Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis

Vol.

174,

No.

January

2,

BIOCHEMICAL

1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

31, 1991

625-630

SALIVARY HISTATIN AS AN INHIBITOR OF A PROTEASE PRODUCED BY THE ORAL BACTERIUM BACTEROlDES GINGlVALIS Makoto

“Oral

Nishikatal, Takashi KanehiraZ, Hourei Oh”, Hiroshi Mariko Tazaki”, and Yoshinori Kuboki”

‘(:entral Research Division and Departments of 2 Preventive Biochemistry, School of Dt>ntistry, Hokkaido University,

Received

December

12,

Tani2,

Dentistry Sapporo

and .Japan

060,

1990

examined the effect of histatin 5 from human parotid saliva on various Histatin 5 strongly inhibited a trypsin-like protease produced by Hacteroides gingivalis with an I(:50 value of 55 nM. Clostripain was also inhibited (lC50=800 nM). Activities of other proteases were not affected significantly. Because B. gingivalis is a suspected periodontal pathogen and its proteolytic enzymes have been considered to be associated with periodontal tissue destruction, it is suggested that salivary histatins play a role as a preventive against periodontal disease. 0 1991 Rcademlc fre5.5, Inc. We

proteases.

Histatins unusually acid

arc

high

residues

a

family

content in

of

salivary

of hjstidinc

histatin

characterized

polypeptides

residue,

which

comprises

saliva

activities

( 1).

and

a histamine

ical

role

In this examined strong suspected

Although

against

study, the

histatins

releasing cavity

to obtain effect

inhibitor

of

Abbreviations: Bz, thiobenzyl ester: aminoethanesulfonic propanesulfonate.

antibacterial

mutans

and

29% of the

from

remains

to be elucidated.

further histatin

a pathogen

rat

information 5 on from of adult

various

of

Lho

(2)

and

Candida

activity

of a protease

of being

one

have

Streptococcus

in the oral

their amino

5 (Asp-Ser-flis-Ala-Lys-Arg-His-His-Gly-Tyr-l,ys-Arg-

Lrs-P~~e-His-Glu-l,ys-His-His-His-Ser-t~is-Ar~-Gl~-‘~yr), in

hy

mast

ceils

on the

(41,

periodontal

Eingivalis, disease

(3)

respectively, their

function and

histatins

antifungal

albkans,

proteases

Bacteroides

ma.jor

true

of

histatins,

found an

biolog-

oral

it

to

we he

bacterium

(5).

benzoyl; Z, henzyloxycarhonyl; pNA, pnitroanilide; OEt, ethyl ester: TES, N-trisf hydroxymethyl)methyl-Zacid: CHAPS, 3-l (3-cholamidopropyl)dimethyiammonio]-l-

SBzl,

a

Vol.

174,

No.

2, 1991

BIOCHEMICAL

MATERIALS

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

Materials: The following enzymes were obtained as indicated: bovine chymotrypsin (type Il), porcine elastase (type I), human thrombin (3313 r&l units/mg protein), papain (twice crystallized) and clostripain (132 units/mg protein) (Sigma, St. Louis); Flavobacterium meningosepticum prolyl endopeptidase (Seikagaku Kogyo, Tokyo); Clostridium histolyticum collagenase (CLSPA grade) (Worthington, Freehold); bovine trypsin (Boehringer, Mannheim). Preparation of Histatin 5: reported elsewhere. Briefly, separated by hydroxyapatite urea. Fractions containing a into individual components by the main peak was found to this study. Its concentration

The detailed purification procedure will be pooled parotid saliva from healthy adults was column chromatography in the presence of 6 M mixture of his&&ins were pooled and separated reversed-phase HPLC. The component eluted as be histatin 5 by sequence analysis and used in was determined by amino acid analysis.

Purification of B. gingivalis Trypsin-Like Protease: The whole envelope fraction of B. gingivalis 381 prepared as described previously (6) was homogenized with a Teflon homogenizer in 20 mM phosphate buffer (pH 7.3) containing 1% CHAPS, 25 mM 2-mercaptoethanol and 5 mM methylguanidine. Methylguanidine was added to prevent autodigestion of the protease. The homogenate was centrifuged (110,000 x g, 1 h) and the supernatant was subjected to gel filtration (all chromatography done at 4°C) on a column of Bio-Gel A-0.5m (Bio-Rad) equilibrated with the same buffer as above except that CHAPS and methylguanidine concentrations were 0.5% and 1 mM, respectively. Elution with this buffer was monitored by measuring the enzymatic activity toward 0.2 mM BzArg-pNA at pH 8.5. Pooled active fractions were then applied to a column of DE-52 (Whatman) equilibrated as above. Active fractions eluted by NaCl linear gradient elution were pooled, diluted J-fold and subjected to the same DE-52 column chromatography. The overall yield of the activity was 58%. SDSpolyacrylamide gel electrophoresis of the fraction with the highest activity gave a single but broad protein band around 85 kDa under reducing conditions. This fraction was used in this study after removal of methylguanidine by dialysis. The purified protease, like trypsin, hydrolyzed only those synthetic substrates containing arginine or lysine. The arginine-containing substrates were hydrolyzed more rapidly than the corresponding lysine analogs. The Km value for the hydrolysis of Bz-Arg-pNA by this enzyme was determined to be 1.9 ,Y M at pH 8.5 and 25°C. Other enzymatic properties were essentially the same as those reported for the partially purified protease from B. gingivaljs (6). Since our purified protease resembles trypsin in cleavage specificity, we will refer to this protease as ‘trypsin-like protease’ in this report. Assay of Protease: Protease activity was measured with specific synthetic substrates (6-14). Hydrolysis of substrates was started by addition of 5 ,U 1 of an enzyme solution to 0.5 ml of thermostated (25 “C) 50 mM TES-NaOH (pH 7.5) which contained a substrate and, if necessary, histatin 5. In addition, the assay mixture contained 10 mM CaClz (collagenase assay), 1 mM dithiothreito1 (papain and clostripain assays), 5-5’-dithiobis(2-nitrobenzoic acid) (trypsin and thrombin assays) and 50 mM 2-mercaptoethanol (B. gingivalis trypsin-like protease assay). Papain and clostripain had been activated by 10 mM dithiothreitol before added to the substrate solution. Hydrolysis was then followed at 25°C by continuous monitoring of the change of absorbance at 410 nm (p-nitroanilides), 412 nm (Z-Lys-SBzl), 324 nm (furanacryloyl-Leu-Gly-Pro-Ala) and 254 nm (Be-Tyr-OEt). Enzyme concentration was estimated by normality titration (trypsin and CL-chymotrypsin) (15, 16), spectrophotometrically (elastase and papain) or on the weight basis assuming that the enzymes are pure (thrombin, prolyl endopeptidase, collagenase and clostripain). 626

Vol.

174,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS Effect

of histatin

5 on

initial

rates

of substrate

(Fig.

1).

The

B.

Obviously

substrate

from

Fig.

10 ,u M histatin plete.

5.

almost

protease 1, the

B.

was

investigated

presence

about

Km for

0.1

protease

gingivalis

of clostripain

was

inhibited.

were

was also Other

comparing

by

absence

and

employed

and

moderately

was

of histatin

about the

0.5

other

inhibited

5

Km

for

proteases.

completely

remarkable

but

protease

activities

by

not

comwere

unaffected. The

inhibition

of the

as

a function

investigated 1 uMM, histatin The

al.

in the

concentrations

Inhibition

Collagenase

proteases

hydrolysis

trypsin-like

gingivalis

various

5 could

concentration

B.

of inhibit

of

protease

gingivalis

histatin

histatin the

B.

and

clostripain

5 concentration

(Fig.

further Even

2).

protease

completely

the

protease

activity

02

0

gingivdis

5 at which

was

at

(curve is

half

of

Trypsln Thrombln Chymotrypsi

n

Elastase Proly1 endopept

idase

Collagenase Papa

in

Clostripain B.glngivalis protease

01

0

ACTIVITY50

Fig.

1. Effect of histatin presence of 10 WM histatin absence of histatin 5. as follows: trypsin (0.26 (4fiM); o-chymotrypsin succinyl-Ala-Ala-Ala-pNA pNA (0.1 mM); collagenase papain (69 nM)/Bz-Arg-pNA B. gingivalis trypsin-like (1 PM). Fig. 2. Inhibition of clostripain (c) by histatin was assumed to be 100. (b), and 80 ,uM (c).

100

0.01

HISTATIN 01

15 (PM)

Protease activities in the 5 on various proteases. 5 are shown as percentages of the activity in the Protease/substrate pairs and their concentrations are nM)/Z-Lys-SBzl (5 ,uM); thrombin (3.0 nM)/Z-Lys-SBzl (3.2 nM)/Bz-Tyr-OEt (0.26 mM); elastase (0.17 ,uM)/ (0.1 mM); prolyl endopeptidase (9.2 nM)/ Z-Gly-Pro(0.1 fi M)/2-furanacryloyl-Leu-Gly-Pro-Ala (50 ,U M); (0.3 mM); clostripain (8.4 nM)/Bz-Arg-pNA (80 KM); protease (concentration not determined)/ Bz-Arg-pNA gingivalis Protease The concentration

B.

5.

trypsin-like protease (a and b) and activity in the absence of histatin 5 of Bz-Arg-pNA is 1 UM (a), 10 tiM

627

10

Vol.

174,

the

No.

2, 1991

uninhibited

ICso

value

Values

value for

the

of 55 nM

(0.53

Km) and

clostripain

(lC50) B.

(5.3

from

protease 200

Km)

estimated

BIOPHYSICAL

estimated

a) and

10 ,uM

AND

was

gingivalis

(curve

was

(curve

BIOCHEMICAL

to

(curve

Bz-Arg-pNA, be

800

the

varied

nM

RESEARCH

curves

with b)

in

with

were

80

Fig.

substrate

The

with

The

,uM

2.

concentration.

obtained

respectively.

nM

COMMUNICATIONS

(0.1

IC5o Km)

1 ,uM

value

for

Bz-Arg-pNA

c).

DlSCUSSlON Although 4),

little

In

the

several

is

known

present

study,

we

examined

found

that

histatin

tease

with

an

u M, which bition the

most The

to

be

tion.

type

not

activity of inhibition

according and

the

taken values

is

protease

for

properties. cleavage

example, specificity

arginine

over

on

the

molecular

proteases,

and

trypsin-like

gingivalis

IC5o

to experimental

pro-

values

concentrations

in this

(2-

specifically.

various

Lower

2).

observed

B.

would well

study

be

below

difficulties.

equation

The

seems

Ki

on

to

1

inhi-

be

far

so

histatin were

histatin

histatin

concentra-

(Ki)

the

lC50

V are

the

using

where

v and

hydrolysis,

respectively,

5 concentration. calculated

to

5 appears

substrate

constant

substrate

200 nM,

because

lysine

of

values

similar

dependent

of inhibition

I is

both

was

by

~=VS/lStKm(ltl/Ki)l,

rates and

protease

gingivalis

value

values

that

clostripain, For

lC50

of 55 nM and

noteworthy

and

for

Km.

histatins

at substrate

owing

of the

maximum

concentration

IC50

It

to the

B.

reported

of histatin.

to determine

,YM was

done

been interact

5 and

of

(Fig.

done

protease

We tried

Bz-Arg-pNA

erence

was

of

histatin

55 nM

have

histatins

functions

inhibitor

was

because

observed

strict

of

competitive

values

using

value

gingivalis

pot,ent

molecule(s)

between

experiment

B.

of histatins

to understand

5 is a strong

actually

of the

what

interaction

IC50

if the

activities

as to with

basis,

obtained

biological

be

36

S is

A value

of

1.9

nM

32

nM

and

respectively. 5

inhibits

these

two

enzymes

are

to that (6, 17).

628

enzymes

8.

share

sulfhydryl-dependent

of trypsin

trypsin-like

gingivalis

but

show

similar

enzymatic and

a marked

have pref-

a

Vol.

174,

No.

The

we infer the

protease histatin

BIOCHEMICAL

protease-inhibitory

However, inhibits

2, 1991

B.

from

site

5 essential

consideration The with

mechanism the

forming for

the

is

can

be

histatins

for

the

complete

assuming

that

all

activities with

produced

preventive

evaluate

histatin

trypsin-like

periodontal

histatin

said

tissue

against content the

by

significance

human

saliva

have

the

disease. the

of hi&&ins

191,

A survey

degree

of periodontal

in this

al.,

the

into

greatly

unpublished). amount

of

protease

major

considered

on

in

activity.

are

histatins

to the

residue

varies

trypsin-like

same inhibitory

been

5

taking

sufficient

gingivalis

have

(18,

et

contains

histatin

enzymes. saliva

M (Oh

activities

and

destruction

10e5

B.

collagenolytic

gingivalis

and

in human

acid

lysine

of both

present.

binding

amino or

at

that

by

The

residue

of

study

arginine

of

clear

clostripain

of histatins

inhibition

periodontal in saliva

acid

COMMUNICATIONS

not

this

complex.

an order

species

B.

in

is probably

that

and

5 is

probably

amino

roughly

it

Since

for

RESEARCH

histatin

obtained

and

of a mixture

but

of

data

binding

preference

individuals

BIOPHYSICAL

a noncovalent

the

concentration

Therefore,

limited

protease

gingivalis

active

AND

may the

protease

to be associated play

a role

correlation

disease

as a

between

is required

to

disease.

ACKNOWLEDGMENTS We thank Prof. F. Yoshimura of Aichi-Gakuin University, Nagoya, for providing us with cells of B. gingivalis 381. We also thank Drs. S. Abe and R. Muramatsu of Nippon Mining Co., Tokyo, for excellent amino acid sequence analysis of histatin 5.

REFERENCES 1.

2. 3. 4.

5. 6. 7.

8. 9.

Oppenheim, F.G., Xu, T., McMillian, F.M., Levitz, S.M., Diamond, R.D., Offner, G.D., and Troxler, R.F. (1988) J. Biol. Chem. 263, 7472-7477. McKay, B.J., Denepitiya, L., Iacono, V.J., Krost, S.B., and Pollock, J..J. (1984) Infect. Immun. 44, 695-701. Pollock, J.J., Denepitiya, L., McKay, B.J., and lacono, V.J. (1984) Infect. Immun. 44, 702-707. Su$iyama, K., Suzuki, Y., and Furuta, H. (1985) Life Sci. 37, 475-480. Slots, J., Bragd, L., Wikstrom, M., and Dahlen, G. (1986) J. Clin. Periodontol. 13, 570-577. Yoshimura, F., Nishikata, M., Suzuki, ‘I?., Hoover, C.l., and Newbrun, E. (1984) Arch. Oral Biol. 29, 559-564. Green, G.D.J., and Shaw, E. (1978) Anal. Biochem. 93, 223-226. Hummel, B.C.W. (1959) Can. J. Biochem. Physiol. 37, 1393-1399. Folk, J.E., and Schirmer, E.W. (1965) .J. Biol. Chem. 240, 181-192.

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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