Secretion of unhydroxylated chick tendon procollagen

BIOCHEMICAL

Vol. 67, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SECRETION OF UNHYDROXYIATSD CHICK TENDON PROCOLLAGEN Sergio

A. Jimenez*

of Medicine,

Department

Medicine

and Ronald

University

of Pennsylvania

and Philadelphia

General

Philadelphia,

Received

September

Yankowski

Pa.

School

of

Hospital

19104

23,1975

SUMMARY: The secretion of unhydroxylated procollagen at 37O by isolated chick tendon fibroblasts independent of protein synthesis was examined, The data showed that intact molecules were secreted and that their degradation was an extracellular event. The kinetics of secretion indicated that most of the secreted procollagen appeared in the medium during the initial 30 min following inhibition of protein synthesis and only an additional 35% reached the extracellular space in the subsequent 90 min. The pattern of secretion suggested the existence of an intracellular binding site for the unhydroxylated molecules which was saturated during the early period of secretion. It is speculated that such a binding site could be the enzyme prolyl hydroxylase which has a high affinity for unhydroxylated procollagen at 37O. INTRODUCTION One of the crucial of appropiate assembled lysyl but

prolyl on the

residues it

ribosomes.

secreted

weight

degradation

that

of collagen

and that

normal

temperatures

body

unhydroxylated

were

were

conformation

* To wham correspondence

while

rate

present

secreted

was required

should

proteins

be addressed.

cells

are not

has been normal

intracellularly

indicated

for

suggested

secretion.

and

was synthesized

was suggested

molecules

for

are being of prolyl

and accumulated

is crucial

and it

chains

procollagen

by the inhibited

unhydroxylated

the hydroxylation

hydroxylation

and it

hydroxyproline

(6,7)

is

nascent

When the enzymatic

of the secreted

products

biosynthesis

residues

at a normal

peptides

have shown

collagen

and lysyl

Characterization

molecular

helical

during

was inhibited,

was not

(l-4).

studies

steps

that

only

that

only

(3-5).

Recent

the thermal triple that

small

stability

helical a triple

In the present

at

Vol. 67, No. 2, 1975

study

we have

under

conditions

BIOCHEMICAL

further

examined

at which

the

AND BIOPHYSICAL

secretion

the molecules MATERIALS

RESEARCH COMMUNICATIONS

of unhydroxylated

were

not

triple

procollagen

helical.

ANDMETHODS

Fibroblasts were obtained from 17-day701d chick embryo tendons as previously described (2). Subsequently 10 cells/ml were incubated at 37O in Krebs medium containing 2% fetal calf serum, 25 ug/ml asiorbic acid and 0.5mM o, or dipyridyl. After 20 min preincubation C proline was added and the incubation was continued for another 60 min. Further protein synthesis was then inhibited by addition of cycloheximide. The cells were separated from the media by centrifugation and then they were resuspended in fresh media containing cycloheximide and o, o' dipyridyl and the incubation was continued at 37O. Aliquots were obtained at the intervals indicated and were centrifuged at 1200 xg for 10 min to separate cells fra the secreted proteins present in the media. Cells and media were prepared and chromate aphed on SDS* - Agarose columns as reported previously (8). The 5%C-hydroxyproline content of the collagen samples labeled with 14C proline was assayed by a specific chemical procedure (9). The radioactivity of the medium and intracellular proteins was determined directly after extensive dialysis against running tap water. RESULTS As shawn previously, blasts

with

cellular

0.5 mu

at 37O was examined,

that

to that

was evident

weight

than

* Abbreviation:

that

of the

in the

about

fragments

in the medium

labeled

of collagen

SDS; sodium

eluted small

the main peak

was found

alpha-chains;

sulfate

736

procollagen

appeared In the

appeared over

which

at various

(Fig.

weight broader

had

inhibition

lA)

in a position

molecular

in the area

proteins

dcdecyl

columns,

of 14c-

amounts

120 min following

present

found.

and intra-

40% of the protein

in SDS-Agarose

were

synthesis

fibro-

unhydroxylated

in the

for

tendon

no detectable

of the radioactivity

no radioactivity

and the majority

that

and no evidence

chick

C-labeled

was secreted

At 90 min however,

molecular

14

of

chromatographed

of procollagen

essentially

smaller

secretion

at 30 min most

isolated

containing

When the proteins

were

was present. small

it

synthesis.

intervals

found

resulted

intracellularly

of protein time

dipyridyl

of procollagen

when the

accumulated

of the

CY, O'-

accumulation

hydroxyproline.

incubation

it

was

comparable fragments and scane

180 min sample, of elution to have

50% of the

of procollagen a molecular recovered

weight radio-

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

6-

4-

2

i 30

40

50 Fraction

39 number

40

50

Fig. 1. SDS-Agarose gel chromatography of secreted and intracellular labeled proteins at various time-periods following inhibition of protein synthesis. Cells and medium were prepared for chromatography and aliquots containing approximately 200,000 dpm were chramatographed as described previously. Marker B rat skin collagen eluted at fraction 30 and ci rat skin collagen eluted at fraction 35. A, Medium proteins; B, Intracellular proteins.

activity

eluted

proteins

were

near similarly

was identical eluting

periods,

in the region

early

phase

the rate very

lB),

the major

of secretion

decreased

volume.

When the

the pattern

portion

of the

No evidence

markedly

first

secreted

the

intracellular

of the labeled

chromatograms protein

to suggest

subsequent

proteins

at a rapid

and in the 65% of the

cells

labeled

occurred

30 min and only

in the

from

of the

secretion

Approximately

in the

recovered

(Fig.

of procollagen.

at which

slow.

medium

effluent

intracellular

was found.

The kinetics

the

examined,

at all

degradation

were

the end of the

subsequent

secreted

and medium,

2) showed

At about

90 min the

appeared

amount

constant

for

an

30 min

secretion

35% of labeled

The total

remained

rate.

radioactivity

an additional

90 min.

(Fig.

was in the

proteins

of radioactivity the duration

of

experiment. DISCUSSION Previous

presence

of

studies

showed

LT., @- dipyridyl

that

tendon

or under

fibroblasts anaerobic

737

incubated conditions

in the (3-5)

synthesized

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

Time

RESEARCH COMMUNICATIONS

(mln)

Kinetics of secretion of unhydroxylated procollagen at 37O. FibroFig.2. blasts were incubated at 37' at a concentration of 107/ml in 10 ml of zebs After 20 min 5uCi/ml of C medium containing 0.5 mM c1, W- dipyridyl. proline was added and the incubation was continued for 60 min. Cycloheximide was then added to a final concentration of 100 ug/ml and the cells were separated from the media by centrifugation. The cells were resuspended in 10 ml of fresh Krebs medium containing 0.5 mM CL, CY.'- dipyridyl and After resuspension the incubation was continued 100 ug/ml cycloheximide. at 37O and 1.5 ml aliquots were obtained at 10, 30, 60, 90 and 120 min after addition of cycloheximide. The aliquots were centrifuged, the cells were resuspended in distilled water, aggitated in a vortex and then dialyzed against running tap water. The media were directly dialyzed against running tap water. After dialysis aliquots were counted in a triton-toluene scintillant.

unhydroxylated retained

in

The results

and only It

the media.

degraded

elute

The unhydroxylated

intracellularly

present were

procollagen.

in a position

here

indicate

comparable in

the media

at

This

is

similar

cultured degradation

fibroblasts of these

(10).

only

that

to that

are present result

that

and that

columns, 37O.

molecular

was suggested

intracellularly presented

small

degradation

findings

tendon of

our results

738

products

were molecules

products

were

molecules

of procollagen

molecules

was apparently

the unhydroxylated

of isolated to the

weight

unhydroxylated

Furthermore,

unhydroxylated

procollagen

secreted.

which

on the SDS-Agarose fibroblasts Ramaley suggest

does not

occur

incubated

et.

al.

that intra-

in the

BIOCHEMICAL

Vol. 67, No. 2, 1975

cellularly

as suggested

but

earlier,

the present

study,

the

immediately

after

protein

synthesis

synthesis

was not

studies

protein

without

additional

these

studies,

preparation the

washing

the

secreted

could

saturated

at the

initial

period

transport

of the

excess

diminishes site

for

of unhydroxylated

inhibited,

study. and the

Also

for

the possibility

in fresh

medium

cells

were

incubated

seems possible

that

remaining

the cell

from

in

may have been responsible

rates.

During

are rapidly

secreted

for

that

there is

the

to the extracellular

binding

sites,

then

on the Previous are

a composite

the

pro-

secretion

rate

of the binding

studies

on the kinetics

not

directly

of the

comparable

synthesis

of both

rapid space.

affinity

protein

of degradation

change

permits

of intracellular

reports

This

which

amount

(4,ll)

are

procollagen

and the

dependent

period

in the medium

and therefore

molecules

that

the early

the possibility

unhydroxylated

molecules.

data

indicate

significantly.

with

of secretion

collagen

molecules

decreases

the

In the previous kinetic

It

themselves

is reached

and becomes

the unhydroxylated

the present

and the

be consistent

of the available

markedly

secretion

tion.

that

In

In the previous

activity

unhydroxylated

point

equals

inhibited

of secretion

site

collagen

was inhibited.

molecules

binding

When the equilibrium

and resuspended

at two distinct

an intracellular

event.

molecules.

radioactive

of secretion

RESEARCH COMMUNICATIONS

an extracellular

of the unhydroxylated

occurs

30 min the rate

in the rate

washed

cells

of secretion

process

of secretion, and after

by the

of the

secretory

is

and protease

or released

degradation

were

it

and resuspension.

collagenase

The kinetics the

cells

AND BIOPHYSICAL

of to

was not

synthesis

and secre-

secreted

molecules

was not

can only

be speculated.

excluded. The nature We suggest very

high

been

shown

Additional

that

of the

intracellular

prolyl

hydroxylase

affinity

for

to form

stable

support

for

non-triple

binding

may be the binding helical

complexes this

site

possibility

procollagen

with

site (12-14)

unhydroxylated is

739

apparent

since

and it

substrates fran

it

experiments

has a has (15).

is

Vol. 67, No. 2, 1975

which

explored

the

erature

below

these

experiments

rate

when

the prolyl triple

affinity

secretion

the

the

conformation,

of unhydroxylated

enzymes

increased

involved

may excert affinity

is

the

in the similar

when

likely

that

It

intracellular

of such enzymes

for

Since

at a faster

is

the more rapid

of in a

rate

of

due to the decreased

can also

under

the collagen

In

the affinity

be speculated

post-ribosomal effects

(14,16).

the substrate

at 24O is

substrate.

binding

at a temp-

was secreted

at 24O.

procollagen for

procollagen

procollagen

diminishes it

RESEARCH COMMUNICATIONS

of the molecules

was maintained markedly

of the enzyme

of collagen

temperature

unhydroxylated

temperature

hydroxylase

AND BIOPHYSICAL

of unhydroxylated

the denaturation

helical

secretion

other

BIOCHEMICAL

that

modifications

circumstances

where

exist.

ACKNOWLEDGMENTS We thank Dr. N. A. Kefalides for valuable criticism. We acknowledge the expert assistance of Mrs. P. Frontino and Mrs. C. Porter. Supported by the Philadelphia General Hospital Research Fund and the Barsumian Fund. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

COOPER., AND LASH, J. (1966) Science =,92. JWA, K., PRCCKOP, D-J., Acta 240,358. DEHM, P., AND PROCKOP, D.J. (1971) Biochim,Biophys. RAMALEY, P.B. AND ROSENBLOOM, J. (1971) Fed. Eur. Biochem. Sot. Letters Is, 59. JIMENEZ, S.A., DEHM, P., OLSEN, B.R., AND PROCKOP, D.J. (1973) J. Biol. Chem. 248,720. (1974) Eur. J. Biochem. 43,221. UITTO, J., AND PROCKOP, D.J. JIMENEZ, s.A., HARSCH, M., AND RCSENBLOOM, J. (1973) zochem. Biophys. Res. Ccrmmun. 52, 106. (1973) Biochem. Biophys. Res. Commun. 52,115. BERG, R., AND PRCCKOP, D.J. JIMENEZ, S.A., DEHM, P., AND PRCCKOP, D.J. (1971) Fed. Eur. Biochem. sot. Letters 2,245. (1966) Anal. Biochem. 15, 77. JWA, K., AND PRCCKOP, D.J. RAMALEY, P.B., JIMENEZ, S.A., AND ROSENBLOm, J. (1973) Fed. Eur. Biochem. Sot. Lett. 33,187. MARGOLIS, R.L., AND LUSNS, L.N. (1971) Arch. Biochem. Biophys. 147,612. (1973) Biochemistry z,3395. BERG, R.A., AND PRCCKOP, D.J. MURPHY, L., AND ROSENBLOOM, J. (1973) Biochem. J. 135,249. JIMENEZ, S.A., HARSCH, M., MURPHY, L., AND ROSENBLOOM, J. (1974) J. Biol. Chem. 249,448O. JWA, K., AND PROCKOP, D.J. (1969) J. Biol. Chem. 244,6486. JIMENEZ, S.A., AND YANKCWKI, R. (1975) Fed. Proc.x,563.

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