Mature collagen crosslinks

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATURE COLLAGEN CROSSLINES Department

Received

October

Norman R. Davis and Oksana M. Risen of Oral Biology, University of Alberta, Edmonton, Canada, T6G 2H7

15,1974

SUMMARY: During incubation with physiological buffers at 37', as well as during in vivo maturation, native collagen fibers display a progressive increase in tensile strength and insolubility. This is paralleled by a progressive loss of reducible, intermolecular crosslinks. The experiments described in this paper indicate that nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible crosslinks transforms them into more stable, non-reducible crosslinks. Indeed, modification of lysine/hydroxylysine residues completely blocks this transformation, while modification of his, arg, glu and asp is without effect. On the basis of these and other experiments, tentative structures are proposed for the stable crosslinks. INTRODUCTION: collagen since

Formation

molecules these

ubility

endow

to their

crosslinks

reaction

between

hydes,

as indicated

lysine

These collagen

progressively into

the fibers

in

figure

more stable,

(I,

residues

crosslinks,

form

non-reducible

crosslinks

PCH (OH) CH2NHCH2C

rather

and in vitro deduced

that

they

of undetermined

~(~H)c~,NHcH-CH(~H)P

I

: FOIWATION

OF REDUCIBLE

COLLAGEN

CROSSLINKS.

to

6, 7).

in native

aging,

III

FIGURE

by

alde-

(5,

rapidly

PCH(OH)CH,N-CHCH(ON)P

(0) P #-H

produced

can isomerize

are

they

I I

are

transformed

structure

PCH(OH)cHO

PCH(OH)CH2N-CHCH2P

inter-

dehydro-hydroxy-

intermediate

PcH2C”O‘rPC”‘““)CH2NH2~

I

and insol-

and lysine-derived

crosslinks

therefore

fibers,

formed

an a-hydroxy-aldimine,

maturation

tropo-

strength

2, 3, 4) to be aldimines

an enaminol

during

of collagen

The initially

1) via

Bailey

(5).

is

between

the mechanical

One of these

which

figure

However, lost

with function.

1.

(II),

crosslinks

the maturation

or hydroxylysine

electrophilic

fibers.

in

been shown

(III,

reducible,

step

biological

have

lysinohydroxynorleucine the a-amino-ketone

intermolecular

a critical

crosslinks

essential

molecular

is

of stable

(8).

The

Vol. 61, No. 2, 1974

present that

experiments crosslink

residues

were

performed

transformation

Calf

bone

and dentin

collagens

buffer

under

vitro

"aging"

of toluene

filtered

off

dry)

5 mM acetate this

procedure

leaves

while

0.05

modification

M tris). NaCl and -in

dry)

also

also

reversibly

blocks (pH 7.0)

modification

citraconic

anhydride titrated

0.7 ml of citraconic

his, for

M NaCl

at pH 8.5

to pH 8.5 anhydride

for

the his

and hyl

residues (11).

724

all

tris). (600

at pH 7.0 in hours

at 20'.

was regenerated at 20'

of lys

added,

one

1 ml of

of collagen

eighteen

(10).

by This

intact.

was achieved Samples

in 7 ml of water

were

for

with

(pH 7.4

(10)

one hour

and hyl

1 ml of

at 8.5 + 0.1

by treatment

derivatives of lys

bath,

was

and the acetylated

0.15

ethanol

0.5 M borate

treatments

performed,

10 percent

1972).

the collagen

on an ice

one-hour

blocked

(Davis,

in

The pH was held

with

(GEE) , or

by the following

while

buffer

with

ester

previously

Then,

repeatedly

the ethoxycarbonyl

were

ethyl

10 ml of 0.1 M diethylpyrocarbonate containing

by treatment

was equilibrated

borate

were

modified

acetylated

Four additional

1 M hydroxylamine

Reversible with

dry)

at once.

and washed

with

procedure with

mg, blotted

with

at 4' in the tris

as described

was removed.

all

residues

buffer

treatment

treatment

to yield

extraction

(pH 7.4,

(EDC) and gly

at O", pH 8.5 + 0.1 were

Lys and hyl

Since

stored

were

completely

ice-cold

of 5N NaOH.

anhydride

mg, blotted

in subsequent

(600 mg, blotted

was added

by addition

collagen

and used

of collagen

and the buffer

anhydride

acetic

crosslinks

1.0 M NaCl

were

were

in 30 ml of fresh,

acetic

1973)

collagen

by repeated

with

and NH Cl, 4

residues

Collagen (pH 8.5)

stirred

decalcified

carbodiimide

carbodiimide

Lys and hyl

buffer

(Davis,

of nucleophilic

obtained

and asp residues

the above

procedure.

proposal

bond of the reducible

extracted

N-ethyl-NFdimethylaminopropyl with

addition

the

studies.

The glu

hour

were

thereby

a layer

to test

crosslinks.

M EDTA at pH 7.4 and then

The insoluble

in order

double

non-reducible

METHODS:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

involves

to the electrophilic

more stable,

0.5

BIOCHEMICAL

of collagen

and stirred

at once.

by (600 rapidly

The pH of the

Vol. 61, No. 2, 1974

vigorously NaOH.

BIOCHEMICAL

stirred Within

mixture

ten

was maintained

to fifteen

minutes,

reacted.

For more extensive

anhydride

at pH 8.5 + 0.5 was repeated

hyl

residues

could

37'

overnight

of free

with

four

days

(1 ml/30

lates

all

unmodified His

1.04

residues

ment continued

amino

point,

formate

with

pH 3.5

were

for-mate

All

the

0.1 M formate

in 0.15 removed

with

in acetylated,

lys

and

(pH 3.5)

at

completely

M borate

by stirring (pH 10.5)

and irreversibly

for

cyanoethy-

residues. by stirring

anhydride) Every fresh

analysis

citraconylated,

was determined

in 0.075

modified

at 5'.

acid

by stirring

the dark were

and extent

with three

200 mg samples

or four

days the

0.3 M iodoacetamide, revealed

protecting

of

10 ml of 0.3 M iodoacet-

groups

0.3 M iodo-

and the

a maximal

600 mg of citraconic

M NaCl

of reducible

treat-

carboxymethylation

were

removed

anhydride

at 37'

treated

in 0.1 M triethylamine When all

from

lys/hyl

as previously

crosslink

and controls

(pH 7.4 in 0.05

at intervals

and asp residues

(13).

removed

collagens

RESULTS AND DISCUSSION: glu

0.7 ml of citraconic

(pH 11)

the arg was blocked, by treatment

the citra-

at 37'

overnight

(11).

unmodified

determination,

or

(11).

groups

The rate collagens,

were

the citraconyl

days in

protecting

at 37'

with

collagens

560 mg of 1,2-cyclohexanedione

one to six

cony1

or thrice.

(12)

citraconic

buffer

Arg was modified

for

twice

residues

This

of collagen

until

with

and lys untreated

was replaced

At this

collagen

with

and hydroxylysine

with

solution

in pH 3.5

had hydrolyzed

treatment once,

M acrylonitrile

lysine

in pH 7 phosphate

of his.

hyl

mg of collagen).

(pre-treated

acetamide

the anhydride

by treatment

treated,and

collagens

amide

all

of 5N

(11).

diethylpyrocarbonate

collagen

at pH 8.5 + 0.5 by addition

modification,

be regenerated

The content

these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

reduction described

As indicated

of collagen

M tris with

transformation

was determined or phosphate

after

in modified incubation Samples

buffer).

NaBH4 and reducible

crosslink

(14). in figure

has no discernible

725

2, modification effect

of his, on reducible

arg, cross-

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REDUCIBLE CROSSLINKS Fradion of initial cnnc.l 1.0 0.8 0.6 0.4 0.2 b 12 I8 24 Day8 FIGURE 3 REDUCIBLE CROS5LINKTRANSFORMATlON RAXS --- Collagens mdlfied with acrylonltrllea. acetic anhydride. , ethoxyformlcanhydrlder. and citraconic anhydrldeoto block lysine and hydroxylyslne residues. --+Collagens madified with citraconic anhydrlde. tested for blockage of transformation, and then de-modified at DH 3.5

link

transformation

extent

since

as unmodified

specific

of reducible

collagen

samples

were

in which

as in untreated

held

normally pletely

blocked

maleic

blocks

3).

one lys/hyl

and hyl

Control

3).

groups

same

blocks

acetic the

trans-

in which

and temperature

reagents,

were

with

experiments

composition

modification

crosslinks

modification

anhydride)

indicates

is

blocked;

transformed

Modification

in which

citraconic residues

of lys that

and hyl

when lys

but when the

to pH 3.5 formate,

all

of

yielded

at the

eleven

anhydride irreversibly

citraconyl

group

at pH 3.7,

with

produces

a collagen

which

displays

lys/hyl

yields

same rate

cyanoethylated. regeneration

lys

residues

the same kinetics

are

maleyl)

groups transform

and three

hyl

1000 were

the remaining

However, of the

residues

com-

Acrylonitrile per

with

citraconic

collagens

crosslinks.

a product

726

(or

the de-modified

of reducible

with

and hyl

citraconyl

of as few as eight

the transformation

of collagen with

without

reversible

by exposure

(figure

treatment

(figure

the reducible

transformation

are removed

and to the

collagen.

(or

blocked,

same rate

completely

at the pH, solvent but

Furthermore, anhydride

oflys

or acrylonitrile

crosslinks

above modifications,

collagens

age at the

modification

diethylpyrocarbonate

formation

the

collagens

collagens.

In contrast, anhydride,

these

eleven

previously twenty-

removal

of the

lysfhyl

residues,

of crosslink

transform-

Vol. 61, No. 2, 1974

ation

as does unmodified

ethylated

lysfhyl

by blocking

resulting

adjacent his

employed

expect here

no effect

three

transformation

on the reducible

lys/hyl

residues.

C-terminal

crosslink

arg

would

residues,

trans-

Indeed, are

all

perturbations,

with

appear

nucleophilio

the bulky

that

groups have

lysfhyl

attack

by sterically

three

residues.

Such modifications

therefore

and not

perturbation

acidic

to steric

residues

cyano-

of crosslink

and three

sensitive

by preventing

crosslinks,

steric

of eleven

transformation.

It

on transformation.

that

inhibition

accessible

crosslink

of twenty-one

the total

were

of these

presence

unlikely

and the

transformation

to inhibit

blocks

for

crosslink

blockage

the

therefore

from modification

crosslink

one would

is

responsible

of the (xl chain,

collagen

hyl

It

to the N-terminal

residues

ation

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

despite

collagen-

residues.

groups

formation

If

BIOCHEMICAL

modific-

of lys

perturbing

and/or

the

collagen

structure. These

results

links

in vitro

philic

addition

at 37', of lys

outlined

in figure

11 is

the electrophilic

4.

crosslink

nucleophilic

addition

necessarily

yield

with

can join

Crosslink

suggest

as well

hyl

The only

III

of lys

products

V may also

is

react

transformation

normal

residues reactive

centre

These

up to three further

PCH(XICH$=CHCHlXIP I or II

with

electrophilic

while

which

PCHlXICS$W~CfOIP

PCHIXlCt$VH )CHCHIXIP PCHlXG+NH ,“a or b PCH(XIC+NHZ __ -50

FIGURE 4: FORMATlON OF STABLE COLLAGEN CROSSLINKS.

727

centre Hence,

must are not

tropocollagen

PCHIXlCk+jNHt __

I and

the GO bond.

crosslinks,

nucleophilic

bond as

reactive

crosslinks

another

nucleo-

crosslinks

reducible

different

X = H or OH.

in

cross-

double

the only

carbon

to these

involves

in reducible

the electrophilic or hyl

of reducible

maturation,

to the

of the C=N bond,

IV and V.

together

that

as during

and/or

carbon

in reducible

NaBH4,

strongly

reducible

molecules. lysthyl

residue,

Vol.61,No.2,1974

expelling

BIOCHEMICAL

water

crosslink.

to form VI,

Crosslinks

tensile

disruption

collagen crosslinks

extensive

lys

I,

II

to I,

on three resulting

would

thirty

treatment

cent

collagens

linked

by IV,

crosslinks yield

V or VI.

smaller

prior

treatment

lys/hyl

residues

released

lys/hyl

residues,

which

reducible

crosslinks

appropriate igating structure

these

from should

ACKNOWLEDGEMENTS: Canada for financial

collagen The authors support,

the

stable

in

specific order

for

to provide

lys/hyl.

regenerated. How-

and to 70 perNaBH4

peptides

cross-

these

mature

these,

peptides

will

after

thermal

treat-

identification

in amounts

are

acids

blockage

then be labelled

when in vitro

crosslinks.

containing

crosslinks

cross-

in 0.1 M HCl for

soluble

Furthermore,

could

that

of mature

and only

permit

the more

be cleaved

1000 residues

crosslinks

be released

reagent

possibilities

of stable

reducible

should

released,

radioactive

these,

crosslinks.

should

found

dilute

yield

V and VI are

fiber.

at 70'

of peptides

because

containing

they

CNBr cleavage

Identification

the mature

to thermal

to cold,

and

molecules,

some reducible

HCOOH could

be possible

peptides

ment to cleave

per

Hence,

in 70 percent

should

of II/III

to be stable

HCOOH at room temperature.

reduced

hyl

appear

that

heated

stabilize

IV,

to the

is

are

of the bifunctional

we have

at pH 7.4 regenerates

the crosslinks

ever,

II/III,

they

precursors

crosslinks

stability

Indeed,

residues

crosslink

transformation

acids.

and

because

tropocollagen

additional

little

up to 0.19

if

V and VI suggest

by hot

contains

minutes,

Even thermal

give

of IV,

and III

which

from

hydrolytic

IV and V could

between

different

as a tetrafunctional

simply

of crosslinks

Finally,

act

thermal,

crosslinks

any equilibrium

residues

and III

II

aged bone,

formation

RESEARCH COMMUNICATIONS

could

resist

reducible

crosslinks.

The structures back

than

the

crosslinking

which

and VI would

by forcing

to favour from

links

IV

better

fibers

derived

a gem-diamine

Furthermore,

more stable.

AND BlOPHYSlCAL

of all

free

lys/

of the nucleophilic

by thermal

treatment.

equivalent

to the

by treatment We are

more direct

with

currently

evidence

The

an invest-

for

the

crosslinks. are grateful to the Medical and to Annelise Gottrup for

728

Research technical

Council of assistance.

Vol. 6 1, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Bailey, A. J. and Peach, C. M. (1968), Biochem. Biophys. Res. Commun. 33, 812. Tanzer, M. L., Mechanic, G., and Gallop, P. M. (1970), Biochim. Biophys. Acta 207, 548. Biochem. Biophys. Res. Mechanic, G., Gallop, P., and Tanzer, M. (1971), Commun. 2, 175. Biochem. Biophys. Res. Commun. 45, Davis, N. R. and Bailey, A. J. (1971), 1416. Robins, S. P., Shimokomaki, M., and Bailey, A. J. (1973), Biochem. J. 131, 771. Biochem. Biophys. Res. Commun. Eyre, D. R. and Glimcher, M. J. (1973), 52, 663. Davis, N. R. (1973), Biochem. Biophys. Res. Commun. 3, 914. Bailey, A. J. (1968), Biochim. Biophys. Acta 160, 447. Biochem. Biophys. Res. Commun. 48, 1656. Davis, N. R. (1972), Melchior, W. B. and Fahrney, D. (1970), Biochemistry 2, 251. Dixon, H. B. F. and Perham, R. N. (1968), Biochem. J. 109, 312. Riehm, J. P. and Scheraga, H. A. (1966), Biochemistry 5, 93. Liu Woo-Hoe, Dsiega, Haynes, D. T., and Feeney, R. E. (1968), Biochemistry 1, 2886. Davis, N. R. (1973), Biochem. Biophys. Res. Commun. 52, 877. Harkness, M. L. K. and Harkness, R. D. (1965), Nature 205, 912.

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