Identification of collagen α1(I) trimer in embryonic chick tendons and calvaria

Vol. 78, No. 4, 1977

BIOCHEMICAL

IDENTIFICATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF COLLAGEN a,(I)

TRIMER IN

EMBRYONIC CHICK TENDONS AND CALVARIA

Sergio

A. Jimenez*,

Milton

Benditt

Department

and Ronald

of Medicine,

of Pennsylvania.

School

September

13,

Bashey,

Yankowski

of Medicine,

3700 Hamilton Pennsylvania

Received

Reza I.

Walk,

19174

University

Philadelphia,

U.S.A.

1977

SUMMARY: Collagen with a molecular composition [a (I)], has been identified in acetic acid extracts from lathyritic chic k embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development. INTRODUCTION Although consist

it

has been well

of two CL (1) 1

to indicate a,(I)

that

chains

collagen

were

b,(I)

they

*

of

their

greater

molecules

(1)

evidence

has appeared

In these

have different

in normal should

Copyrighf 0 1977 by Academic Press, Inc. All rights of reproduction in any forrrt reserved.

studies

gingiva

tissues

of these

molecules

culture

in normal

salt

recent

collagen

collagen

amounts

the role

To whom correspondence

collagen

that

fractional

presence

interstitial

in tissue

time

molecules

during

Although

collagen. stration

the first

These

contain

(Z-6).

such as inflamed

I3 can be demonstrated

Type I collagen

conditions

chondrocytes

we show for

and calvaria.

ition,

certain

tissues

that

and one a 2 chain

be detected

either

from pathologic report

chains

under

could

established

(4)

tumors

molecules

with

residues not

tissues

[al(I)]3 obtained In this

a composition chick

tendons

characteristics at neutral

is

of

(3,6).

such as embryonic solubility

of three

fibroblasts

or

of hydroxylysine

embryonic

the sources

(2,5),

precipitation

molecules

composed

pH. than

apparent

as

suggests

that

than In addType I yet,

demon-

they

be addressed.

1354 ISSN

0006-291X

Vol. 78, No. 4, 1977

might

BIOCHEMICAL

be important

in the process

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of differentiation

MATERIALS

and development.

AND METHODS

Seventeen day old embryonated eggs were purchased from Shaw Hatcheries (West Chester, Pa.) and were maintained in a moist atmosphere at 37" until Twenty mg of 6 amino-propionitrlle dissolved in 0.1 ml of sacrificed. sterilized saline was injected into the chorio-allantoic membrane and the Tendons and calvaria were dissected and embryos were sacrificed 48 hr later. The tissues were extracted with they were rinsed in cold distilled water. The extracts were 0.5 N acetic acid at 4" for 72 hr with gentle shaking. The clear aspirated and clarified by centrifugation at 30,000 xg for 30 min. supernatants were dialyzed against 0.5 M acetic acid and after extensive the extracted collagens were precipitated by slow addition of solid dialysis, were collected by NaCl to a final concentration of 0.9 M. The precipitates centrifugation and resuspended in 1.0 M NaCl, 0.05 M Tris-HCI buffer pH 7.4 at 4". Differential NaCl precipitation was performed as described by Trelstad et al (7) and collagen fractions were obtained at increasing NaCl molarities. The Collagens precipitating at 1.71 M and at 2.5 M NaCl were removed. supernatant remaining after removal of the 2.5 M NaCl was brought up to 4.0 M with solid NaCl. The precipitate obtained was collected by centrifugation and it was resuspended in and dialyzed against 0.15 M NaCl 0.05 M Tris-HCl pH 7.4 at 4°C. The suspension was then clarified by centrifugation and collagen was re-precipitated at 2.5 M and 4.0 M NaCl as described above. Sodium dodecyl sulfate disc gel electrophoresis was performed on 5% polyacrylamide gels as described previously (8). Samples were denatured with 1% sodium dodecyl sulfate in the presence and absence of reducing agents by After electrophoresis the gels were stained with heating at 100" for 3 min. Coomassie Blue for 12 hr and subsequently de-stained in a solution of acetic Stained gels were scanned at 560 nm on a Gilford spectrophotoacid-methanol. meter fitted with a linear transporter. Determination of the area under each peak on the recorder tracings was made using a planimeter. For aminoacid analyses, aliquots under nitrogen in 6 N HCl for 18 hr of the hydrolyzate, the individual in a single column of resin eluted viously described (9) in a Beckman

of collagen suspensions were hydrolyzed and after evaporation and re-suspension aminoacids were separated by chromatography following a modification of a method preaminoacid analyzer.

RESULTS AND DISCUSSION When acetic examined the

stained

acid

by sodium

extracts

dodecyl

from

sulfate

electrophoretograms

lathyritic

chick

polyacrylamide quantitated

of a 1 to a chains were not 2:l 2 an excess of al chains was demonstrated.

tropocollagen sent

molecules

in addition

determine

composed

to molecules

the nature

of these

with

entirely

disc

as expected These from a

a composition

molecules

gel

fractional

1355

1

and calvaria

it for

a2.

that

Type I collagen

suggested

polypeptides (al)2

and

was found

pure

results

of

were

electrophoresis

by planimetry,

the ratios but

tendons

were

that also

pre-

To further

NaCl precipitation

was per-

Vol. 78, No. 4, 1977

TABLE I.

BIOCHEMICAL

ratios

Relative

Collagen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of al to a2 chains

M NaCl precipitate

2.5 M NaCl re-precipitate of 4.0 M NaCl collagen

fraction

4.0 M NaCl re-precipitate of 4.0 M NaCl collagen

fraction

(a)

(b)

Average

of 5 determinations.

(cl

Average

of 3 determinations.

Cd)

Average

of 2 determinations.

As shown previously

molecules extracts ratio

with

result

rived

that

from Type III

When the collagen with

polypeptides molecules precipitated

with

only

3.6:1(')

2.3:lcd)

2.7:Gd)

13.1:dd)

collagen

composed

remaining

of 2:l

were

a greater 1).

1356

and the unchanged.

probably

not

de-

in 0.15

Type I collagen.

was further

precipi-

of a 1 over purification

excess

Further

was accomplished

4 M NaCl was re-suspended

of the

at 2.5 M NaCl contained

at 2.5 M NaCl

I and Fig.

al chains

was essentially

and was therefore

containing

[al(IIl)13

of collagen

precipitated

in solution

(Table

fraction

al polypeptides

Collagen

a fraction

only

a minor

of

Precipitation

in the supernatant

in a ratio

was obtained containing

3.2:lcb)

Type III

the excess

collagen.

4 M NaCl

2.4:1

at low NaCl concentrations.

and a2 polypeptides

tated

(7)

remaining

indicated

2.3:1

of acetic acid extracted lathyritic described by Trelstad (7) and aliquots were dodecyl sulfate-polyacrylamide gels. After the gels were scanned, and the areas under and a2 chains were quantitated with a

1.7 M NaCl removed

of polypeptides

This

5

is precipitated

(a)

fractions

Calvaria

10.8:dd)

Fractional NaCl precipitation collagen was performed as electrophoresed on sodium staining and de-staining, peaks corresponding to al planimeter.

formed.

collagen

Tendon

Fraction

0.9 M NaCl precipitate 4.0

in various

a

2

of

when the collagen M NaCl,

0.05

M Tris

HCl

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

m

1357

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B

Figure

2.

pH 7.4,

Photograph of Coomassie Blue stained gels of p urified [al( collagen from tendon and calvaria. (A) 2.5 M NaCl preciplta ed tendon collagen containing predominantly Type I collagen; (B) tendon and (C) calvaria [a (I)] before reduction of disulfide bonds; (D) tendon and (E)lcalvzria [al(I)]3 after reduction of disulfide bonds.

and solid

NaCl was added

in precipitation 2.3:1

which

to that

this

and a highly a

at least

1O:l

chains

close

after

entirely

a2

of a collagen is

in solution tration

1

chains

step was then

I),

fraction

collagen,

we examined

as described

a

precipitated

and a

1

again

above represented of disulfide (7,10,11).

1358

chains

resulted

in a ratio

was not that

remaining

at 4 M NaCl concen-

which

to a2 ratios it

2

This

The collagen

of collagen

the possibility

molecules

of 2.5 M.

Type I collagen.

The al

the effects

of these

both

and in some cases

To exclude

2).

purified

behavior

of pure

was obtained.

(Table

(Fig.

to a concentration

containing

purified

molecules

phoretic

C

contained

almost

in these

fractions

was

possible

to detect

any

the tropocollagen

either

Type III

bond reduction Comparison

or Type IV

on the of electro-

electro-

Vol. 78, No. 4, 1977

TABLE II.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Aminoacid composition of collagen fractions re-precipitated 4 M NaCl compared to other collagen polypeptides(a)

Aminoacid

3-Hydroxyproline 4-Hydroxyproline

Tendon

Calvaria

1.5

ra,(I)lp

al(T)(c)

1.8

2.3

i.0

with

u,(rI)(d)

2.2

110

104

108

102

103

46

45

45

42

42

Threonine

19

19

20

19

26

Serine

31

30

39

29

26

70

76

77

78

a7

Aspartic

Glutamic

acid

acid

Proline

119

117

109

118

115

Glycine

331

330

334

330

329

Alanine

113

119

111

129

104

Valine

13

14

18

14

16

Methionine

7.1

Isoleucine

10

Leucine

24

Tyrosine

2.4

6.0

7.5

8.2

9.0

a.7

6.1

22

23

3.5

2.2

20 2.0

11 7.8 26 2.2

Phenylalanine

13

13

12

14

15

Lysine

27

25

24

30

13

Hydroxylysine

11

13

11

Histidine Arginine Hydroxylysine Lysine

(a) (b) (c) (d)

2.8 49

0.41

Residues per 1000 residues. Pepsin treated TSD4 collagen Chick bone collagen (15). Chick sternal cartilage (15).

5.0

2.5

48

46

0.52

0.46

(3).

1359

5.1 2.0 51

0.17

23 2.0 50

1.77

Vol. 78, No. 4, 1977

phoretograms ences

BlOCHEMlCAL

of reduced

in collagen

of reducing

agent.

held

and urn-educed

in the distribution

no increase

The migration authentic

migrating

bonds

were

derived

as shown previously

similar

to those

collagens chains present here

of Type II

II).

in hyaline contained

collagens by other

these

The presence

peculiar

feature

clear number

hydroxylase al(I)

systems

as well

has not

been previously

molecules.

but

it

of lysine than chains

that

residues

capable

shown to our

but

their

knowledge.

1360

residues.

to be a constant

This

may result

content

primary of being

Tropocollagen

have been demonstrated (2-6)

of lysine

residues structure

in these con-

hydroxylated

in several

Since

The mech-

molecules

presence

in

of hydroxy-

of lysine their

Type I collagen.

as in some tumors

similarity

carbohydrate.

hydroxylation

is likely

isolated fractional

striking

appears

such as increased

increased

exclusively

during

another

or hydroxylysine-linked

of al(II)

molecules

in hydroxylation

collagen

that

These

behaviour

of lysine

of these

cartilage.

to similar

hydroxylation

the

than

are

employed

collagen

the increase

here

some

the tissues

[a,(I)],

electrophoresis

which

composition

of contaminating

the

characteristics

for

of three

gel

of these

responsible

posed

amounts

that

these

Although

obtained

the aminoacid

of

that

(11-14).

fractions

unlikely

is

resemble

is

anism

lysyl

collagen

In addition

cross-links

the

and it

of increased

a greater

mobility

not

[al(IV)]3.

procollagen

or

is almost

derived

tains

the possibilities

Type IV collagen

Type II

or

were

the same as that

collagen

collagens

is not

[ol(III)]3

In addition,

lysine

molecules

not

was

in the absence molecules

chains

and disc

structural

and there

of al(I)

(3,4).

and distinctive

chains

collagen

was about

(15),

any differ-

more the composition

closely

NaCl precipitation

were

electrophoretic

any significant

authors

between

either

reveal

of y-chains

the

excluded

collagen

cartilage

however,

that

they

of the c11 -rich

resembled (Table

from

have less

of the characteristics

showed

result

not

polypeptide

polypeptides

This

did

the position

and therefore

chains.

polypeptides

in

results

of the reduced al(I)

molecules

of the individual

These

by disulfide

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

&

com-

vitro

in normal

embryonic

by

tissues

tissues

appear

BIOCHEMICAL

Vol. 78, No. 4, 1977

to contain

greater

that

presence

their

capability

which

and maturation. u,(I)

trimer

amounts

of these

in diseased is normally If

this

molecules

tissues lost

suggestion

may be indicative

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

during is

of cell

than

adult

and tumors

it

is

possible

may be due to expression

the process confirmed,

tissues,

of tissue

demonstration

of a

differentiation of collagen

de-differentiation.

ACKNOWLEDGEMENTS We acknowledge the assistance of Ms. Esther Lobb. NO 1 HV 4 2982 and USPH Grant HL-19128. NIH Contract 1. 2. 3.

Supported

in part

by

REFERENCES PIEZ, K.A. (1967) In Treatise on Collagen (Ramachandran, G.N., editor) Vol. I, pp. 207-252, Academic Press, New York. MAYNE, R., VAIL, M.S., AND MILLER, E.J. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 4511-4515. LITTLE, CH.D., CHURCH, R.L., MILLER, R.A., AND RUDDLE, F.H. (1977) Cell lo, 287-295.

4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15.

Chem. 251, 5464-5471. NARAYANAN, A.S., AND PAGE, R.C. (1976) J. Biol. 16, 865. BENYA, R.D., PADILLA, R., AND NIMNI, M.E. (1977) Biochemistry MORO, L., AND SMITH, B.D. (1977) Arch. Biochem. Biophys. 182, 33-41. TRELSTAD, R.L., CATANESE, V.M., AND RUBIN, D.F. (1976) Analytical Biochem. 2, 114-118. JIMENEZ, S.A., AND ROSENBLOOM, J. (1974) Arch. Biochem. Biophys. 163, 459-465 Biophys UITTO, J., JIMENEZ, S.A., DEHM, P., AND PROCKOP, D.J. (1972) Biochim. Acta 278, 198-205. 13, 3459-3467. CHUNG, E., KEELE, E.M., AND MILLER, E.J. (1974) Biochemistry KEFALIDES, N.A. (1971) Biochem. Biophys. Res. Comm. g, 226-234. LAYMAN, D.L., MCGOODWIN, E.B., AND MARTIN, G.R. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 454. BELLAMY, G., AND BORNSTEIN, P. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 1138. .JIMENEZ, S.A., DEHM, P., AND PROCKOP, D.J. (1971) Fed. Eur. Biochem. Sot. Lett. 17, 245. MILLER, E.J. (1971) Biochemistry lo, 1652-1659.

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