An irreversible tissue inhibitor of collagenase in human amniotic fluid: Characterization and separation from fibronectin

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Pages 11951201

16, 1981

AN IRREVERSIBLE TISSUE INHIBITOR OF COLLAGENASE IN HUMAN AMNIOTIC FLUID: CHARACTERIZATION AND SEPARATION FROM FIBRONECTIN Judith Aggeler, Eva Engvall, and Zena

Werb

Laboratory of Radiobiology and Department of Anatomy, University of California, San Francisco, California 94143; and the La Jolla Cancer Research Foundation, La Jolla, California 92037 Received

May 1, 1981

SUMMARY: Soluble fibronectin isolated from human plasma and amniotic fluid by gelatin-Sepharose affinity chromatography was tested for inhibitory activity against specific collagenase secreted by human and rabbit= fibroblasts. The fibronectin preparation derived from plasma showed little inhibition, but the one derived from amniotic fluid contained potent inhibitory activity against collagenase. This activity was separated from fibronectin on a DE-52 cellulose column and did not cross-react with antibodies to fibronectin. The inhibitor was a glycoprotein that was partially purified from amniotic fluid by concanavalin A-Sepharose affinity chromatography. Inhibition was irreversible and enzyme activity was not recovered after reaction with latent or activated collagenase by either trypsin or organomercurial treatment. Collagenase activity nective

tissues

are

is difficult

resistant

to degradation

experimental

conditions in culture.

in

cartilage,

plasma,

(l-71.

glycoprotein

proteolytic

sites

Biswas et al. (101 tested fibroblast

(81.

fibronectin

glycoproteins

associated

them from degradation Fibronectin

is a

large

human

amniotic

to

pro-

We report that fibronectin

prepara-

fluid contained a potent collagenase inhibitor

could be separated from fibronectin

ADbreviations: APMA, concanavalin-A-Sepharose.

for its ability

and found that it inhibited collagenase directly,

without binding to the collagen substrate. from

of

cultures

that binds to collagen very near the cleavage site for collagenase

tect collagen from degradation

tions

have been identified

muscle and in skin fibroblast

proteins may protect

masking

con-

by active collagenase even under

It has also been suggested that a variety

by binding to and

in viva, and certain --

Collagenase inhibitors

and smooth

bone,

with connective tissue matrix

(9).

to demonstrate

that

itself.

4-aminophenylmercuric

acetate;

Con-A-Sepharose,

0006-291X/81/111195-07$01.00/0 1195

Copyrighf @ 1981 by Academic Press, Inc. All righfs of reproduction in any form reserved.

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MATERIALS AND METHODS Cell culture. Rabbit synovial fibroblasts were cultured in Dulbeccols modifmEwmedium containing 10% fetal calf serum, as previously described (11). Conditioned medium containing collagenase was obtained by incubating flasks of confluent cells (2 x 106) for 72 hr in serum-free medium containing 100 rig/ml 12-0-tetradecanoylphorbol-13-acetate, which stimulates secretion of latent collagenase by fibroblasts (12,13). Human collagenase was obtained from medium conditioned by human rheumatoid synovial cells, which spontaneously secrete large amounts of collagenase activity in culture (14). Determination of collagenase activity and collagenase inhibitor activity. Collagenase activity-was determined by meGring release om radioactive peptides from ‘c-labeled collagen fibrils (15). (O%e unit of collagenase activity degrades 1 pg of collagen per min at 37 C.) Latent collagnase was activated by incubating medium with 10 ug/ml trypsin for 30 min at 22 C; activation was stopped by the addition of 20 ug/ml soybean trypsin inhibitor. (One microgram of soybean trypsin inhibitor inhibited 1.1 ug of trypsin in an azocasein assay (16).) To prevent nonspecific loss of inhibitor by adherence to glass tubes, inhibitor and activated collagenase were added directly to the assay buffer overlying the collagen fibrils (100 ug per tube) and incubated at 22’C for 30 min. The tubes were then transferred to 37oC and incubation continued for 2-4 hr. Collagenase inhibitor activity was determined by calculating either the number of units of collagenase inhibited per milligram of protein in a preparation or the percent inhibition of collagenolysis. To determine reversibility of inhibition, samples containing collagenase-inhibitor complexes (preincubated at 22’C) were reactivated with 50 ug/ml trypsin or with 1 mM APMA (17). Trypsin reactivation was stopped by adding 50 ug/ml soybean trypsin inhibitor. Purification of fibronectin. Fibronectin was isolated from human plasma and from humar-amniotic fluid by a two-step purification process. First, the starting material was passed over a gelatin-Sepharose column and eluted with 4.5 M urea in 0.05 M Tris-HCl buffer, pH 7.5, as previously described (18). Part of the urea eluate was then fractionated by binding to a DE-52 cellulose column and eluting with a urea-NaCl gradient (19). Salt and urea were removed from all samples before assay by passage over a Sephadex G-15 column in 0.01 M Tris-HCl, pH 7.0. Lectin binding g inhibitor @i amniotic fluid. Pooled human amniotic fluid was bound to Con-A-Sepharose equilibrated with 0.2 M NaCl and 0.05 M Tris-HCl (pH 7.6), and protein was eluted with 50 mM a-methylmannoside; l-ml fractions were collected. Protein was measured spectrophotometrically (A Collagenase inhibitor activity was determined as described mg/ml). Fibronectin and at least four unidentified serum proteins were detected in the Con-A-Sepharose eluate by double immunodiffusion against antiserum to human fibronectin and to serum proteins. RESULTS Fibronectin,

a large

collagen-binding

glycoprotein,

purified by a one-step process involving gelatin-Sepharose phy (16).

We tested soluble fibronectin

fluid

this

by

method

for its ability

lagenase.

The plasma preparation

amniotic

fluid contained potent

inhibitor

in the gelatin-Sepharose

commonly

has

affinity

been

chromatogra-

isolated from human plasma and amniotic to inhibit the action of mammalian

showed little inhibitory

inhibition,

activity

eluate of amniotic

1196

but

(Fig. 1A).

the

one

colfrom

The collagenase

fluid was separated

from

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0 I 1.00

1.

0.5

Protein Concentration

(mg/mi)

Fig. 1.

Lack of inhibition of fibroblast collagenases by fibronecttn from plasma or amniotic fluid. Amniotic fluid and plasma were partially purified by gelatin-Sepharose affinity chromatography followed by fractionation of the 4.5 M urea eluate on a DE-52 cellulose column to isolate fibronectin. Media from cultured human or rabbit fibroblasts activated before addition of inhibitor and assayed by incubation.with collagen fibrils. Fibrils were incubated with A) 0.39 units of human collagenase and gelatin-Sepharose eluate from human plasma (0) or gelatin-Sepharose eluate from human amniotic fluid (Oh B) 0.37 units of rabbit collagenase and gelatin-Sepharose eluate from human plasma (01, or gelatin-Sepharose eluate from human amniotic fluid fe), or fibronectin purified from human amniotic fluid by DE-52 chromatography (ml. human

fibronectin

tory activity reduced itself

passage over a DE-52 cellulose column.

by

of the fibronectin

from

was not the inhibitor

The inhibitor

Collagenase inhibitory

appeared to act directly

was less

when

fibronectin

was incorporated

directly

directly

the

gelatin-Sepharose

on the

was greatly fibronectin

that

activity

preparation

to the medium containing

was not

inhibited

Although most of the is

fibroneetin

amniotic

inhibitor

(Twenty-six collagenase

incorporated

protein

(181, the collagenase inhibitor

1197

inhibition and

substrate than when it was

0.42 units of

fluid

because

containing

collagenase.

to the medium, but only 0.29 units when

Sepharose

enzyme,

into the collagen fibril

fluid preparation

the amniotic

fibrils.)

(Fig. 1B).

eluate, indicating

inhibi-

from the DE-52 column under the conditions used.

recovered

added

peak eluted from the DE-52 column

of the gelatin-Sepharose

that

The collagenase

eluted

micrograms of when

added

into the collagen from

gelatin-

was also enriched in

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Fig. 2. Inhibition of fibroblast couagenase by amniotic fluid glycoproteims). Pooled human amniotic fluid (50 ml) was bound to Con-A8epharose (0.2 M NaCl, 0.05 M ‘Iris-HCl), and glycoprotein was eluted with 50 mM o-methylmannoside; l-ml fractions were collected. Aliquots (150 ul) of the Con-A-Sepharose eluate were incubated with 0.55 unit8 of activated rabbit fibroblast collagenase. Protein concentration (mg/ml) (---); % collagenase inhibition (H ).

this preparation. activity

per

Pooled mg

amniotic

protein,

fluid

contained

5 units

whereas various gelatin-Sepharose

tained 20-115 units of inhibitory

activity

per mg protein,

of

inhibitory

preparations

con-

representing a 4-

to

23-fold enrichment. Because several inhibitors teins

(3,20),

we

attempted

of metalloproteinases partial purification

from amniotic

fluid by Con-A-Sepharose

eluted

the

from

lagenase inhibitory and

18 were

protein,

column activity

206

in

are known to be glycoproof the collagenase inhibitor

affinity

chromatography.

(Fig. 2).

The specific activities

of fractions

14

of collagenase inhibited per mg

which represented a 41- to 46-fold increase over that of

amniotic

fluid.

diffusion,

and a number of other proteins, as shown by sodium

polyacrylamide

was

two major peaks that coincided with peaks of col-

and 228 units, respectively,

Fraction

Protein

14 contained fibroneetin,

the

starting

as shown by double immunododecyl

sulfate-

gel electrophoresis.

Collagenase is often secreted in a latent

1198

form

that

requires

activation,

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Table

BIOCHEMICAL

I.

AND

Irreversibility

of

Isolated Collagenase

from

8lOPHYSlCAL

Collagenase Human

treatment

RESEARCH

Inhibition

Amniotic

by

COMMUNICATIONS

Inhibitor

Fluid

Collagenolysis

Inhibition

Unactivated Activated

(10

ug/ml

Inhibitor

trypsin)

100.0

--

31.7

fja.3

30.9

69.1

33.5

66.5

3.8

96.2

27.8

72.2

added

Reactivated

(SO ug/ml

trypsin)

(1 mM ARMA) (Inactivated; trypsin

inhibitor treated

Activated inhibitor

(trypsin) added

added;

; trypsin-treated

Collagenase inhibitor affinity chromatography Collagenase from rabbit Collagenase-inhibitor and Methods.

from

fibroblasts complexes

and it has been suggested that To test whether

(21,22).

inhibitor the

from amniotic

organomercurial

amniotic

fluid 145 was used at were reactivated

was partially purified by ug were used in each assay. 0.55 units per assay. as described in Materials

latent collagenase is an enzyme-inhibitor

collagenase bound to

we activated

collagenase,

human

on Con-A-Sepharose;

rabbit

behaved

The

reactivated

it with

enzyme-inhibitor

treatment

unactivated activated

with

either

trypsin

was

or APMA (Table I).

The inhibitor

teinase sensitive because pretreatment inhibit

either

(latent) collagenase was incubated with inhibitor, by trypsin treatment.

latent

trypsin

or

complex did not behave like

latent collagenase and no recovery of collagenase activity second

like

collagenase with trypsin, incubated it with the

fluid, and then APMA.

inhibitor

complex

after

observed

a

In addition, after it could

not

be

itself did not appear to be pro-

with trypsin did not alter its ability to

collagenase (Table I). DISCUSSION Glycoproteins

lagen

and

are commonly associated with the fibrillar

elastin in the extracellular

from degradation

network

matrix and may protect

(8), as well as aiding in cell adhesion and

II99

these

of

col-

components

recognition

(23).

Vol. 100, No. 3,198l

BIOCHEMICAL

We tested purified fibronectin tion by mammalian This

suggests

AND

BIOPHYSICAL

for its ability

near the specific cleavage site, does not play an

of mammalian

teinase inhibitors

tissues (l-7).

inhibitors

from human amniotic

Of particular

Murphy et al. (20) recently fluid that may be identical

purified preparation

described

in

a

interest are the metalloproof molecu-

isolated a similar inhibitor to

ours,

although

they

to be trypsin sensitive, whereas our partially

was not destroyed by trypsin treatment.

It seems

found

puri-

unlikely

that

can be responsible for collagenase latency by forming a complex

with secreted, active enzyme, because recovered after the enzyme-inhibitor

we

that

active

collagenase inhibitor

out a new aspect of the stringent

developing organism.

found

enzyme

was not

complex was formed.

The secretion of an irreversible points

been

have

from bone and smooth muscle that are glycoproteins

lar weight 28,000 (3).

these inhibitors

important

of collagen degradation.

Recently, a number of collagenase

fied inhibitor

collagen from degrada-

that steric hindrance, caused by binding of a large glycoprotein

role in the regulation

their

to protect

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collagenase and found that it was not a collagenase inhibitor.

such as fibronectin

variety

RESEARCH

into amniotic

fluid

control of collagen degradation

by the

Not only can biological systems regulate collagen degrada-

tion by altering synthesis and secretion of collagenase, by producing the enzyme in a latent form, and by storing this latent

enzyme bound to collagen, but

can also produce reversible and irreversible

proteinase inhibitors

nonspecific

and specific for collagenase.

processes,

localized

collagen

processes as differentiation

According to

degradation

the

necessary

and tissue remodeling

that are both

balance

during

they

such

of

important

may take place without

collagenase escaping into surrounding tissues and causing pathologic

these

active

damage.

ACKNOWLEDGEMENTS This work was supported by grant AM 26693 (Eva Engvall) from the Department of Health and Human Services, by the U.S. Department of Energy, and by a Graduate Fellowship from the National Science Foundation (Judith Aggeler).

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Kuettner, K.E., Sable, L., Croxen, R.L., Marczynska, B., Hiti, J., and Harper, E. (1977) Science 196, 653-654. Murphy, G., Cartwright, E.C., Sellers, A., and Reynolds, J.J. (1977) Biochim. Biophys. Acta 483, 493-498. Cawston, T.E., Galloway, W.A., Mercer, E., Murphy, G., and Reynolds, J. J. Personal communication. Woolley, D.E., Akroyd, C., Evanson, J.M., Soames, J.V., and Davies, R.M. (1980) Biochim. Biophys. Acta 522, 205-217. Hiti-Harper, J., Wohl, H., and Harper, E. (1978) Science 199, 991-992. Nolan, J.C., Ridge, S., Oronsky, A.L., Slakey, L.L., and Kerwar, S.S. (1978) Biochem. Biophys. Res. Commum. 83, 1183-1190. Shinkai, H., Kawamoto, T., Hori, H., and Nagai, Y. (1977) J. Bioehem. 81, 261-263. Jones, P.A., and Werb, Z. (1980) J. Exp. Med. 152, 1527-1536. Kleinman, H.K., MeGoodwin, E.B., Martin, G.R., Klebe, R.J., Fietzek, P.P., and Woolley, D.E. (1978) J. Biol. Chem. 253, 5642-5646. Biswas, C., Hynes, R., and Gross, J. (1979) Fed. Proe. 38, 836. Werb, Z., and Aggeler, J. (1978) Proc. Natl. Acad. Sci. USA 75, 1839-1843. Aggeler, J., Mainardi, C., Kramer, J.L., and Werb, Z. (1979) J. Cell Biol. 83, 435a. Brinkerhoff, C.E., McMillan, R.M., Fahey, J.V., and Harris, E.D., Jr. (1979) Arth. Rheum. 22, 1109-1116. Woolley, D.E., Glanville, R.W., Crossley, M.J., and Evanson, J.M. (19751 Eur. J. Biochem. 54, 611-622. Glimcher, M.J., Francois, C.J., Richards, L., and Krane, S.M. (1964) Biochim. Biophys. Acta 93, 585-602. Gordon, S., ‘,berb, Z., and Cohn, Z.A. (1976) In In -- Vitro Methods in CellMediated and Tumor Immunity (B.R. Bloom and J.R. Davm, I$. 341-352, Academic i%%s?? York. Sellers, A., Cartwright, E., Murphy, G., and Reynolds, J.J. (197’7) Bioehem. J. 163, 303-307. Engvall, E., and Ruoslahti, E. (1977) Int. J. Cancer 20, l-5. Ruoslahti, E., Engvall, E., Hayman, E.G., and Spiro, R.G. (1981) Biochem. J. 193, 295-299. Murphy, G., Cawston, T.E., and Reynolds, J.J. Personal communication. Reynolds, J.J., Murphy, G., Sellers, A., and Cartwright, E. (1977) Lancet ii, 333-335. Harris, E.D., Jr., and Vater, C.A. (19801 In Collagenase 5 Normal and Pathological Connective Tissues (D.E. Woolley and J.M. Evansos1, z 37-63, Wiley, New York. Grinnell, F. (1978) hit. Rev. Cytol. 53, 65-144.

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