Animal collagenases: Specificity of action, and structures of the substrate cleavage site

Vol. 61, No. 2, 1974

ANIMAL

Jerome

BIOCHEMICAL

COLLAGENASES: STRUCTURES OF

Gross, John

SPECIFICITY SUBSTRATE

THE

Elvin Harper*, H. Highberger,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Edward Glare

OF ACTION, CLEAVAGE

D. Harris, Corbett and

Jr.*, Andrew

AND SITE;:

Peter H.

A. Kang

McCroskery.

Developmental Biology Laboratory, Department of Medicine at the Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; Department of Medicine, Dartmouth Hitchcock Medical Center, Hanover, New Hampshire: Department of Medicine, VA Hospital and University of Tennessee, Memphis, Tennessee Received

October

lo,1974

SUMMARY: Two highly purified animal collagenases, one of the rabbit V2 ascites cell carcinoma growing in isolated from the media of tadpole tissue cultures (Y chains from chick and rat skin collagen, and the chick skin cul chains at one, and the same peptide Gly-Ile bonds exist elsewhere in the oi chain they A unique collagenases one in

quarter each

of

(1,

their

the of the

tadpole

2) is length

three

collagenase Nagai

one

feature

and

leucine

and from

tadpole

collagenase

two

native

however,

Harris AM

of

action

production

of a single

intact

(Y chains,

of most

collagen

first

of the

known

cleavage

molecule

demonstrated

from homogenates and the second isolated non helical peptide CB7 from Although two other not cleaved.

at a specific

from for

animal

the

locus

the

COOH-terminus

prototype

enzyme,

the

(3).

*
mode

of the

colleagues

termini

substrate

the

derived muscle cleaved GNBr bond. were

and 35506

(4)

reported

isoleucine collagen at

NH2-terminal

pH in

the

in

and

release

by

this

residues,

molecules

neutral

resulted

the

20°C.

release

solution Using

purified and

following denatured

of NH2-terminal

Memorial #644 of the Robert W. Lovett Supported by grants (AM Deformities. from the U.S. P.H. S., The Hoffmann and by the National and New Hampshire

Harper and AM

are the recipients 34222 from the

enzyme

three

of

glycine

attack

by

COOHpurified

collagen

as

leucine, Group 3645,

a

isoleucine, for AM

the Study 14780

LaRoche Co., chapters

of Research Career Development National Institutes of Health.

The of the

BIOCHEMICAL

Vol. 61, No. 2,1974

phenylalanine,

alanine, a purified

collagenase

carcinoma

(6)

liberating

Harris

with fragments

nodule

collagenase. McCroskery

is

both

a poor

collagen

contamination

has

now

high

end

it

site

in

This

the

7.6,

ation

the

(ZO-50%

accomplished had

a specific

5000

times

the

injecting

activity

0.005

M

CaC12,

saturation)

followed

Final

fractionation

agarose

of the

(Bio (mg original

and by by

Gel

collagen

A-l.

sequence

that

the

latter

reflected

the

Highly

NH2-terminal

in

of

thigh

freeze1,

sulfate

chromatography

resulting hr

per purified

DEAE

chromatography

mg

of

buffer

fractionon

enzyme

ascites of

0. 1 M Tris-HC

ammonium

V2

muscle

were

sieve

per

unique

bromide

the

homogenates

The

the

homogenates

into

molecular

collagenases

enzymes.

from

by

tadpole

cyanogen at

these

exchange

606

large

rheumatoid

around

sequences

purified

homogenate.

and

and

and

extracted

degraded

to

. (4) probably

cells

5).

as

reduced

locus

The

ion

gelatin,

single

purified

was

that

collagenase

by

(6).

fragments

purified

acid

tumor

previously

length

that

tumor

acid

was

locus

protease(

released

collagenase

cell

and

LY chains

fragment

that

a single

and

rabbit

amino

amino

ascites at

observed

synovial

other the

rabbit

noted

of Nagai

of

the

after

same

with

reports

activity

the

(5)

attacked

proposed

at

et al.

quarter

previously weakly

results

collagenolytic

on

that

(6)

study

three

rheumatoid

purification

described

A-50.

had only

enzyme

to

formed as

was

paper

containing

Sephadex

(7)

molecules,

tumor

and

Krane

helical

rabbit

thawed

and

triple

The

rabbits

quarter

earlier

possible

terminal

adult

one

gelatin

of

COOH

carcinoma

collagen

purified

tadpole

of the

cell

pH

and

degree

made

peptides.

denatured

associates

the

of

cleavage

and

partially

and

substrate;

The

of

collagen, by

Harris

homogenates

native

and

native

peptide

cleaves

both

Recently

from

characteristic

(Y chains.

compared

valine.

obtained

cleaved

the

from

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was preparation

enzyme)

tadpole

over tail

fin

Vol. 61, No. 2, 1974

collagenase

was

of Harper

et

saturation

precipitate)

limb

of the

affinity

BIOCHEMICAL

prepared

from

(8)

includes

al.

first

which

the

two

chromatography

graphy

on

bromide

purified

substrate

ratio stat

was

for

extensively

time

more

enzyme

further Since

with

pg

purified

the

larger amount presumably lyophilized,

T,

for

denatured

CNBr

rabbit

by

SDS

white

and

~2

chains

of

acid,

cul

lyophilized

was

incubated

rise

in

and

chromato-

chains mixed

mixture

was

cyanogen (12). in

alkali

cleavage

reaction

chicks

by

were

a maximum further

by

Leghorn chains

from

mixture

without

and

analyzed

this

0.005

M

column lesser

into amount

starting the

about

amino

C and

Incubation and CaC12

were

an

enzyme-

at

37°C

in

addition

(at

about

3

dialyzed

prepared

for

was

carried

out

either

of

The

a low

weight

molecular

region acid

composition

607

the

sequencing

lowest

of the

using

oi-CB7

or

al-CB8

reaction

products

material. molecular

cleaved and

enzyme

37”

bulk

at

tumor

of

it was

fragment,

starting The

this

with

and

material.

rabbit

reaction

26 hours.

of undigested

by

the

9. 0 mg

for

cleavage

electrophoresis

COOH-terminal for

22”

temperature

peptides.

polyacrylamide

undigested from

was

collagenase

7.6,

a Bio-Gel

peptide was

acetic

o2

ascending

purified

homogeneous

reaction

samples

at

tumor pH

on

the

added

oi-CB7

negligible

0. 1 Tris-HCl

tionated

M

(~1 or

The

(9).

old

prepared

method

(O-30%

further

columns

Purified

After

was mg

0. 01

was

monitored

and

then

ol

the

fractionation.

collagenase run

into

and

by

precipitation

3 week

were

periods.

Twenty

against

of

separated

(rl-CB7

1:80,

various

incubation).

without

skins

collagenase

of about

reached,

hours

and

tadpole

was 4B

(11).

ol-CB8

Purified

a pH

and

medium

on agarose.

peaks

from

(lo),

culture sulfate

collagen-Sephadex

carboxymethylcellulose peptides

day

chromatography

collagenase

collagen

and

third ammonium

by

on

Acid-extracted prepared,

crude

followed of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ol-CB7

sequenced.

was 200 in were

frac-

another The weight was

lowest fragment desalted,

Vol. 61, No. 2, 1974

Automated

sequential

890B

sequencer

were

dissolved

Quadrol by

back

hydrolysis,

the

shown

was

to

the

in

low

an

automated

weight

ol-CB7 line

(rl-CB7

(14)

and

3,

Table

from

established

both

cleavage

and

calf

(15)

site,

sequentially

10%

in

the

SP-400

fragment

isolated

chick

(16)

Since

SEQUENCES

AT

1 1)

Calf

Skin

2)

Chick

3)

(RT)*::’

Chick

4)

(Tp)::“>:

5)

6)

Skin

Ile

there

after

mixture

unique of

sequence

12 amino

at the one

instru-

analyzer

the

beginning only

CC-45

reaction

sequence

is

accomplished

acids

previously

Gly-Ile

sequence

I

ANIMAL

COLLAGENASE

CLEAVAGE

SITE

2

3

4

5

6

7

8

9

10

11

Ala

Gly

Gln

Arg

Gly

Val

Val

Gly

Leu

Hyp

12 Gly

Ala

Gly

Gln

Arg

Gly

Val

Val

Gly

Leu

Hyp

Gly

ol-CB7

Ile

Ala

Gly

Gln

Arg

Gly

Val

Val

Gly

Leu

Hyp

Gly

Chick

ol-CB7’

Ile

Ala

Gly

(TP)

Chick

crl

Ile

Ala

Gly

Gln

Arg

Gly

Val

-

Gly

Leu

-

Gly

(TP)

Chick

cu2

Leu

Ala

GUY

= Rabbit

c~l-CB7::

the

was

the

Ile

::iSequence skin, and Highberger, ;:*RT

ol-CB7::’

the

samples

with

acid

gave

skin

TABLE NH2-TERMINAL

from

to

The

Beckman

amino

collagenase

identical

Gly-He.

5AH

model

(13).

degraded residues

model

and

Begg

of

on

is

and

a Beckman

Identification

tumor

This

with

Edman

JEOLCO

purified 1.

performed of

water

Program.

molecular

chick

method

glass-distilled

Cleavage

also

in

degradation

chromatography

and

in

in

gas-liquid

ment

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

according

Single

The

BIOCHEMICAL

taken from the sequence Corbett, Tumor

that for Kang

published by Fietzek et chick s’kin was obtained and Gross.

Collagenase;

Tp

= Tadpole

608

al.

(16) from

Collagenase

for entire unpublished

(rl-CB7 data

of calf of

BIOCHEMICAL

Vol. 61, No. 2, 1974

in

al-CB7

chick an

the

skin

order

(~1 type

artifactual

a free

with

tadpole

was

identical

cleavage

site

crl

an

collagenase;

no

Analysis

(line

the

chick

the

Table

obtained chick

yielding

a predominant

predicted

from

We

have

shown

divergent

species,

molecule

in

bond; the

the

Gly-Ile

Thus

collagenases enzyme,

is

of the

NH2-terminal

in

al-CB8,

made

to

cleave

was

noted.

the

unresolved, chick

one

peptide

terminal

substrate

the

the

blocks

apparent (Table

three

product of

the

1, was

residues

NH2-terminus

the

rabbit

skin l/4

the

tumor

01

chain

length

amino

fragterminus

positions

8 and

that

these

are

line

2).

also

accomplished,

beginning

of

11 in identical

Cleavage

with

the

of

leucine

as

(4).

or cul-CB7.

peptide specificity The

identical.

one

reacted

side

of the

at

collagenase

purified

in

is

pi-CB7

of

form

the

or

was

chick

end

residues, it

mammalian

adjacent

and

probably

resulting

with

of

acid

tadpole

highly

native

the

near

products

two

studies

and

cleaves

this

amino

skin

with

that

the

located

reaction

were

sequence

homologous

CYZ chain.

tumor

both

residues

calf

product (17))

from

for

(rl-CB7’,

of a lactone

obtained

Although

earlier

skin

sequence

sequence

the

site

deamidated

instead

(18).

cu2 chains

cleavage

chick

pyrrolidone-carboxylic

from

purified

a partially

of

1).

from

I);

sequence,

those

(either

unfractionated

cyl chain

skin

with

was

side

Purified

present

sequence

5;

intact

also

degradation

of

either

known.

three (Table

effort

a single

ment

the

first

cul-CB7 is

on

homoserine and

the

Gly-Ile

now

(rl-CB7

carboxyl

of

chain,

yielded

of

with

acids

is

collagenase

Since

of

chains

variant

containing

the

of amino

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tadpole

denatured

animal and

one

as

isolated In

collagenases amphibian,

Gly-Leu

in for

these

the

the

same

single

collagen peptide

enzyme

cleaved

of the

different that

widely

specific

region

like at

one

tadpole

same two

collagenase, (Y chain

at

the the

two

cleaved

a chains

addition,

609

from

isolated

animal from

the peptide

mammalian bond

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H al

610

Vol. 61, No. 2, 1974

and

does

not

The site

is

al

we

chain

residues,

it

other cul-CB8

species. chick calf

unique

the

in

other

of

been

in

the We

very

and

rat

(rl-CB8 that

of

and

III

be

the

region

predict

to

region

we

expect

the

cleavage

entire

There the

is

chain

in

chain

CY chain

but

varying

in

length

of

rat

species between

of

ai-CB7

essentially

no

reason

to

suspect

characteristics

that

of

animal

sequence

the

of

in

this

ol-CB7

determined.

region

in

region

synthetic

specific

those

collagenases

to be

site

helix

from

found

yet

A study

and

from

different the

of

of

Gly-Ile

in

qualitatively

in skin

a single

ol-CB7

cleavage

susceptibility

and

in

the

relative

1,052

surrounding

reasons

of the

the

in

Gly-Ile

region

for

CYI-CB7.

site

homologies

acid

in

in

ol-CB6

site

likely amino

sequences that

be

seems

~2

crl

physical

should

the

skin

Those those

cleavage

surrounding

close

identical.

particular

to

also

the

of the

those

the

from

It

the

homologous

this

are

that

of

that

and

in

molecule.

cleavage

residues),

anywhere

calf

from

glycine.

the

with

namely,

of cri-CB7

for

homologous

collagens

homologous

region

side

repeated

region

differ

for

(279

the

Apparently,

either

not

peptides

sequences

affinity

closely

are

this

(15)

(rl-CB8

cleaved.

on

sequence skin

of

would

they

containing

enzyme.

however

calf

cul-CB7,

not

cul chain,

and

regions a specific

either

two

reported.

whereas

of

sequenced,

site

peptide,

is

that

These

except

previously

10 residues

the

20-residue

(16)

as

bond,

comparing

1).

cleavage

have

in

worth

examination

in

will

the

position

simple

II

that

cul-CB6

every

are

know

The

both

and

action

regions

yet

enzymes,

the

(Figure not

both

for

is

known

CNBr

peptide

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

scissions

residue

for

a Gly-Ile

Since

in

268

required

have

multiple

a substrate

not

the

make

isolated

containing is

BIOCHEMICAL

CYZ-CB5 in

substrates amino

acid

residues

profitable.

BIBLIOGRAPHY 1. 2. 3.

Gross, Harris, Gross,

in J.. E.D., J. and

the Harvey Lectures, Jr., and Krane. Nagai, Y., Proc.

S.M., Nat,

1974, in press. N.E. J. Med., Acad. Sci. U.S.

611

type

in 54,

Press. 1197

(1965)

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. 9.

Nagai, Y., Lapiere, C.M. and Gross, J., Proc. Sixth Congr. Biochemistry II: 120 (1966) Harris, E.D. Jr., Faulkner, C.S. II, Wood, S. Jr., Biochem. Biophys. Res. Commun. 48: 1247 (1972) McCroskery, P.A., Wood, S. Jr., and Harris, E.D. Jr., Science -’182 70 (1973) Harris, E.D. Jr., and Krane, S.M., Biochim. Biophys. Acta 258, 566 (1972); Harris, E. D. Jr., J. Clin. Invest. 2973 (1972). -51, Harper, E., Bloch, K. J. and Gross, J., Biochemistry 10. 3035 (1971). Eisen, A.Z. and Jeffrey, J. J., J. Clin. Kvest. 50, 2056 Bauer, E. A., -

10.

(1971). Kang,

4. 5. 6. 7.

11. 12. 13. 14. 15. 16. 17. 18. 19.

20.

A-H.,

Nagai,

Y.,

Piez,

K. A.

and

Gross,

J.,

Biochemistry

2,

509

(1966). Piez, K.A., Eigner, E.A. and Lewis, M.D., Biochemistry 2, 58 (1963). Piez, K.A. and Gross, J., Biochemistry 8, 1506 (1969) Kang, A.H., Edman, P. and Begg, G., Eur. J. Biochem. 1, 80 (1961) Pisano, J. J., Bronzert, T. J. and Brewer, H.B., Anal. Biochem. -’45 43 (1972). Fietzek, P.P., Rexrodt, F. W., Hopper, K.E. and Kuhn, K., Eur. J. Biochem. 38, 396 (1973). HighbergerJ.H., Corbett, C. M., Kang, A.H. and Gross, J., in preparation. Butler, w.T., Piez, K.A. and Bornstein, P., Biochemistry, 3771 (1967). Kang, A.H. and Gross, J., Biochemistry Click, E.M. and Bornstein, Balian, G., Balian, G., Click, E. M., Hermodson, 11, 3798 (1972). vonder Mark, K., Rexrodt, vendt, P., 30: 169, 1972. -

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9, PT, M.A. F.,

796 (1970). Biochemistry and and

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K.,

(1971); Biochem.

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