Intramolecular cross-link of chick skin collagen

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INTRAMOLECULAR CROSS-LINK Andrew

H. Kang+,

OF CHICK SKIN COLLAGEN*

Barbara

Faris

and Carl

Franzblau*

Developmental Biology Laboratory, Departments of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts.

ReceivedJune

4, 1969 Summary

Purified chick skin collagen constituent chains were examined for the content of the aldol condensation product of two residues of a-aminoadipic b-semialdehyde after reduction with NaBT4 and alkali hydrolysis. The single chains, ctl and c&Z, contained none whereas their cross-linked dimer, 812, contained one equivalent. These findings were confirmed by the similar studies performed on the peptides derived from the cross-link region of collagen by CNBr cleavage. Data presented here strongly indicate that the aldol condensation product is the intramolecular cross-link. Definitive full

understanding

knowledge

after

the

truded

of time

synthesis

into

extracellular

skin

collagen

(Bornstein

collagen

(Kang

near

deaminated

and Piez,

et al.,

al well

occur

tissue.

the

crosslink.

was not

the presence

obtained.

with

sodium

(Paz et al.,

and CQ chains as the

Using

and the cross-linked

cyanogen

bromide

(CNBr)

dimer peptides

become for

is ex-

formation on rat

and on chick lysyl

and that

however,

residues oxidatively they

then

the chemistry

Lent

et al. (1969) have Aproduct (ACP) of two residues

condensation

these

the studies

1968)

residues

This

and hydrolyzing

is

chains

evidence,

shown to be present 1969).

and the molecule

two specific

polypeptide

in elastin.

borohydride been

molecule

et al.,

that

Recently,

of the aldol

in the collagen

Recent

Rojkind

indicate

Direct

requires

One of these

cross-links.

of adjacent

of collagen

is complete

to form a-aminoadipic&-semialdehyde to form

same ACP has also collagen

that

1966;

1969b)

of cr-aminoadipic(J-semialdehyde elastin

maturation

chains

covalent

the NH2-termini

of the cross-link described

changes

connective

interchain

skin

condense

dependent

of polypeptide

of intramolecular,

located

of physiologic

was achieved the protein

in small procedures, of these, derived

amount

by reducing in

2 N NaOH.

in calf

we examined

the purified

the 812 component, from

The

skin as

the cross-link

* This is publication #478 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. This work has been supported by Grants from the National Institutes of Health (AM 3564 ; AM 07697) and the RGK Foundation. + Senior Investigator of the Arthritis Foundation at the Massachusetts General Hospital. * Established Investigator of the American Heart Association. 345

Vol. 36, No. 3, 1969

region

BIOCHEMICAL

of chick

skin

intramolecular adipic

collagen.

cross-link

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In this

in chick

paper,

skin

we report

collagen

is

evidence

that

the

indeed

the ACP of a-amino-

according

to the procedure

&-semialdehyde. Materials Chick

skin

collagen

of Kang et al.

(1969a).

chromatography

of gently

as described tides

from

sors,

al-CBl

analyzed

et al.,

as the peptide were

described

for

ninhydrin

activity

in the

counter

employing

That

portion

reactivity

individual

with

precur-

was performed

of the al,

amino

--et al. (1965). of the eluent

(Bray,

and their

a2 and out in

(1968). acid

In all which

in fractions

was determined

solution

CNBr pep-

NaBT4 was carried

et al.,

was collected

fractions

Bray's

Reduction

by

on CM-cellulose

of the Bl2-CBl,

on a Technicon

by Burns

was employed.

peptide,

by Gallop

obtained

collagen

and CXZ-CB~~'~,

1969b).

performed

were

Preparation

fragments

as described

analyses

chains

solubilized 1969b).

CX~-CB~A'~

region, (Kang

the gradient device

and f~2

and the cross-linked

previously

acid

al,

(Kang et al.,

M EDTA, pH 9.0,

ploying

a2,

and a2-CBl, as well

Amino stream

Purified

the cross-link

p12 chains 0.001

and purified

heat-denatured,

elsewhere

as described

and Methods

was isolated

analyzer

em-

cases

a split

had not

been

of1.3

in a liquid

ml. Radio-

scintilation

1960).

Results The al

and a2 chains

tained

from

chick

of the preparation normal

and the 017 component.

lathyritic

chick

skin

collagen

in a sealed

corresponding It

is

in al Imole. per chain

collagen

by the @ components.

Each was.reduced hours

skin

alkali-resistent

from

the data

ob-

was obtained

in 2 N NaCH at 110'

The radioactivity

and the reduced the

were

contamination from

above.

vial.

that

possible

and hydrolyzed

to e-hydroxynorleucine

clear

and a2 chains

The 812 component

as described

separately

The al to minimize

reduced

ACP is

in the

ACP ire,given

present

for

22

fractions in Table

I.

in 812 and not

only

The specific activity of the reduced ACP was 2.1 x lo6 dpm per or a2. Based on the ninhydrin reaction, there was 0.4 residue of reduced ACP two tyrosyl (both

residues

al

and a2,

812 component, hydrolyzed

there

reduced

in the reduced

in 812. 0.8

812 is

then

also

observed

chromatogram

for

the reduced

ACP upon amino

acid

The presence

residue

shown

of reduced

hydrolyzed

disappear.

ACP has been

is

there are two tyrosyl residues per 0: 1969b) and four tyrosyl residues per

see Kang et al., is

ACP region

and phenylalanine,

Since

in elastin

812.

If

in 6 N HCZ‘, the counts

Similar (Lent

$12 component in Figure

ACP per

1.

acid et al.,

lability

Both al

which

of the

1969).

in the region

the alkali-

The amino between

and 02 did

not

occur

reduced acid

isoleucine contain

analysis. of the ACP in 812 and its 346

absence

in al

and ~0 suggests

any

BIOCHEMICAL

Vol. 36, No. 3,1969

TABLE I.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Total Counts in s-Hydroxvnorleucine

and the Reduced ACPa

e-Hydroxynorleucine

Reduced ACP 260

22,000

al a2 812 fS12 acidb

16,850 4500

200 26,800

(200

(200

The amount of protein was a. Total counts per 10 mg of protein. calculated from amino acid analyses assuming four residues of tyrosine, eighteen residues of valine and twenty-seven lysine residues per 01chain. b. Hydrolysis in 6 N HCl was preceded by hydrolysis in 2N NaOH.

LEUCINE

I .o 0.0

? F

0.6

I I

96

I

PHENYLALANINE 1

I

II

I

98

100

102

I

I

104

I

I

I

106

I

100

I

I

110

I

II

112

f RACT/ON NUM0ER Figure 1.

The amino acid chromatogram of reduced $ in the tyrosine region where the reduced ACP is et2uted.

strongly that ACP is the cross-linking amino acid. This conclusion is further supported by the data obtained from studies on the CNBr peptides derived from the cross-link region of the collagen molecule. The CNBr Peptides from the Cross-link Region, c~l-CBl~'~, cz2-CBIAld and j313-CBl. Examination of these peptides, derived from the NH2-terminal region 347

Vol.36,No.3,

1969

of collagen

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chains

and cr2-CBIAld

contained

the presence no counts

by CNBr cleavage,

or ninhydrin

eluted.

Both

reduced

&2-CBl

hand,

the

is

tyrosyl

residues ACP.

amounts

of the reduced

digest

is

than

lished

acid

where

showed

a relatively

1969b),

been observed

The values

one normally

finds

(-)

REDUCED

obtained after

for

reduced

There

large

peak

there

is

there

0.4

of three of the

decreased

of an elastase

ACP were

of elastin

the

are

residue

However,

reduction

reduced

for

profile

that

value. after

reduction

fact

were

ACP normally

and radioactivity

406 of the theoretical

ACP have

indicating

peptides.

the

Based on the

(Kang et al.,

only

al-CBIAld

c-hydroxynorleucine

region

2.

results.

in these

chromatogram

in Figure

in fq2-CBl This

of elastin.

lower

@12-CBl

amino

given

reduced

in the

following

for

&semialdehyde

peaks

On the other ACP.

the

a radioactive'peak

of a-aminoadipic

reduced

gave

significantly

(C. Franzblau,

unpub-

data).

TYROSINE

ACP

0.4-

- .___ - --_-__-______--o,,

1 100

I

I 102

I

I 104

I

I 106

I

I 108

I

I 110

I

I 112

I

I 114

I

'0 116

FRAC T/ON NUMBEff Figure

2.

The tyrosine and radioactivity

region of the amino acid chromatogram profile of reduced f312-CBl.

Discussion Spectral

analyses

the cross-link and Pies, 1969b)

containing

1966;

Rojkind

suggested

exact

structure

isolation

benzothiazolone from rat @12-CBl,

peptide, et al.,

the presence

1968)

and chick

of an &B-unsaturated

of the cross-linking

and characterization

semialdehyde amounts

of the N-Methyl

from

in calf

intramolecular The presence

elastin skin

acid

skin

collagen

was not

(Lent

et al.,

1969),

(Paz et al.,

1969)

and its

established

then.

of cr-aminoadipic presence

stimulated

of

(Kang --9 et al However, the

aldehyde.

of the ACP of two residues

collagen

cross-link

amino

hydrazone derivative skin collagen (Bornstein

The 8-

in small

us to explore

the

of collagen.

of stoichiometric

amounts 348

of

the ACP in the

cross-linked

812

Vol. 36, No. 3, 1969

component

but

evidence

BIOCHEMICAL

its

that

absence

in the

the ACP is

data

obtained

from

link

region.

The acid

the

Although

the studies

is

only

that

common to rat

skin

chains,

al

This

of the

lability

chick

indicates

single

cross-link.

and a2,

is further

CNBr peptides

skin

derived

explaLns

the

studies

on rat

skin

and chick

skin

in this

study,

was examined

and chick

of intramolecular

skin

collagens

(Kang

Hence we suggest acid

the cross(Lent -et to identify

failure

collagens. available

cross-link et al.,

that

in vertebrate

by the

previously

study

collagen

strong

from

present

the mechanism

M 0, manuscript in preparation). molecular cross-linking amino

provide supported

of the ACP as described

in the al. 2 1969) and observed the cross-link in the previous evidence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

formation

1969b;

the ACP is

Rojkind, the

intra-

collagens.

References Bornstein, Bray,

P.,

G.A.,

Burns,

Anal,

J.A.,

Gallop,

P.M.,

Kang,

H.,

Henson,

E.,

J.

of Chromat.

and Schneider,

and Gross,

J.,

Biochemistry,

A.H.,

Piez,

K.A.,

and Gross,

J.,

submitted

M.A., M.,

A.L.,

(1965). Biochemistry

in

press

(1969a).

to Biochemistry

for

publi-

(1969b). Smith,

B.,

Salcedo,

L.,

Faris,

B.,

and Franzblau,

C.,

Biochemistry,

(1969).

Lent,

Gallop,

20, 310

(1968). K.A.,

R.W.,

Rojkind,

(1960).

and Kaeser, O.O.,

5, 340 (1966).

Piez,

in press Pas,

1, 279

C.F.,

Blumenfeld,

Biochemistry

A.H., cation

Lent,

K.A.,

Biochem.

Curtis,

7, 2409 Kang,

and Piez,

P.M., Rhi,

R.W.,

Faris,

Biochem. L.,

B.,

Franzblau,

Biophys.

and Aguirre,

Res. M.,

J.

349

C.,

Blumenfeld,

Corenun. Biol.

34,

229

Chem. 243,

O.O.,

and

(1969). 2266

(1968).