A component of normal human serum which enhances the activity of vertebrate collagenases

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

February 14, 1978

Pages

637-645

A COMPONENT OF NORMAL HUMAN SERUM WHICH ENHANCES THE ACTIVITY Jo Louise

Seltzer:

Arthur

OF VERTEBRATE COLLAGENASES

Z. Eisent

John J. Jeffrey+and

Joseph

Feder*

Division of Dermatology, Department of Medicine Washington University School of Medicine+ and Corporate Research and Development Monsanto Chemical Company* St. Louis, Missouri, 63110 Received

January

3,1978

Summary: The activity of vertebrate collagenase is increased by approximately 3-fold in the presence of saturating amounts of a macromolecule found in normal human serum. The activities of collagenases from human skin, rat skin, and tadpole tailfin are all markedly enhanced in the presence of this molecule, but activities of bacterial collagenase, trypsin, chymotrypsin, thermolysin, and a gelatin-specific neutral protease from human skin are unchanged. The enhancer itself has no proteolytic activity and does not change the normal cleavage products of human skin collagenase. The collagenase enhancer is an extremely stable molecule. It is resistant to heat, to extremes of pH at physiological temperature, and appears to be protein in nature. Of particular interest is the requirement that the collagen substrate be in fibrillar form in order for the enhancer to be effective. Collagenase ability is

to initiate

a key enzyme

modify over

its

(l-4)

protein

the enzymatic

breakdown

found

effect

only

in tissues.

serum

enzyme activity

component

important of collagenase

for

several

molecule

when the

is

These

findings

could

have

in its

collagenase

molecules

regulation

enzymes

which

of collagen derived

is

a potent

turn-

both

have not the

enhancer

to vertebrate

substrate suggest

(6-13),

we report

specific

important

unique

from

have now been described.

paper,

collagen

is

Because

inhibitors,

(5)

In this

which

remodeling,

in the

human serum which

This

activity.

its

exist

normal

tissue

fibroblasts

any proteases.

from

collagenase

cultured which

for

macromolecule

be most

protease, of collagen.

of connective

A number

level.

and from

demonstrated

by this

neutral

could

activators,

exerts

an extracellular

in processes

activity

at a local

serum

tion

is

is

that

implications

for

as yet

existence

been

of a

of vertebrate collagenases

in the fibrillar

enhancement

However,

and

conforma-

of co1 lagenase the modu lation

of

in vivo.

0006-291x/78/0803-0637$01.00/0 637

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Materials Trypsin, chymotrypsin, thermolysin, collagenase type I, heparin and dextran Chemical, St. Louis, Missouri. Preparation

RNase, DNase, pronase, clostridial sulfate were obtained from Sigma

of Enhancer

Crude collagenase enhancer was prepared from a 4% solution of human serum in 0.05 M Tris, pH 7.5, 0.15 M NaCl which had been treated in one of two ways to inactivate or remove serum protease inhibitors. In most cases, serum was heated at 800C for 15 minutes to inactivate the inhibitors. In addition, ammonium sulfate fractionation of unheated serum yielded an inhibitor-free precipitate (O-30% saturation) which contained the crude enhancer. Preparation

of Human Skin

Collagenase

Medium obtained from explants of human skin in serum-free was the source of collagenase, as previously described (14). lagenase was subjected to ammonium sulfate fractionation and chromatography on CM-cellulose using the method of Stricklin resulting enzyme preparations are purified about 130 fold. Crude rat skin and in these preparations was fully active. collagenases were obtained from organ culture (16)(17), and purification. Assay

organ culture The crude colthen purified by et al. (15). The All the collagenase tadpole tailfin used without further

Procedures

Collagenase activity was determined by the release of soluble peptides from [14C]glycine-labeled reconstituted native collagen fibrils (17). The collagen was allowed to gel at 37°C for at least 24 h to complete the aggregation process. All collagen preparations employed had specific activities of approximately 30,000 cpm/mg. Incubation of the fibrils with 0.01% trypsin resulted in the release of less than 8% of the total radioactivity, indicating that the collagen fibrils were in helical conformation. A typical reaction mixture contained 200 pg [14C]glycine-labeled collagen fibrils (approximately 6,000 cpm) in a gel volume of 50 ~1, and 175 ~1 total volumes of 0.05 M Tris, pH 7.5, 0.005 M CaC12, containing 5 pg purified human skin collagenase with or without enhancer, Enhancer dilutions were always added in a volume of 50 ~1, containing 50 pg protein or less. After an appropriate period of incubation, from 1 to 4 h at 37"C, the assay was terminated by centrifugation at 12,000 x g for 10 minutes, and the entire supernatant counted in a liquid scintillation spectrometer. Gelatin specific protease was assayed by the method of Harris and Krane (18) using [l4C]labeled heat-denatured collagen as the substrate. Activities of trypsin, chymotrypsin, and thermolysin were assayed by measuring trichloroacetic acid soluble peptides resulting from proteolysis of casein(l9). RESULTS When protease fractionation

inhibitors

or as a result

were of heat

removed

from

inactivation,

638

human serum either a non-dialyzable

by salt molecule

Vol. 80,No.

3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ccdlo 8”0584 Heat 4 mated !32lUfll

TIME

(hrs)

Fiqure 1. The effect of serum enhancer on the collaqenase catalyzed igestion of collagen. Purified collagenase (5 ug protein) was incubated with [ 18 Clglycinelabeled collagen with and without 50 ~1 of 4% human serum which had been previParallel reactions were terminated by centriously heated at 80°C for 15 min. and the solubilized collagen counted in a fugation at the times indicated, scintillation spectrophotometer.

remained the

which,

amount

treated

of collagen

serum

collagen

on the rate

ase were

that

affected

Of particular

interest

when the collagen

substrate

significantly

hydrolysis

Both

enhancement crude

and highly

by interaction was the

with observation

was in the

native

639

increase

was constant

this that fibrillar

of labeled

of 2-4

over

purified

of heat

At saturating

on the enhancement.

was a maximum activity

this

increased

1 and 2 show the effect

collagenase-catalyzed

of concentration

of collagenase. equally

collagenase,

Figures

of the

of serum there

3 demonstrates

centration

to human skin

solubilized.

and the effect

concentrations Figure

when added

fold.

a 3-fold

human skin

concollagen-

molecule. enhancement

occurred

form.

When collagen

only

BIOCHEMICAL

Vol. 80, No. 3, 1978

7

I

I IO JJI 4%

Figure 2. Effect of concentration collagenase activity. Increasing skin collagenase (5 pg protein)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

/

20

30

40

HEAT

TREATED

SERUM

.\D 50

ADDED

of heat-treated serum on the enhancement of amounts of 4% heated serum were added to human and the reaction incubated for 90 min. at 37°C.

TABLE I ENHANCEMENT OF COLLAGENASE ACTIVITY

ON COLLAGEN FIBRILS

AT 27°C AND 37'C

Incubation Temperature 27°C 37°C

558

1698

3.0

I

I

I

Fibrils were formed at 37"C, incubated at 27°C. The same preparation of collagenase and enhancer were used at both temperatures. The assay at 27°C was incubated for 18 hours, while that at 37'C was incubated for 2 hours. The reaction at 27°C was terminated with 20 mM EDTA and the gel incubated at 37°C for 5 min. before centrifugation.

in solution

(27°C)

be demonstrated. ture

(27"C),

was employed, However,

the degree

at 37°C (Table

I).

of collagenase

activity

no enhancement

when collagen

of enhancement

Furthermore, on the

fibrils produced

in the fibrillar

presence substrate

640

of collagenase were

activity

could

used at the same tempera-

was identical

to that

of serum enhancer, at 27°C were

the

observed the products normal

Vol. 80, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5.

COLLAGENASE *ENHANCER

1

20

40

JJI COLLAGENASE

60

(.09mg

protein/ml)

Figure 3. Enhancement of collagenase activity as a function of enzyme concentration. 50 ~1 of 4% serum heated to 80°C for 15 minutes was added to the concentrations of collagenase indicated and the reaction incubated for 90 minutes at 37°C. Figure 4. Densitometric scans of acrylamide gels showing soluble cleavage products from collagenase incubated at 27°C with fibrillar collagen. The increase in product detected by the scans was equivalent to the observed increase in activity as shown in Table I. a) Collagenase alone. b) Collagenase and enhancer.

3/4-l/4

cleavage

fragments

previously

(Fig.

4 a,b).

Thus,

lagen

molecule

in the presence

selves

displayed

added

simply

mately

dextran

sulfate

their

failed

effect to affect

collagen

Bovine

assay

mixture,

purified

maximum enhancement

could

them-

not

be

serum albumin,

of collagenase (ZO),

the activity

when

to increase

preparations

on mouse bone collagenase appreciably

col-

or gelatin.

failed

enhancer

(14)

in the

preparations

collagenase

effect.

collagenase

cleavages

either

of purified

partially

produced

human skin

Enhancer

toward

up to 0.5 mg per

4 ug of protein) unlike

enhancer.

protective

whereas

for

no additional

activity

as a protein

activity,

Furthermore,

of the

of the activity

in concentrations

collagenase

produced

no proteolytic

The enhancement explained

collagenase

reported

(approxiactivity.

heparin

of human skin

and collagen-

ase. The specificity As shown in Table

of the II,

enhancer

concentrations

was examined of enhancer

641

using which

a variety fully

of proteases.

activate

human skin

BIOCHEMICAL

Vol. 80, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II EFFECT OF COLLAGENASE ENHANCER FROM HUMAN SERUM ON COLLAGENASES FROM VARIOUS SOURCES AND ON SELECTED PROTEOLYTIC ENZYMES

Enzyme Source

Enzyme Alone

Enzyme + Enhancer

Fold

Increase

cpm - Blank Human Skin Collagenase Rat Skin Collagenase Tadpole

Collagenase

Bacterial

3096

2.4

398

758

1.9

508

953

1.8

1279

Collagenase

Human Skin Gelatin Specific Protease

2531

3114

1.2

744

0.8

-

974 O.D.

Trypsin

0.405

0.381

0.9

Chymotrypsin

0.561

0.555

1.0

Thermolysin

0.864

0.864

1.0

In the fic protease), heat-treated trypsin and 0.5% casein enhancer is collagenase.

assays on ['4C]collagen and [14C]denatured collagen (gelatin-specienzyme was incubated for 90 minutes at 37°C with 50 ~1 of 4% serum. In the trypsin, chymotrypsin and thermolysin assays, 5 vg chymotrypsin and 0.25 vg thermolysin were incubated with 2 ml of and 300 ~1 of crude enhancer for 30 minutes. This amount of 5-fold in excess of that necessary to fully activate human skin

Blanks are complete absence of enzyme.

collagenase tadpole

were tailfin

however,

showed

In contrast in organ (Table

also

capable

mixtures

of increasing

collagenases.

Bacterial

no significant

activation

to human skin cultures

II).

reaction

Finally

collagenase,

of human skin neither

with

substrate

the activity collagenase

from

642

skin

Clostridium

neutral

was unaffected nor

rat

protease

by the

thermolysin

and

histolyticum,

of heat-treated

a gelatin-specific

chymotrypsin,

in the

of crude

in the presence

(unpublished)

trypsin,

incubated

serum. found

serum enhancer was affected

Vol. 80, No. 3, 1978

BIOCHEMICAL

by concentrations

of heat-treated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

serum capable

of fully

stimulating

human skin

collagenase. The collagenase

enhancer

80°C for

20 minutes.

interval

at 100°C.

0°C or 37"C,

However, Exposure

extremely

stable,

90% of the

activity

to extremes

had no effect.

RNase, trypsin, with

is

during

pH 2-12,

was resistant

and thermolysin

100% activity

was lost

of pH, from

The enhancer

chymotrypsin

retaining

the

for

same

1 h at either

to digestion

but was destroyed

at

by DNase,

by incubation

pronase. DISCUSSION Protein

disparate

activators catalytic

have now been described activity.

phosphodiesterases(6),RNA and galactosyl for

polymerases

transferase

a proteolytic

first

time,

but

it

ase,

since

the

products

most

appears 1ikel.y

this

In view

of the

promote

unlikely

substrate,

proper

of the fact it

molecule

at which

proposed

for

the

is

activators

are

the

enhancement

of a number

the specific occurs.

unknown

collagen-

to those

Furthermore,

the

own.

Thus,

the

is of

fibrillar

the enhancer locus

en-

it

properties

acts

to

in the collagen

Such a mechanism

of DNA-dependent

at

identical

kinetic

that

to be the

of the

requires

to speculate

cleavage

is

specificity

of its

by changing

enzyme with

collagenolytic

the

(8,9)

appears

of the enhancer

activity

tempting

of the

here

of the enhancer.

that

AMP

has been described

none

collagenase

no proteolytic

cyclic

however, described

activated

acts

for

of

hydroxylase

affects

absence

molecule

alignment

it

of enzymes

phenylalanine

of action

that

of the

to possess

that

(7),

and the molecule

by the enzyme in the

collagenase. form

is

a variety

describedarethose

To date,

The mechanism

this

hancer

(10).

enzyme,

such entity.

produced

The best

for

has been

RNA polymerases

(21-23). It presence form.

should

be emphasized

of serum enhancer The human skin

that is

collagenase

the

increase

in collagenase

not due to conversion used

in these

643

activity

of a zymogen

studies

contains

to its

in the active

no proenzyme

(15)

Vol. 80, No. 3, 1978

Furthermore, with

BIOCHEMICAL

repeated

enhancer

attempts

preparations

The collagenase protein acid. that

molecule

preliminary

suggests

of the collagenase view of the

local

role enhancer

requirement

an important level,

both

role

has led

the

for

and the cells is

fibrillar

in the

in normal

presently

control

not shown).

stability

is

other

to heat

and to

conclusion

Additionally,

of relatively

low

(unpublished).

responsible unknown.

for It

the production

seems,

collagen,

that

of tissue

remodeling

and in pathological

with

tentative

in nature.

enhancer daltons

proenzyme

some properties

us to the

protein

20,000

(data

such as its

partly that

weight-approximately

The physiological

play

at least

collagenase

;*esults to share

to date,

by pronase

evidence

molecular

appears

described

is

purified

gave negative

The inactivation this

to activate

enhancer

activators

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

especially

the enhancer

in

could

processes

at a

states.

Acknowledgements We wish technical Service

to thank Mr. Dennis Nagy and Mr. Keith assistance. This work was supported Grants AM-12129 and HD-05291.

Krummenacher for by United States

their excellent Public Health

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

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Eisen, A.Z., Bauer, E.A., and Jeffrey, J.J. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 248-251 Werb, Z., Burleigh, M.C., Barrett, A., and Starkey, P.M. (1974) Biochem. J. 139, 359-368. I. (1973) Eur. J. Biochem. 36, 473-481. Ohlsson, K., and Olsson, Woolley, D.E., Roberts, D.R., and Evanson, J.M. (1975) Biochem. Biophys. Res. Comm. 66, 747-754. Bauer, E.A., Stricklin, G.P., Jeffrey, J.J., and Eisen, A.Z. (1975) Biochem. Biophys. Res. Comm. 64, 232-240. Wells, J.N., and Hardman, J.G. (1977) Adv. in Cyclic Nucleotide Research 8, 119-143. Chamberlin, M.J. In RNA Polymerase, Ed. Losick, R., and Chamberlin, M. (1976) Cold Spring Harbor Laboratory. Huang, C.Y., and Kaufman, S. (1973) J. Biol. Chem. 248, 4242-4251. Huang, C.Y., Max, E.E., and Kaufman, S. (1973) J. Biol. Chem. 248, 4235-4241. Brew, K., and Powell, J.F. (1976) Fed. Proc. 35, 1892-1898. Kuo, W.N., and Kuo, J.F. (1976) J. Biol. Chem. 251, 4283-4286. Kuo, W.N., Shoji, M., and KUO, J.F. (1976) Biochim. Biophys. Acta 437, 142-149 Peters, S-P., Coyle, P., Coffee, C.J., Glew, R.H., Kuhlenschmidt, M.S., Rosenfeld, L. and Lee, Y.C. (1976) J. Biol. Chem. 252, 563-573. Eisen, A.Z., Jeffrey, J.J., and Gross, J. (1968) Biochim. Biophys. Acta 151,

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