Isolation and characterization of A peptide containing the site of cleavage of the chick skin collagen α1[I] chain by animal collagenases

Vol.

89,

July

12, 1979

No.

1, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

202-208

ISOLATION AND CHARACTERIZATION OF A PEPTIDE CONTAINING THE SITE OF CLEAVAGE --__I_ OF THE CHICK SKIN COLLAGEN CHAIN BY ANIMAL _____ __- al[I] -___COLLAGENASES" John H. Highberger,

Clare

Corbett

and Jerome

Developmental Biology Laboratory, Medical Massachusetts General Hospital and Department Harvard Medical School, Boston,Massachusetts Received

June

Gross

Services of Medicine 02114

4,1979

SUMMARY: A 36-residue peptide containing the bond cleaved by animal collagenases -__ was isolated from a digest of chick skin collagen nl-CB7 by Staphylococcus V8 protease. This cleavage site peptide, in contrast to the 36-residue al-CB2, showed no tendency to renature to the triple helical form, as monitored by molecular sieve chromatography and the determination of circular dichroism spectra. These results provide a direct demonstration that the conformation of the al[I] chain immediately around the collagenase cleavage site in the native molecule must be of a lower degree of helicity than other portions of the chain. This is considered to be an important factor in the collagenase specificity, in providing access to the sensitive bonds, but enzyme binding sites, probably located in the adjacent region(s) of maximum helicity,are also considered necessary to produce the maximum reaction rate. 1N_TRODLJCTION: As the collagenases 285,000,

chains

at a point

peptide

bonds

Gly-Leu

in

in a highly about

a-2

is well

it

highly

known that resistant

of sensitive

cleavage

site

fore

structural

specific

manner

the

chain

the

variation,

at a single from

and Gly-Leu coiled

action

Amino

of the

the

collagen

leading

in each

amino

Residue numbering The numbers cited additional triplet

begins with the first residue of in this paper have been corrected in al-CB7.

0006-291X/79/130202-07$01.00/0 Copyright 411 rights

@ 1979 by Academic Press. Inc. in unv form reserwd.

of reproduction

202

three The

(2). chain,

of the

and one

have

helix

molecule

of the

determinations

that

the

minimal

are met and that

helical

disruption

ren-

inaccessa-

around

shown

*This is publication No.779 of the Robert W.Lovett Memorial Group Diseases Causing Deformities. This work was supported by National Health grant AM 3564 and a grant from Hoffmann-La Roche Company 1

a-l

because

(6.7)

to localized

of the

terminus

structure

triple

of molecular

affected.

sequence

species

molecule

in each

are not helical

acid

of collagen,animal

locus

of most proteases,

of two different

formation

helical

are one Gly-Ile

tightly

bonds.

degradation

triple

length

775-7761)

to the

in collagen for

the native

Gly-Ile

peptide

requirements primary

Other

physiological

c 1 eave

(residue

(3,4).

in the

75% of the

cleaved

It

bility

step

(EC.4.24.7)(1)

weight

ders

first

thereat

the

for the Study of Institutes of

the helical portion of the chain. for a recently discovered (5)

Vol.

89,

No.

cleavage

site,

suggested might

of

that

to

the

Recently

V8

has

and

the

791,

of which

the

probable

the

native In

al-CB7, which the

is

previously

the

thus

should

been

cleavage

site

permitting

access

and the

nearest

same of

the

the by

of

by

with

of

give

peptide

an

chain

enzyme

each in

helix-coil

is are

which

transition

CNBr

the

of

side

peptide

peptide

opportunity

around

S.aureus

collagenases on

the

from

cleavage

animal

the of

a pep-

chain

the

a 36-residue

those

would

the

residues

Study

isolate

ill[I]

to

cleaved

in

center.

size,

of

glutamyl

result

comparison

to

essentially bond

the

possible

(al-CB7)

restricted

would in

be

chain

the

present

and

the be

communication helix-coil

referred

by AND

describe

transition to

transition

reported

we

herein

as

the of

characterization and

METHODS:

of

(xl-CB7

of

Sherman

of

al-CB2 assessing

cleavage

site

in

of,

cleavage chick rat

S.

aureus

from

site skin

skin

(CS) collagen

collagen

this

chick

skin

36-residue

oil-CB2 ctl-CB2

peptide For

peptide. are has

collager

comparison also

des-

previously

(10).

Preparation

by

isolation

characteristics

properties

Piez

the

of

Chick

Skin

Chick skin collagen was prepared from 3-week chicks, and al-CB7 and al-CB2 were isolated from al-CB2 was further as described by Kang et al.(ll). and Sherman (10). An analysis of the peptide as sequence (12) is shown in Figure 2. Digestion

of

cleavage

the

the

these

stability

A similar

MATERIALS

has

COMMUNICATIONS

molecule.

helix-coil

been

of be

and

nearly

exactly

it

glycyl-isoleucyl

peptide,

of

region

nature,

collagenase

can

at is

helical

will

cribed.

animal

776,

cleavage

this

that

segment

and

site

properties

of

Since

collagenase

(lo),

site

775

It

the

coiled

evident

action

residues

the

become

whose

between

specificity. in

tightly

RESEARCH

bonds.

(9), bonds.

this

BIOPHYSICAL

structure

less

appropriate

protease

755

or

AND

in

helical

the

glutamyl

at

the

the

of

a factor

sensitive

it

containing

a digest

be

a "loose",

enzyme

tide

BIOCHEMICAL

cannot

(2.,8) be

the

1, 1979

V8

Collagen

nl-CB2

and

al-CB7

old White Leghorn lathyritic the CNBr digest of this collagen, purified as described by Piez used is given in Table I, and its

Protease

al-CB7 was dissolved in 0.05 M NH4HC0 , pH 7.80, containing 0.05% Na azide, at a concentration of approximately 1 mg/m 2 . The solution was heated in a bath at 50" for 20-30 min. After cooling, S.aureus V8 protease (9)(Miles Laboratories) dissolved in the same buffer at a concentration of 1 mg/ml was added in amount sufficient to make an enzyme to substrate ratio of 1:30. The solution was placed

203

Vol.

89,

No.

1, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TAELE I AMINO ACID ANALYSES OF CHICK SKIN COLLAGEN CS PEPTIDE AND al-(I)CB2 CS Peptide HYP ASP Thr

2.49

5.34

1.12

0.12

0.85 0.77 3.82 3.33 12.12 3.44

Ser

Glu Pro GUY

Ala Val Ile Leu Phe

1.03 4.25 6.20 12.58 3.49

1.61 1.52 0.85

HY~ LYS

0.93 1.05

0.13

Arg Hse Total Residues Results than

Chick Skin Collagen CXI-CB2

0.1

*Includes

1.10 1.91

0.98

36

0.63" 36

are given as residues residue found. homoserine lactone.

per peptide.

A blank

indicates

less

at 37" for approximately 24 hours. At the end of this time another similar aliquot of enzyme was added and the solution was incubated at 37" for an additional 24 hours. It was then frozen and lyophilized. The enzymatic digest was fractionated on a 2.5 x 110 cm previously calibrated column of Sephadex G-50s in 0.03 M Na acetate, pH. 4.8 at 35". A typical chromatogram is shown in Figure 1. Eight reasonably well-separated peaks were obtained, of which Peak 7. corresponding to a molecular weight of 3,150, appeared to be the CS peptide. Fractions belonging to this peak were pooled, desalted on Biogel P-2 in 0.1 N acetic acid and rechromatographed on Biogel P-10 in 0.1 N acetic acid. The amino acid analysis of the CS peptide thus purified is given in Table I. Amino Acid Analyses: Samples for amino acid analysis were hydrolyzed under nitrogen for 24 hours at 110" in glass distilled constant boiling HCl. The analyses were run on a Beckman up-dated 121 analyzer with Autolab System AA Integrater, using the buffer system of Trelstad and Lawley (13). No corrections were made for destruc tion of amino acids during hydrolysis. Sequencing the CS Peptide: In order to be certain that the peptide isolated from Peak 7 of the S. aureus protease digest of nl-CB7, as described above, was in fact the CS peptide, it was sequenced, using the methods already described (5). The sequence is shown in Figure 2. Chromatography of Renatured ul-CB2 andoftheCS Peptide: crl-CB2 and the CS pep~__ tide were subjected to renaturing conditions by dissolving in 0.015 M potassium acetate, pH 4.8, at a concentration of 5 mg/ml, and maintaining the solution in a bath at 5' for 3 days. The solutions were then chromatographed on a 1.5 x 60 cm column of Biogel P-10 at the same temperature and in the same solvent. The column effluents were monitored by continuous determination of absorbance at 226 nm. Determination of Circular Dichroism Spectra: Circular dichroism (CD) spectra of the CS peptide and of al-CB2 were determined using a Cary Model 60 Recording Spectropolarimeter fitted with a Model 6001 CD accessory. The solutions were made

204

Vol.

89,

No.

1, 1979

BIOCHEMICAL

AND

200

BIOPHYSICAL

250

EFFLUENT

RESEARCH

300

VOLUME,

COMMUNICATIONS

350

ml

Fl&ure 1. Elution profile of S. aureus V8 protease digestion of 30 mg chick skin collagen (rl-CB7 (see text for details) on a 2.5 x 110 cm column of Sephadex I;-50s in 0.03 M Na acetate,pH 4.8, at 35". Flow rate was 21 ml/hr and fractions of 2.8 ml were collected. The horizontal bar indicates how fractions were pooled for isolation of the cleavage site peptide. Tritiated water eluted in this run at 491.5 ml.

Gly-Pro-Ala-Gly-Pro-Arg-Gly-Leu-Hyp-Gly-Pro-HypGly-Ala-Hyp-Gly-Pro-Gln-Gly-Phe-Gln-Gly-Pro-HypGly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-Ser-Gly-Pro-Met a

Lys-Gly-Ser-Hyp-Gly-Ala-Asp-Gly-Pro-Ile-Gly-AlaHyp-Gly-Thr-Pro-Gly-Pro-Gln-Gly~Ile-Ala-Gly-GlnArg-Gly-Val-Val-Gly-Leu-Hyp-Gly-Gln-Arg-Gly-Glu b Figure skin

2. collagen

Amino acid sequences CS peptide. Arrow

at the concentrations 0.15 M potassium the acetate buffer. 0.15 in a this mined were

AND

The

of

described adjusted

above to

chick

skin collagen bond cleaved

and renatured pH 4.8 with

nl-CB2 animal

by

(13); b, collagenases.

in the same way, a trace of sulfuric

except acid

chick

that replaced

After renaturation the solutions were quickly diluted about 30 times with M potassium fluoride,pH 4.8, in order to bring the readings on scale, and jacketed cell maintained at 4.5". After determination of the CD spectrum temperature, and the spectrum was redeterthe temperature was raised to 45", after establishment of equilibrium. The concentrations of the solutions determined by amino acid analysis of aliquots of the dilutions measured.

RESULTS

kept

fluoride

of: a, indicates

P-10

renaturing under

shown

by

chick

skin

used

DISCUSSION:

chromatograms,obtained

under

cold placed at

Piez

solutions

for

3 days,

conditions

the

same

and

Sherman

collagen

when

conditions,areshown

ctl-CB2

(10)

for

is

eluted

of were in

the

rat at

run

Figure

skin

a position

205

ctl-CB2 on 3.

collagen

and

of

the

a gel

filtration

It

seen

is

crl-CB2,

corresponding

CS peptide, column that,

the to

bulk a trimer,

as

first

of

the due

Vol.

89,

r

No.

I

BIOCHEMICAL

1, 1979

I

I

I

Trimer

1

I

I

AND

BIOPHYSICAL

COMMUNICATIONS

I

Monomer !

I

RESEARCH

0

P .p a:

-'

3 s . 8 Q v

-2

6 .

-3

i I

I I I

: ’I \ I I I \ I

s -4

: ’ ’ : ’ I ‘b’

-5 I 190 EffiUENT

03

VOLUME,

Chromatography Figure 3. on a 0.9 x 53 cm column of pH 4.8, at 5". Flow rate Dashed lines, collected. renaturation are given in

of

renatured solutions Biogel P-10, 200-400 was 46.0 ml per hr, crl-CB2. Solid lines, the text.

Figure 4. Circular dichroism equilibrated under renaturing Dashed lines, 1x1-CB2, Solid

to

the

association

small

fraction

elutes tion

of

the

the

the in

total the

molecules

remains

in

monomeric

form,

the .c

monomer and

I 230

I 250

I

LENGTH,

_

nm

of al-CB2 and of the CS peptide mesh, in 0.15 M sodium acetate, and fractions of 2.3 ml were CS peptide. Details of the prior

into the

I

shows

the

CS peptide, each (45") conditions. are given in the text.

triple

helical

form.

The

no

trace

of

the

form.

Only

CS peptide

of

a peak

a

however

in

the

posi-

trimer.

made 4,

WAVE

spectra of crl-CB2 and of (4.2" ,4.5") and denaturing lines, CS peptide. Details

3 monomeric

observation

spectra,

large

of

completely

This

Figure

of

I 210

I

04

ml

’ ’

as

was described

plotted

negative CS peptide

as

does

The

residue

effect not.

by

above.

mean

Cotton

confirmed

at Very

the

results

results

of

these

ellipticities. 197 little

nm,

determination measurements

Whereas characteristic

difference

306

renatured of

is

observed

the

collagen between

of are

the

CD

shown

I-rl-CB2 triple spectra

in

shows

a

helix, for

CS

Vol.

89,

peptide

No.

1, 1979

solutions

give

curves

exposed

indicative

Reference in

Table

in

behavior

not

high

helix. one

whole

of

results

perturbation

in

also

molecule

conditions.

that

the

their

and

which only

6,

the

is

for

Both

assume

the

proline

very

the

in

reaction

than

describing

protein

the

presentwork

i?rl[I]

difference of

similar

imino

to

that

the

16 of

and of

position

the

of

the

to

12

CS peptide,

hydroxyproline,

conformation

of

amount

In

Y position

helicity

the

the

adjacent The

native

helical

the

completion

the

isolation

of

of that

serum. is

the

this

polyproline CS peptide

triplet,

in

the

is

but

that

the

the

enzyme

gelatin

(denatured

collagen)

work

supports the

homologous

possible

that in

of

peptide contains

this

J. J.

and and

Lapiere, Nagai,

Y.

C.M.(1962), (1965),Proc.

specificity,

207

region

in

sites,

probably for

cleaved

maximum much

more

interpretation. Kleinman

binding

conformational

rat

et

al

(16)

skin

collagen

site

for factor

the

appeared, al-CB7. cell

demonstrated

case.

Proc. Natl.

a

We believe

REFERENCES Gross, Gross,

of

site

locus.

is

site

presence

cleavage

necessary

from the

the

also

this

paper

this

binding

are

(15)

the

molecule

cleavage The

collagenase

region(s),

peptide

important

in

collagen

chain. in

at

native

collagenase

the

residues

helical

this

this It

also

factor

collagen of

of

susceptibility

bond,

that

bulky

the

the

portions

of

important

fully

in

around

other

collagenase

sensitive

fact

showed

than

an

that

immediately

distribution

is to

demonstration

chain

to

acce.ss

workers

ment

a direct

contribute

rates.

After

1. 2.

al-CBZ,

the

together

are

at

of

content

hydroxyproline. which

that

reason

differing

3 of

in

to

hydroxyproline

are

to

that

compared

behavior

prolines

the

conformation

providing

slowly

the also,

provide

of

located

to

4 unhydroxylated

degree

this

probably

proline

amount

COMMUNICATIONS

(45")

as

show

transition

6 of

noting,

a lower

that

These

worth

2,

very

total,

force

the

may

is

denaturing

CS peptide,

Figure

itself,

acids to

of

and

RESEARCH

form.

the

helix-coil

the

of

(14)

of

peptides

conformation

unique

random

coil

BIOPHYSICAL

chain.

'These

of

the

two

of

only

al[I]

(4.2")

sequences

imino

is

renaturing

molecule

enough

It

to

the

one-third total

AND

composition

whose

collagen

however,

is

the

al,-CB2,

whole

the

the to

of

residues,

is

and

In

the

is

to

I,

acids.

of

BIOCHEMICAL

Natl. Acad. Acad. Sci.

Sci.USA 48:1014-1022. USA 54:=97-1204.

attachin

Vol.

3. 4. 5. 6. 7. 8. 3. 10. 11. 12. 13. 14.

15. 16.

89,

No.

1, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Nagai, Y., Lapiere, C.M. and Gross, J. (1974). Proc. Int. Congr.Biochem. 6th. 2, 170. Gross, J., Harper,E., Harris, E.D.,Jr., McCroskery,P.A., Highberger, J.H., Corbett, C. and Kang, A.H.(1974), Biochem. Biophys.Res. Commun.61:605-612. Highberger, J.H., Corbett. C., Kang, A.H. and Gross, J. (1978), Biochem. Biophys. Res. Commun. 83:43-49. Fietzek, P.P., Rexr0dtTF.W.. Hopper, K.E., Kuhn, K, (1973). Eur. J. Biochem. 38:396-400. gghberger, J.H., Corbett, C., Kang, A.H. and Gross, J.(1975), Biochemistry 14:2872-2881. Weiss, J.B.(1976), Int. Rev. Conn. Tissue Res. 7:101-157. Houmard, J. and Drapeau. G.R.(1972), Proc. NatlAcad. Sci. USA 69:3506-3509. Piez, K.A. and Sherman, M.R.(1970), Biochemistry 9:4129-4133; 41Ti-4140. Kang, A.H., Piez, K.A. and Gross, 3. (1969), Biochemistry 8:1506-1514. Kang, A.H. and Gras, J. (1970), Biochemistry _9.:796-804. Trelstad, R.L. and Lawley, K.R. (1976). Anal. Biochem. 70:287-289. Gross, J., Highberger, J.H., Johnson-Wint, B. and Biswas,~ C. (1979),& Collagenase in Normal and Pathological Connective Tissues. eds, D.E. Woolley and 3.M. Evanson, John Wiley and Sons, Ltd, Chichester, England, in press. McCroskery, P.A., Wood, S. Jr., and Harris, E.D., Jr. (1973), Science 182:70-7 Kleinman, H.K., McGoodwin, E.B., Martin, G.R., Klebe, R.J., Fietzek, P.P., and J. Biol. Chem. 253:5642-5646. Woolley, D.E. (1978).

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