

molec-

conditions

(Fig.

2). Attempt Fig.

1C shows,

lyzing of

the

HA To

Bio-Gel

A-0.5m

ciative

synthesis

could

protease

activity

was

accompanied

learn

whether in

in the

conditions.

The

properties

presence

tried

to HA

irreversible this

activities:

As the

by the

protease

purified

HA

from also

failed

separate

the

preparation

was

protease

compound 337

will

(Fig.

due the

3).

The

to separate two

published

with

a trace prepara-

HA

and

filtration the

activities

treated

HA

of hydro-

to

Gel

other.

inhibitor be

activity

was

preparation,

each

chromatogram

protease

activity

of CHAPS

separated

purified

of

the

of CHAPS

we

site-directed and

be

presence

Next,

the

in the not

shown).

active

and

by PAGE

activities

(not

HA

protease

was analyzed

protease

itie

separate

Bz-Arg-pNA.

contaminating

tion

tive

to

the on

two

activ-

under

disso-

the

[AC-3HlALCK elsewhere)

radioac(the and

Vol.

BIOCHEMICAL

178, No. 1, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

u Purification of P. gingivalis HA. (A) Gel filtration on a Bto-Gel A-0.5m column (3.1 x 74-cm). (B) Ion-exchange chromatography on a DE-52 column (1.0 x 6.4 cm). (C) Affinity chromatography on an arginine-cellulose column (1.0 x 6.4 cm). Elution buffer: (A) 20 mM phosphate buffer (pH 7.3)/ 0.1% CHAPS/25 mM 2-ME/l mM methylguanidine; (B and C) the same as in A except that methylguanidine was excluded. Fraction volume: (A) 20 ml; (B and C) 4 ml. All chromatography steps were done at 4OC. Fractions were assayed for HA activity, protease activity (lo-p1 aliquot, IO-min incubation) and protein content (estimated from fluorescence of tryptophan). Fractions pooled are indicated by the bar. The first HA peak in A seemed to contain aggregates and therefore was not fractionated further.

subjected

to

stained

band

urement

of

taining

3%

SDS-PAGE.

As

were

detected

excised

fractions

Protosol

rated

exclusively

strate

that

the

and the protease

Fig. at

the

of

(DuPont-NEN)

into

the

4 shows, same

the

position.

stained

gel

confirmed

stained

protease

activity

is

activities

are exhibited

and

radioactivity

that

Direct suspended the

band

(not

shown).

not

due

to

by the same 338

a Coomassie radioactivity in

radioactivity

These

contamination,

molecule.

bluemeas-

Econofluor was

resu I ts i.e.,

conincorpo-

demonthe

HA

Vol.

178,

No.

BIOCHEMICAL

1, 1991

AND

COMMUNICATIONS

kDe 66-e

i

*i

45Jo-

02

RESEARCH

r=

kDa 94BB-

BIOPHYSICAL

HA

Protsase

I

0

SDS-PAGE of buffer system with 5% 2-ME.

1)

30-

I I I 1I 1’ ’

Act.

BPB-

FicLz. Laemmli reduced

45--.

Act.

(A,,,)

purified HA. The electrophoresis (10) in 12.5% polyacrylamide The gel was silver-stained.

E&J-.& PAGE of purified HA. The using the Davis buffer system (11) presence of 0.6% CHAPS. One lane R-250. The other was excised (5 mm) a spatula in 0.5 ml of 50 mM Tris-HCI incubation of the suspension at 4OC assayed for HA activity and the whole tease activity (30-min incubation).

0.1

slab

a

0 4

was performed gel. The

using sample

the was

electrophoresis was performed in duplicate in 5% polyacrylamide slab gel in the was starned with Coomassie brilliant blue and each gel fraction was pulverized with (pH 8.5) containing 50 mM 2-ME, After for 18 h, the supernatant (100 ul) was of the remainder was assayed for pro-

Fia. 4. SDS-PAGE of [Ac-3H]ALCK-treated HA. The purified HA, after removal of methylguanldine by dialysis, was incubated with 50 uM [AC-~H]ALCK at 25OC for 30 min in 20 mM phosphate buffer (DH 7.3) containina 0.5% CHAPS and 1 mM The incubation inactivated protease activity by 94%. The reaction mix2-ME. ture was dialyzed against dilute HCI and tyophilyzed. SDS-PAGE was performed One lane was stained with Coomassie blue (a) and in duplicate as in Fig. 2. the other was subjected to fluorography (b).

Effects protease est

of

99%

at

0.1

it

by

81%

though

protease

inhibitors

ethylmaleimide

of

the

the the 0.04

Salmine

FM.

activities,

amino

compounds

with

and

Benzamidine,

enzyme

Of inhibited

(measured

activity +O.

Various

activities.

inhibitor:

by

inhibitors:

the

activity

protease

much

more

TLCK and

inhibitor

(7)

after

activity

by

and

ALCK,

other

two

activities

than

arginine by

TLCK

at

and

The

iodoacetamide

and

was

the

investigated

339

was

either with

the

pg/ml

activity at

0.2

HA -

inhibited

0.4 both

irreversible reagents

activities.

The

inhibitory.

Sugars

activity. various

25OC)

the

(roughly also

and

strong-

inhibited

thiol-blocking

not

HA

protease

site-directed

both

inhibit

the

preincubation

arginine

the

was not

2

active

inactivated

did

and lo-min

69%

agmatin weakly.

(6)

polypeptide,

diisopropylfluorophosphate

acids

98%

arginine-rrch

methylguanidine,

both

leupeptin

by

Bz-Arg-pNA an

inhibited

examined,

HA

mM

sulfate,

compounds

iserine

Inactivation TLCK

concentra-

and

b

Vol.

178,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

TLCK Fia. 5. Inactivation purified HA, after TLCK at 25OC for and 5 mM 2-ME. bly, TLCK reacted

As

tions.

Fig.

increasing

5

TLCK

least

the

the

tin

to

of

a

HA

these

in in

the

the

protease

37OC

with

found

to functrons

the

sole

site

as for

investigated

substrate

to

an

the

hydrolyzed (Bz-Arg-OC2H8)

100

80%

FM)

could

and of

HA

and

can

be

or

at

was wells

the

leupepof

the

(&fold inhib-

showed was

(6-day

total

its

of

strongly

that

negative chosen

and

incubation

protease

findings

that

detected

hemagglutination

measured of

be

resuspension

concentration

These

factor

HA

Addition

the

was

erythrocytes.

at

activity

demonstrate

was that

substrate-binding

the

site

is

attachment.

substrates

small

activity

From

About

the

with

activity,

hemagglutination

highest

parallel

hemagglutination.

collapse

supernatant

attachment

erythrocyte

with HA

the

the

leupeptin.

Bz-Arg-pNA).

of

of

The with CHAPS Proba-

in

catalytic

protease

above,

with

its

that

hemagglutination.

in

uM

well

unattached

Hydrolysis

ester

0.1

in

mM

No

concentration

of

indicate

that for

showing

described

decreased

results

is essential

wells

As

activity

activities

and

resulted

the

be

HA

the

the

(final

presence

0.5

two

protease site,

titer).

hemagglutination,

1,

cystein

of wells

(nM)

These

erythrocytes

decrease

the

in hemagglutination:

supernatant

agglutinated

ited

shows,

substrate-binding

Role in

clearly

as

COMMUNICATIONS

of HA (0) and protease (0) activities by TLCK. removal of methylguanidine by dialysis, was incubated 1 h in 20 mM phosphate buffer (pH 7.3) containing 0.5% Longer incubation did not cause further inactivation. with 2-ME as well as with the HA.

concentrations.

characterized

RESEARCH

by HA:

synthetic only

The

substrates

those was

synthetic hydrolyzed

340

catalytic and

properties proteins.

substrates much

of As

containing more

slowly

the

shown

HA

were

in

Table

arglnine. than

The the

amide

Vol.

178,

No.

1, 1991

BIOCHEMICAL

Table

1.

Hydrolysis

AND

BIOPHYSICAL

of synthetic

RESEARCH

substrates

Substrate

by purified

Hydrolysis

Bz-Arg-pNA Z-Arg-pNA Bz-Arg-MCA Tos-Gly-Pro-Arg-pNA Bz-Arg-OC2H5

COMMUNICATIONS

HA

rate

100 45 111 155 1.6

Hydrolysis not observed: glutaryl-Phe-pNA, Bz-Tyr-pNA, succinyl-(Alafg-pNA, Z-Gly-Pro-pNA, Bz-DL-Lys-pNA (0.08 mM), Tos-Gly-Pro-Lys-pNA, Boc-Val-Leu-Lys-MCA Substrates (0.04 mM) were incubated with the purified HA at 25OC in 50 Tris-HCI (pH 8.5) containing 50 mM 2-ME. Hydrolysis was followed by continuous monitoring of the change of absorbance at 410 nm (e-nitroanilide), 370 nm (4-methylcoumaryl-7-amide) and 253 nm (Bz-Arg-OC2H5). The rate of Arg-pNA hydrolysis was assumed to be 100.

substrates.

Proteins

bovine

such

fibrinogen

hydrolysates

were

(not

as

also

bovine

serum

hydrolyzed

albumin,

as

chicken

demonstrated

mM the Bz-

ovalbumin

by

and

SDS-PAGE

of

the

shown).

DISCUSSION In -lis

this

381

work

and

we

found

it

arginine-containing lation This

(4).

Vibrio

HA,

a

is

cytes

via

a its

The

(nii)

treatment loss

the of

of

of the

culture

and

the

to

is

hydrolyzes with

as

supernatant

a

per

is

di-

by

(i)

arginine

lnoshita

contact of

residue 1.

gingivalis

341

in

to

erythro-

results

to

et between 381.

al.

(2)

and

related

erythrocytes It

is

to com-

and an

the

binds

(iv)

appreciable

suggested and

not

deter-

with HA

also

or

be

the

in

con-

multimeric,

substrates,

deiminase

of

HA,

interacts

because

inhibited

or

remains

specifically

arginine,

arginine

characteristics

factor

molecule

specuactivity.

Our

arginine-containing

(unpublished).

arginine

surface

HA

and

protease

attachment the

gingiva-

previous

(8).

an

that

be

with

change

Whether

erythrocyte

HA

erythrocytes

as

site

seems

HA

f.

proteins

our

autoaggregate

serves

of

hydrolyze

confirmed

to

to

fraction

to

an

assumed

them

an

HA

agglutinability

importance from

(ii)

able

produces

site.

on

arginine-cellulose, pounds,

is

membrane

which

substrate-binding

site

the

protease

also

protease

residue

substrate-binding

from

substrates,

allowing

one

HA a

substrate-binding

than

mined.

be

metalloprotease,

cystein

more

an

cholerae

thereby

trast,

to

synthetic

erythrocytes,

has

purified

known

the

their whether

HA

Vol.

178, No. 1, 1991

our

HA

for

hemagglutination.

HA

wrth

hydrolyzes

erythrocyte This

reagents

cyte-binding

of p.

BIOCHEMICAL

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

proteins should

be

inactivate

or

peptides

verified

protease

as a prerequisite

by chemical activity

action

modification

without

altering

of

the

erythro-

ability.

Recently,

Li

et

gingivalis

to

Actinomvces

It is possible

that

al.

the

(9)

HA

viscosus obtained

activity-possessing

HAS

part,

of the two

in the cohesion

in

demonstrated

partial by

in the

protease

of

inhibitors

present if

_P. gingivalis,

inhibition

work

any,

and

the

adherence

such

as TLCK.

other

protease

participate,

at

least

in

bacteria.

ACKNOWLEDGMENTS

This from

the

work Ministry

and the Akiyama

was of

supported Education,

in part

by

Science

Grants-in-Aid and

for

Culture

of

Scientific

Japan

(No.

Research 63570865)

Foundation. REFERENCES

1. 2. 3. 4. 5. 6. 7.

Slots, J., Bragd, L., Wikstrom, M., and Dahlen, G. (1986) J. Clan. Per iodontol. 13, 570-577. lnoshita, E., Amano, A., Hanioka, T., Tamagawa, H., Shizukuishj, S., and Tsunemitsu, A. (1986) Infect. Immun. 52, 421-427. Okuda, K., Yamamoto, A., Naito, Y., Takazoe, I., Slots, J., and Genco, R.J. (1986) Infect. Immun. 54, 659-665. Nishikata, M., Yoshimura, F., and Nodasaka, Y. (1989) Microbial. Immunol. 33, 75-78. Yoshimura, F., Nishikata, M., Suzuki, T., Hoover, C.I., and Newbrun, E. (1984) Archs Oral Biol. 29, 559-564. Aoyagi, T., Miyata, S., Nanbo, M., Kojima, F., Matsuzaki, M., Ishizuka, M., Takeuchi, T., and Umezawa, H. (1969) J. Antibiotics 22, 558-568. Shaw, E., Mares-Guia, M., and Cohen, W. (1965) Biochemrstry 4, 221g2224.

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9.

Booth, B.A., Sciortino, C.V., and Finkelstein, Lectins and Agglutinins (D. Mirelman, Ed.), Sons, Inc., New York. Li, J Ellen, R.P., Hoover, C.I., and Felton, 70,

10. 11.

R.A. (1986) In Microbial pp.169-182. John Wiley J.R.

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Laemmli, V.K. (1970) Nature Davis, B.J. (1964) Ann. N.Y.

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342

(1991)

J.

Dent.

&

Res.