10T12 cells which is inhibited by the Bowman-Birk proteinase inhibitor

Vol.

178,

August

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

856-861

Pages

15, 1991

IDENTIFICATION OF A SOLUBLE ENZYME FROM C3H/lOT+ CELLS WHICH IS INHIBITED BY THE BOWMAN-BIRK PROTEINASE INHIBITOR Julie Department

of Animal

M. Fagan

Sciences,

and Lloyd

Rutgers

*Department of Biological Research Laboratories, Received

June

Waxman' New Brunswick,

University,

NJ 08903

Chemistry, Merck Sharp & Dohme West Point, PA 19486

4, 1991

SUMMARY: The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/lOT% transformed fibroblasts. Two serine and a novel neutral proteolytic proteinases, the proline endopeptidase activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/lOTG cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase 0 1991 Academic Press, Inc. from C3H/lOTs cells.

Epidemiological of

cancer

in

occurrence

humans

of

[l-4].

studies

show

Vegetables

breast

inhibitors

leguminous

which

inhibitors

have

carcinogen-induced soybean

diet

decreased rats

added

to

reduced

with

both

trypsin

the

with

diet

liver

of the

colon

carcinomas

$1.50

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

856

or with

in

proof the

proteinase [6,7]. --in

Provivo

were

and fed

the

skin

cancer

[16]

BBI,

when

Soybean-derived

in

[13]. number hamsters

significantly When applied topically, and [ll].

a

was significantly

dimethylhydrazine,

and rectum reduced

[15] [17].

[1,4].

inhibitors

When animals formation

fat

and vege-

Bowman-Birk

chymotrypsin

the

and

rich

carcinogenesis

cancer with

oral

proteinase and

meat cancer

unusually

and the

tumor

treated

benzanthracene-induced

are

[8-141.

mice

in

prostatic

etiology

between

cereals

and

suppress

cancer

significantly

Copyright All rights

not

of

to

--in vitro inhibitors, breast

STI,

0006-291X/91

shown

spontaneous

x-ray-induced

adenocarcinomas but

inhibit

found

rich

diets

[5]

the

of

the serine type

in proteinase

in mice

and in

are

in

consumption

particular,

(STI)

been

the

colon,

inhibitor

transformation rich

in

abundant

as a factor has been

consume

breast,

seeds,

Most trypsin

and

which of

diet

correlation

cancers

populations

(BBI)

teinase

colon

incidence

inhibitors. soybean

identified

A positive

and

a low and

teinase Kunitz

BBI,

[l].

Conversely,

tables

have

size

of BBI,

dimethylbut

not

Vol. 178, No. 3, 1991

BIOCHEMICAL

STI, also suppressed the x-ray transformed fibroblasts tors of chymotrypsin act

[14].

AND BIOPHYSICAL

induced malignant

RESEARCH COMMUNICATIONS

transformation

These studies may indicate

as chemopreventative

agents in certain

in C3H/lOT%

a role

for inhibi-

types of cancer.

Identification of the target enzymes of the proteinase inhibitors which as chemopreventative agents would provide an important first step in

determining the mechanism by which proteinase inhibitors suppress carcinogenesis. A soluble serine proteinase with a mass of 70-kDa [18] was isolated from C3H/lOT)I cells

which was inhibited

examined whether this BBI-inhibitable tidase

(EC 3.4.21.26)

of tissue and cell

by

activity

that has been purified

BBI.

In this communication we is the 70-kDa proline endopep-

and characterized

from a variety

types. METHODS

Materials. Tris, Bis-Tris, dithiothreitol (DTT), acrylamide, and sodium dodecyl sulfate (SDS) were purchased from Research Organics. Sodium borate, dimethylsulfoxide (Me2SO), 9 mi nomethylcoumarin (AMC) and yethoxynaphthylamine (MNA) were from Sigma, and H-diisopropylfluorophosphate ( H-DFP) was obtained from New England Nuclear. Peptide fluorogenic substrates were purchased from Enzyme Systems Products, Cambridge Research Biochemicals and Schweizer-Hall. DEAE-cellulose (DE-52) was obtained from Whatman. Cyanogen bromide-activated Sepharose, Fast Q, S-300, G-50 and columns for fast protein liquid chromatography (FPLC) were from Pharmacia. Purification of the Bowman-Birk Inhibitor. BBI was purified from 50 mg of a crude lyophilized preparation from soybeans (Sigma) based on published procedures-[19]. Following chromatography on Sephadex G-50, only-one region of protein inhibited both trypsin and chymotrypsin. After fractionation of this material on Mono Q with a gradient of NaCl, BBI appeared homogenouson SDSPAGEand eluted as one peak on a Cl8 column using reverse-phase liquid chromatography. The amino acid composition for this material agreed with published values for BBI [19]. Preparation of Cells and Extracts. C3H/lOT$ mouse embryo fibroblast cells were cultured in basal medium (Eagle's) supplemented with 10%heat-inactivated fetal calf serum. After washing the monolayers with ice-cold phosphate buffered saline (PBS) (2OmMNaH PO pH7.4, 150mM JaCl) the cells were scraped from the plastic dishes. 2 C$H/lOT% cells (10 ) were' collected by low speed centrifugation and washed several times with ice-cold PBS to remove serum components. The cells were then suspended in 3 ml of 10 mM Tris-HCl (pH7.4), 1 mM EDTA, 0.25M sucrose and homogenized in a tight-fitting Dounce homogenizer (50 stokes). Unbroken cells and nuclei were removed by centrifugation (6OOxg/8 min 4°C). The pellet was resuspended in sucrose and recentrifuged. The combined supernatants were then centrifuged at 4°C for 90 min at 100,000 xg and the supernatant was retained for further study. Column fractions (100 ul) containing Hydrolysis of Fluorogenic Peptides. enzyme were preincubated with or without BBI (30 uM) for 30 min at 25'C in a reaction volume of 200 ul containing 50 mMTris-HCl (pH8). Peptide substrates dissolved in Me SO were then added (final concentration of peptide was 30 uM and Me2S0 was 51 ), and incubated for 30-60 min at 37°C. The reactions were terminated by the addition of 100 ul 1% SDS and 1.7 ml sodium borate (0.2M, pH9.1). Fluorescence was measured at 3801460 nm and 3351410 nm excitation/emission for AMC and MNA peptide substrates, respectively. by the method of Lowry et al. [20] Other Methods. Protein was quantitated SDS-polyacrylamide gel electrophoresis was performed on with BSA as standard. 12.5% acrylamide slab gels according to the method of Laemmli [21] using molecular weight standards from Sigma. 857

Vol. 178, No. 3, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION An endopeptidase substrate

activity

which

Boc-Val-Pro-Arg-AMC

was

1181. The enzyme had a single was

inhibited

mined

by the

whether

the

this

proline

from

hormone

(22),

endopeptidase insulin

and

BBI

for

30pM

min of

in

of

not

to inhibit

with

cell

Arg-AMC

of

hydrolyzing

activity

(data

gradient

("0.05M

of

were NaCl

NaCl).

1.39 of

of

the

or with at a final

mixture

did

not

enzyme.

was

1.37nmoles

TRH-MNA/30

by this

approxi-

without added

and

(100pM)

to

enzyme

a column

min

in

Figure

one which

Thus

the

BBI

is

likely

-10

0

inhibited

1 shows coelutes

that

these

by BBI,

does

two with

peaks both

activity activities

of

Boc-Val-Pro-

insulin early are

soluble

and eluted degrading in due

the to

0.00 -20

the

inhibit

of DEAE cellulose

0.00 4

a mass

on

hydrolyzed

hydrolyzed

nmoles

and TRH-MNA hydrolyzing It

which

was then 22)

BBI

insulin

cytoplasmic

(O-0.4M).

shown)

has

endopeptidase and

proline

by chromatography (2ng)

substrate,

The

endopeptidase.

applied

activity,

not

BBI

long/ml

an intracellular

a gradient

this

concentrations

the proline

proteins

(TRH-MNA)

The proline

30uM TRH-MNA or

To identify C3H/lOT%

and which

Km for

absence

Higher

hydrolysis appear

(TRH-MNA)

to

isolated releasing

(23).

cells

Enzyme

identical

thyrotropin

polypeptides

C3H/lOT$

Substrate

30 min.

the

BBI.

small

is

and

We deter-

has been

by 1mM DFP was preincubated

37°C. (the

hydrolyzes

cells

'IO-kDa

by BBI.

that

(S-300).

and was inhibited

C3H/lOT%

activity

proteinase

from

filtration

30 min at of

TRH-MNA/30

other

purified

gel

at 37°C for

presence

and

fluorogenic

from

DFP and

proteinase

serine and which

Pyro-Glu-His-Pro-MNA

concentration incubated

cells

insulin

Q and

67-70-kDa (30pM)

serine

trypsin-like

a mass of approximately

inhibitor

a 70-kDa

was partially

Mono

mately

BBI-sensitive

the isolated

with

proteinase

of mammalian

(TRH)

DE-52,

subunit

serine

endopeptidase,

a variety

hydrolyzed previously

10 Fraction

20

30

40

Number

Fig. 1. Fractionation of C3H/lOTQ cell supernatant on DE-52. C3H/lOT% cell supernatant (6 ml) was applied to a 5 ml column of DE-52 equilibrated in 2OmM Bis-Tris (pH7)/0.lmM DTT. The column was washed with the buffer and bound proteins were eluted with a linear gradient of NaCl formed from 30 ml of starting buffer and 30 ml of 0.4M NaCl. 1.5 ml fractions were collected and 100 ~1 assayed for the hydrolysis of Boc-Val-Pro-Arg-AMC and TRH-MNA.

858

salt the

Vol.

BIOCHEMICAL

178, No. 3, 1991

proline

endopeptidase

second

and

elutes

at

smaller

a higher

TRH-MNA (Fig. that

which

much

known of

inhibited

the

isolated

from

Pro-Arg-AMC

(data

hydrolysis

of

C3H/lOT$ by

fraction

15

of Boc-Val-Pro-Arg-AMC BBI

(data 1).

hydrolyze

Succinyl-Ala-Ala-Pro-Phe-AMC,

eluted

both fractions

activity

cells

were

other

which to

column

and

bound

(pH7).

1OOmM HCl.

tested

for

Ala-Pro-Phe-AMC

their

ability

10 and

1OOmM HCl

Succinyl-Ala-Ala-Pro-Phe-AMC.

activity

which

Table

1.

of

elutes

bound

Effect

to

using

hydrolyze

the

two peaks

BBI

(3OuM),

the

70-kDa

for

by peak

enzyme

only

5% the

(fraction

27)

by 79% by BBI (Table their

serine ability

of

pro-

to

substrate

peak

which

of Boc-Val-

chymotrypsin-like

for

also chymo-

Succinyl-Ala-Ala-Pro-

trypsin-like

soluble

Fast

resin

activity

Q.

which

were

step-wise

The eluted

activity

the

in

the

chymotrypsin

None + BBI % Inhibition

TRH-MNA

sub-

(3 pmoles

hydro-

from C3H/lOTs cells

Boc-Val-Pro-Arg-AMC 26

5808

5.5

5536

79

5

100 ~1 of DE-52 fractions 15 and 27 were incubated for 1 h at 37°C in a final volume of 200 ~1 containing 50 mM Tris-HCl (PHB), 30 PM substrate (either TRH-MNA or Boc-val-Pro-Arg-AMC) in the presence or absence of 30 UM BBI.

859

1,

hydrolyzing

Peptide Hydrolysis (pmoles/h) Addition

1,

base

Succinyl-Ala-

1OOmM HCl

isolated

of

1M Tris

Boc-Val-Pro-Arg-AMC with

mM

by 10 ml

5 volumes

was detected or

bro-

(pH7)/50

with

Boc-Val-Pro-Arg-AMC,

Pyro-Glu-His-Pro-MNA

of BBI on proteolytic

with

and

6ml of 0.75M

to cyanogen followed

neutralized

No activity

BBI-Sepharose

with

same buffer

immediately

to hydrolyze

eluted

C3H/lOT$

enzyme

20 mM Bis-Tris

the

eluted

10'

The BBI-sensitive

with

20 ml of

were

from

made of BBI bound

eq.uilibrated with

proteins

(15 mg) were

column

and Pyro-Glu-His-Pro-MNA.

strate

smaller

the

by BBI,

Proteins

The eluates

inhibited

the

fluorogenic

only

not

hydrolysis

and

The

activity

by BBI.

to this [25]

by

the

a broad

found,

a 2ml affinity

was washed

1 mM Bis-Tris

10 and

a

inhibited

Sepharose

The

assayed

to a 2ml column

and applied

NaCl.

and

Although

whether

was inhibited

were

was

enzyme

applied

mide-activated

shown)

NaCl was inhibited

the

proteins

NaCl

not

does

by BBI.

inhibit

trypsin-like

G (24).

hydrolyzing at QO.175M

To purify

of

inhibits column

not

activity

the

and

(30uM)

In contrast,

hydrolyzing

and cathepsin

inhibited

did

(22,23).

hydrolyzing

We tested

shown). were

Since 1). teinases,

Phe-AMC

not

(18),

(Table

substrates

NaCl)

Boc-Val-Pro-Arg-AMC

cells

TRH-MNA

these

(QO.175M

Boc-Val-Pro-Arg-AMC

of

trypsin

to cleave

Boc-Val-Pro-Arg-AMC

concentration

1) or insulin

hydrolyzed

hydrolysis

salt

is peak

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol.

178,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

Proline Endopeptidase

kDa

-7OkDa Enzyme

r----B8I kDa

205 )

+BBI

-BBI

+BBI

’

I -g

,‘

“’

i

97, 66-

66, 45,

4536-

29)

2924-

16,

02

COMMUNICATIONS

03

Fig. 2. SDS-gel electrophoresis of an enzyme purified from C3H/lOT$ cells which is inhibited by BBI. Proteins (0.5pg) which bound to a column of BBISepharose and were eluted with 1OOmMHCl were electrophoresed on a 12% polyacrylamide gel in the presence of SDS. Proteins were silver-stained with a kit from ICN and compared to protein standards of known molecular weight. Fin. 3. The effect of BBI on the incorporation of 3H-DFP into proline endopeptidase and BBI-affinity purified %70-kDa proteinase. BBI-affinitypurified *70-kDa proteinase and proline endopeptidase partially purified by chromatography on Mono Q and gel filtrat on and were incubated at 4°C overnight in 50 mM Tris-HCl (pH8) with 30 pM 3H-DFP in the presence or absence of 30 PM BBI. The mixtures were run on SDS-PAGE (12%) and the gel was autoradiographed. In both cases bands with an Mr=Q67-70-kDa were labelled.

lyzed/h/pg

protein).

of the

purified

Billings

et

polypeptide

The total

material al.

[18],

binding

from

scan 3 H-DFP

of

the

of the to

the

found

serine

proteinase a novel While

there

proteinases cancer

in

is

not

inhibitors

have

enzymes formed

is

is

may be involved in

or

by BBI

the in in

the

the

role

of intracellular

or of

binding the

that

of

extracellular proteinases

that

expression specific

860

in

proteinase

one or more of cellular

BBI acts as a chemopreventative the %70-kDa serine proteinase

investigation.

of

%70-kDa

endopeptidase

BBI and other

suggest

induction

Moreover, by 65% the Consistent

proline

the

that

by

cells.

participation

would

3).

on the

show

the

processing

cancer promotion. Whether types of cancers by inhibiting

not

of C3H/lOT$

observations the

no effect

SDS-PAGE a %70-kDa

enzyme.

Our data is

to

(Fig.

for

activity

either

perhaps

had

3).

cytoplasm

evidence

However,

BBI

(Fig.

5pg.

As reported

BBI blocked

BBI-affinity-purified

inhibited

anticarcinogenic

that

than 2). binds

cells

revealed

and metastasis,

known.

(Fig.

DFP covalently

of C3H/lOT$

here,

in the

good

cancer

phenotype

involved in certain further

that

enzyme present

that

cytoplasm

%70-kDa

was less

at %70-kDa

autoradiogram

with our other findings reported 3 H-DFP to the proline endopeptidase but

recovered

one band

we also

isolated

a densitometric

protein

showed

the

such

transproteins agent merits

Vol.

178,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS We are grateful to Dr. Susan Socher for providing the C3H/lOT% cells used in this study and to Ms. Marilyn Schwartz for her assistance in preparing this manuscript. This work has been supported by research grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and by the New Jersey Agricultural Experiment Station which is supported by State funds. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Grobstein, C. et al. (1982) Diet, Nutrition and Cancer. Committee on Diet, Nutrition and Cancer, Assembly of Life Sciences, National Academy of Sciences, Washington, DC Armstrong, B., and Doll, R. (1975) Int. J. Cancer 15, 617-631. Carroll, K.K. (1975) Cancer Res. 35, 3374-3383. Correa, P. (1981) Cancer Res. 41, 3685-3690. Kunitz, M. (1947) J. Gen. Physiol. 30, 291-310. Birk, Y. (1975) In Methods in Enzymology, Vol. 45, 695-739. Bowman, D.E. (1946) Proc. Sot. Exp. Biol. Med. 63, 547-550. Billings, P.C., St. Clair, W., Ryan, C.A., and Kennedy, A.R. (1987) Carcinogenesis 8:809-812. Corasanti, J.G., Hobika, G.H., and Markus, G. (1982) Science 216, 10201021. Kennedy, A.R., and Little, J.B. (1981) Cancer Res. 41, 2103-2108. Messadi, D.V., Billings, P., Shklar, G., and Kennedy, A.R. (1986) J. Natl. Cancer Inst. 76~447-452. Troll, W., Klassen A., and Janoff, A. (1970) Science 169, 1211-1213. Weed, H.G., McGandy, R.B., and Kennedy, A.R. (1985) Carcinogenesis 6, 1239-1241. J. Collins, M., Birk, Y., Troll, W., and Kennedy, A.R. (1985) Yavelow, Proc. Natl. Acad. Sci. USA 82, 5395-5399. Becker, F.F. (1981) Carcinogenesis 2:1213-1214. Troll, W., Belman, S., Wiesner, R., and Shellabarger, C.J. (1979) In Biological Functions of Proteinases, (H. Holzer and H. Tschesche, Eds.), pp. 165-170. Springer-Verlag, Berlin. Troll, W., Wiesner, R., Shellabarger, C.J., Holtsman, S., and Stone, J.P. (1980) Carcinogenesis 1, 469-472. Billings, P.C., Carew, J.A., Keller-McGandy, C.E., Goldberg, A.L., and Kennedy, A.R. (1987) Proc. Natl. Acad. Sci. USA 84, 4801-4805. D.L.-R., Lin, K.-T.D., Yang, W.-K., and Foard, D.E. Hwang, (1977) Biochim. Biophys. Acta 495, 369-382. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R. (1951) J. Biol. Chem. 193, 265-275. Laemmli, U.K. (1970) Nature 227, 680-685. Dixon, J.E. (1979) Biochemistry 18, Rupnow, J.H., Taylor, W.L, and 1206-1212.

23. 24. 25.

Koida, M., and Walter, R. (1976) J. Biol. Chem. 251, 7593-7599. Nakajima, K., Powers, J.C., Ashe, B.M., and Zimmerman, M. (1979) J. Biol. Chem. 254, 4027-4032. D.E. (1980) J. Chrom. 195, 385-391. Lin, K.D., Hwang, D.L, and Foard,

861