Triple helix formation and disulphide bonding during the biosynthesis of glomerular basement membrane collagen

Vol. 70, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TRIPLE HELIX FORMATION AND DISULPHIDE BONDING DURING THE BIOSYNTHESIS OF GLOMERULAR BASEMENT MEMBRANE COLLAGEN Isabel

F. Williams*,

March

Harwood

&

Michael

E. Grant

Department of Medical Biochemistry, of Manchester, Manchester Ml3 9PT,

University Received

Richard

U.K.

12,1976

Summary: The l4C-collagen s thesised by isolated rat glomeruli during a 2h labelling period with [ I4 Cs"proline was found to be digested by chymotrypsin at 25'C s gesting that it was in a random coil form. After a 4h chase period, the Y 4C-polypeptides were resistant to proteolysis indicating that they were in a triple helical conformation. Gel filtration in sodium dodecylsulphate under reducing and non-reducing conditions suggested that the extent of resistance to digestion by chymotrypsin was closely related to the extent of inter-chain disulphide bond formation. The i4C-collagen was found to be composed of three identical polypeptide chains which were linked by intra-molecular disulphide bonds within the chymotrypsin-resistant triplehelical region of the molecule.

Introduction: Recent collagenous the

component

three

types

Studies rat

investigations

glomeruli

extensions molecule.

with

may be excised

have

extensions

similarly

in these into

triple-helical

demonstrated

in several

feature

of the mechanism

appears

to be the formation

*

Present address: 700 Convention

Copyright All rights

Clinical Boulevard,

synthesising

the presence

of assembly

role

see refs

(1).

(2-4)

and isolated collagen

is

triple-helical I and II

folding

pro-

of the precursor

has been 6,7).

bonds,

An important precursors the

Research Center, Philadelphia General Philadelphia, Pa. 19104, U.S.A. 200

from

peptide

triple-stranded

disulphide

a

of non-collagenous

molecules

of these

distinct

membrane

type

in the

reviews

of inter-chain

0 1976 by Academic Press, Inc. in any form reserved,

of reproduction

of the

contain

tissues

non-collagenous

systems

(for

is

cells

the basement

procollagen

studies

which

lens

secretion

and their

membranes

in vertebrate chick

that

established

molecules

polypeptides

found

after

on cell

basement

IV collagen)

form possessing

Observations

collagens

that

embryonic

h ave demonstrated

in a precursor which

type

collagen

--in vitro

(5)

synthesised

(designated

of fibrillar

conducted

suggest

timing Hospital,

of

BIOCHEMICAL

bl. 70, No. 1,1976

which

is

tendon,

closely

related

cartilage

between

also

described

disulphide

biosynthesis

to triple

and lens

The results

bonding

that

and contains

helix

cells

(7).

here

provide

helix

basement

this

inter-chain

formation

further

and triple

of glomerular

presented

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

comprises

disulphide

bonds

resistant

triple-helical

portion

Materials

and Methods:

Glomeruli

data

on the

formation

during

membrane

collagen

in embryonic

chick

relationship the

(GBM) collagen. only

Evidence

one type

within

is

of polypeptide

the chymotrypsin-

of the molecule. isolated

from

rats

(150

2 log)

by a

combination of sieving and Ficoll-gradient techniques (5) were incubated [ 14 Clproline in Eagle's medium, continually gassed with @ CO,/g5$

with air.

In general

lation

approx.

of proline

and protein

a,o'-bipyridyl 100 &ml

respectively. by centrifugation

resuspended

incubation

in medium

by the

isolated

containing

a further

and an analysis

bonds

in their

subsequent

which

was eluted

(2.0

an aliquot

ml)

with

were

and

and the

[ 14 Clpolypeptides of the

reducing

role

synthesised

of inter-chain investigated

before

agent

and applied

0.1% SDS in 0.02M

collected

in the fractions

were

[14C]proline

by gel

and after

The samples for analysis were treated that the buffer used was O.O2M-Tris-HCl to the gel

Tris-HCl

scintillation was assayed

was omitted buffer

fluid

by the method

the filtration (pH 7.4).

14C was assayed

and total

in a Triton/HCl-containing

[14C]proILine

the glomeruli

[12C]proline

(SDS)-agarose

and in experiments where the (PH 7.4) samples were made O.lM in iodoacetamide column

of

of 1 rJM and

aggregationwere

dodecylsulphate

with mercaptoethanol. previously (5) except

Fractions

by the addition

2h with

100 &ml

glomeruli

on sodium

for

in 5 ml the hydroxy-

4h at 37'C.

of the collagenous

disulphide

inhibited

experiments,

labelling

size

filtration reduction described

for

incubated and then

concentrations

In pulse-chase

continued

The molecular

were

to final

after

were

2h at 37'C

synthesis

and cycloheximide

collected then

3 to 4 x 105 glomeruli

50 - 70 pCi [14C]p ro 1'me for

medium with

by counting

(8).

Hydroxy-

of Juva & Prockop

(9). Whether helical

conformation

proteolysis. periods

or not

After the

glomeruli

the

GBM collagenous

was determined incubation were

with

collected

[14C]polypeptides by their

susceptibility

[14C]proline by centrifugation,

201

for

were

in a triple

to limited appropriate cooled

time to 4'C,

as

Vol. 70, No. 1, 1976

homogenised with (w/v)

AND BIOPHYSICAL

in 2 ml 0.4M NaCl in O.iM

300 G/ml

at 25’c

BIOCHEMICAL

for

a-chymotrypsin

6h.

and the

(Sigma

The reaction

Tris-HCl

buffer,

Chemical

Co. Ltd.,

was stopped

products

of enzymic

and Discussion:

In our

RESEARCH COMMUNICATIONS

by the

digestion

pH 7.4, London,

addition

analysed

and incubated U.K.)

of SDS to 1%

by SDS-agarose

chromatography. Results the

collagenous

incubated

polypeptides

with

chick

for

2h,

polypeptides

tendon

pro-a

chains

when

(5).

Previous

molecular

weights

of tendon

pro-a

and it

initially

synthesised

However,

more detailed

analyses

cells

indicate

in the

range

137 - 145,000

basement larger

membrane than

concluded

that

originally that

co-eluting

with

with

molecular

weights

When the

were

hydroxy[i4C]p

ro 1'ine-containing

(Fig.

synthesised following

la)

that

had attained of 4h,

hydroxy[14C]pro1ine-containing digestion under

and eluted reducing

daltons.

little,

if

conditions

it

a molecular (Fig.

any,

a triple-helical was found

polypeptides with

as small

weight

lb).

These

202

all

molecular

were

virtually resistant

of approx. results

2h with the weight

of the newly conformation.

that

to a

polypeptides

for

at 25'C,

eluted

now be

molecules

labelled

proteolysis

the

somewhat

collagenous

by glomeruli

peptides

indicating

period

140,000

weights

that

must

of precursor

must yield

to limited

it

(5).

by matrix-free

are probably

chains

synthesised

subjected

collagen a chase

pro-a

daltons

indicating

In addition,

conversion

the

have molecular

polypeptides

of

polypeptides

secreted

chains

ahead

120 - 125,000

140,000

therefore

(3,5).

of approx.

14 C-proteins

approx.

that

range

collagenous

were

precursor

tendon

[14C]proline

species

the

(ii)

time-dependent

size

out under

had suggested

pro-a

daltons

the hydroxy[14C]-

was carried

in the

of

glomeruli

column

were

that

size

an SDS-agarose

of the procollagen

proposed

the

that

chains

individual

collagen

was observed from

by the glomeruli

tendon

rat

analyses

was concluded

on the molecular

by isolated

chromatography

conditions

(IO)

it

eluted

reducing

daltons

studies

synthesised

[14C]proline

proline-containing

earlier

However, all

to chymotrypsin 140,000

are

of the

consistent

daltons with

BIOCHEMICAL

Vol. 70, No. 1, 1976

“i

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i

i

2

1

a 20

40

30

50 Fraction

60 No.

70

60

90

14 C-polypeptides Gel filtration on SDS-agarose of collagenous Fig. 1. subjected to limited proteolysis. a) Isolated rat glomeruli were incubated for 2h with [14C]proline and then either treated directly with SDS and mercaptoethanol or incubated with chymotrypsin prior to denaturation and reduction and subsequent gel filtration. b) Glomeruli were labelled for 2h with [l'C]proline and then incubated for a further 4h in the presence of 'cold'-proline. Control and chymotrypsin-treated samples were subjected to gel filtration analysis following denaturation and reduction. n, untreated sample; o - - - o, chymotrypsin-treated sample. The elution positions of dextran blue, tendon pro-a chains (M.Wt. approx. 140000 daltons) and b20 are labelled 1, 2 and 3 respectively.

previous

observations

that

majority

the

and that found assumed therefore helical

even

after

extracellularly that

most

on chick

lens

of intracellular

of the

cells

basement

a labelling (3).

epithelial

period

membrane

of 2h very

In the pulse-chase 14 C-collagen

consistent

with

the

conformation

for

normal

is

findings

203

demonstrated

collagen

is

little

collagen

experiment

it

extracellular

that

secretion

which

collagen to occur

non-helical is

to be

can be

and the results must (7).

be in a triple-

are

BIOCHEMICAL

Vol. 70, No. 1, 1976

“i

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5

i

b

i i ii i

a 20

30

40

60

50 Fraction

70

60

90

NO.

l&cFig. 2. Gel filtration on SDS-agarose of unreduced collagenous polypeptides. a) Elution profiles of labelled polypeptides denatured in SDS and alkylated with iodoacetamide after incubation with [l4C]proline for Elution and for 2h followed by 4h chase (n ). 2h ( .--.I, positions of standards are as in Fig. 1. 14 b) Elution profile of unreduced collagen C-polypeptides following chymotrypsin treatment. Glomeruli were incubated with [14C]proline for 2h followed by a 4h chase and then digested with chymotrypsin as described in text. Sample was denatured and alkylated prior to chromatography with carrier rat tail tendon collagen. Elution positions of rat tail tendon Y, B and a chains are indicated.

Because association the collagen

earlier

studies

of pro-a

chains

triple

of the

on SDS-agarose

under

incubation

collagen

the

pulse-chase eluted (Fig.

2a).

it

conditions were

bonds

not

to examine

polypeptides. revealed

disulphide

weight

aggregates

aggregates could

represent 204

the

bonded

in excess complexes

the

of

state

Gel filtration that

hydroxy[. 14 Clproline-containing

the

between

and the formation

was of interest

GBM collagen

chains

a relationship

disulphide

non-reducing

molecular

These

(6,7),

labelled

experiment

as large

through

helix

of aggregation

had demonstrated

after whereas

a 2h in the

polypeptides of 450,000

daltons

of the hydroxy[14C]-

BIOCHEMICAL

Vol. 70, No.. 1,1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 3. SDS polyacrylamide gel electrophoresis of chymotrypsinresistant l&C-polypeptides from pulse-chase experiment. The peak fractions (33-35) f rom the experiment shown in Fig. 2b were pooled, concentrated, reduced with mercaptoethanol and subjected to electrophoresis on @ polyacrylamide gels (13). Gels were sliced into I mm sections and assayed for radioactivity (hatched histogram). Standards of rat tail tendon collagen and chick tendon procollagen were run on gels in parallel, detected by Coomassie Blue staining and the gels scanned at 620 nm ( - - - ). proline-containing proteins

polypeptides

which

collagenous

are known

components

chase

experiment

prior

to alkylation

polypeptides

obtained

when

(Fig.

appears

helix

migrating

with

However,

digested

with

glycothe

when the pulsechymotrypsin

the hydroxy[14C]proline-containing just

prior

observation

lb)

disulphide

by cystine

residues

tail

contrasts

and provides

the first

III

within

the

located

the results

chromatographed direct

chymotrypsin-

end in this collagen

tendon

with

were

of GBM collagen; type

to rat

polypeptides

bonds

to resemble

respect

in that

the

the type constituent

close

to or within

collagen

obtained

the

(12).

When the peak

acrylsmide

This

association

(1).

sample

filtration,

(Fig.

portion

are linked

SDS-agarose

and the

2b).

inter-chain

helical

IV collagen

membranes

in a position

conditions

for

resistant

triple

and gel

to non-collagenous

in covalent

chymotrypsin-resistant

reducing

a-chains

of basement

now eluted Y-chains

evidence

to be present

was repeated

collagen

under

disulphide-linked

column gel

of chymotrypsin-resistant (Fig.

2b)

was reduced

electrophoresis

in the

region

of pro-a

and analysed

(13) only

one peak

chains

was observed

205

from

by SIhS-poly-

of radioactivity (Fig.

3).

This

the

BIOCHEMICAL

Vol. 70, No. 1,1976

finding

is

component cellular

consistent

with

of GBM comprises matrix

interacts

The heterogeneous

by other

of these

with

(1)

species

other

workers

RESEARCH COMMUNICATIONS

that

the

non-collagenous

(14-16)

collagenous

of cl-chain

of collagenous

covalent

Acknowledgements: Fuud and the Medical

suggestion

a single

population

GBM preparations complexity

the

AND BIOPHYSICAL

which

in the

glycoprotein

polypeptides

and non-covalent

subunits.

extracted

may be a consequence

extra-

from

of the

interactions.

The financial support of the National Kidney Research Council is gratefully acknowledged.

Research

References: Res. (1973) 6, 63-104. (1972) J. Biol. Chem.

1. 2.

Kefalides, N.A. Int. Rev. Grant, M.E., Kefalides,N.A.

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& Prockop,D.J. (1972) J. Biol. Chem. Grant, M.E., Kefalides,N.A. 247, 3545-3551. Kefalides,N.A. & Prockop,D.J. (1973) Grant, M.E., Schofield,J.D., J. Biol. Chem. 248, 7432-7437. Grant, M.E., Harwood, R. & Williams, I.F. (1975) Europ. J. Biochem. J&v 531-540. Grant, M.E., Harwood, R. & Schofield, J.D. (1975) In 'Dynamics of Connective Tissue Macromolecules' (Eds. Burleigh, P.M.C. & Poole, North Holland Pub. Co., Amsterdam. A.R.) pp l-32. Prockop, D.J., Berg, R.A., Kivirikko, K.I. & Uitto, J. (1976) In (Eds. Ramachandran, G.N. & Reddi, A.H.) 'Biochemistry of Collagen* Plenum Pub. Co., New York, in press. Harwood, R., Grant, M.E. & Jackson, D.S. (1974) Biochem. J. &2,

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