- Email: [email protected]
Identification of a major extracellular non-collagenous glycoprotein synthesised by human skin fibroblasts in culture
BIOCHEMICAL
Vol. 71, No. 1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IDENTIFICATION OF A MAJOREXTRACELLULAR NON-COLLAGENOUS GLYCOPRGTEIN SYNTBFSISEDBYBCMANSKINFIBBCBLASTSINCULTURE Christopher H.J. Sear, Michael E. Grant & David S. Jackson Department of Medical Biochemistry,
University
of Manchester,
Medical School, Oxford Road, Manchester Ml3 gPT, U.K. Received
May 12,1976
-: The major protein released into the mediumby humanskin fibroblasts in culture has been shown to be a fucosylated glycoprotein (designated MFGP). Analysis by gel filtration chromatography and polyacrylsmide gel electrophoresis demonstrated that under reducing conditions MFGPhas a molecular weight of approx. 250,000, but occurs as a disulphide-linked aggregate in the medium. Three lines of evidence are presented to establish that MFGPis a non-collagenous molecule. INTRODUCTION: Studies with cultured
cells have contributed
to our uuder-
standing of the synthesis and secretion of both collagenous and proteoglycanrelated
components of the extracellular
matrix.
However, there is au
increasing amount of evidence to suggest that connective tissues also contain non-collagenous structural
glycoproteins
been very poorly characterised
(1).
which, with few exceptions,
In view of the possible importance of
such macromolecules in the development of connective tissues (2) disease processes (3) suitable
we have established optimal culture
for the study of glycoprotein
conditions
incorporate
into a high molecular weight non-collagenous glycoprotein, protein
and in
biosynthesis by human skin fibroblasts
In this paper we demonstrate that such cells
(4).
have
[%]fucose
which is the major
released into the culture medium.
were propagated as described previously cultures maintained in a ch ically defined medium, MAB 87/T were labelled for 12h or 24h with L-[lfucose (10 @X/ml). When fucose-labelled glycoproteins were to be compared with secreted collagenous molecules both L[l-%I]fucose and [U-14C]proline (I @X/ml) were
EXPERIMENTAL:
Abbreviation:
Copyright All rights
Human skin fibroblasts
SDS, sodium fiorlecyl sulfate,
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
379
Vol. 71, No. 1,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
added to the maintenance medium, which acid (50 &ml) aa P-aminopropionitrile
was further supplemented (50 (*g/ml).
with
ascorbic
Non-diffusible radiolabelled macromolecules in the medium and in the cell layer were analysed by SDS-agarose gel filtration chromatography either unreduced or after prior reduction aa alkylation (6). The elution positions of collagenous polypeptides were determined by a specific radiochemical assay c01ultms for hydroxy[%]proline following hydrolysis of the fractions (7). ovalbumin, bovine serum were calibrated using the following standards: albumin and rat tail tendon collagen OL- end P-chains. The %-macromolecules in the medium and cell layer were analysed by using a separating gel 10 cm 10% SDS-polyacrylamide gel electrophoresis (8) The proteins in the medium were aa containing 8$ (w/v) acrylamide. concentrated prior to analysis by precipitation with (NRk)2SOh at i'?$ saturation in the presence of 25 mM EIYTA, 10 mM N-ethylmaleimide and 2 mM Gels were stained with either Coomassie phenylmethylsulphonyl fluoride. Brilliant Blue B or by the periodic acid-Schiff procedure (!I), and the distribution of [%]f ucose-labelled molecules determined by slicing and assaying for radioactivity. RESULTS: our
stocks)
Glycoprotein was examined
non-diffusible proteins
biosynthesis by studying
the
After
macromolecules. present
by human skin
i? the medium were
incorporation
a 24h labelling
aualysed
fibroblasts
(line
BF3 of
of [%]fucose period
by SDS-agarose
into
the %-glycogel
filtration
a
20
40 FRACTION
60 NO.
Fig. 1: Gel filtration on SDS-agarose (Bio-Rad A-5m) of macromolecules labelled with [%I -fucose synthesised by cultured skin fibroblasts. Glycoproteins (a 3 released into the medium, (b) resent in the cell ES!; The elution ositions of collagen a- and P-chains 98,000 and 196,000 and the void volume of the column (57 ml) are amowed. nnreducea sample; (0 - 0 - 0 ) reduced sample.
380
.
BIOCHEMICAL
Vol. 71, No. 1,1976
chromatography (Fig.
la).
accounting for 253% of the column.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In the absence of reducing agents the major peak,
of the total
radioactivity,
eluted at the void volume
However, when the labelled mediumproteins were chromato-
graphed following
reduction
and alkylation
the radioactivity
at the void
volume was markedly diminished and a new major peak appeared (peak M of Fig. The molecular weight of this major species was estimated from six
la).
experiments as 250,000 t 25,000 by extrapolation based on the elution
positions
from a calibration
of standard proteins.
Similar results were
obtained when the cells were labelled with [i-i4C]fucose identical
curve
and essentially
chromatogrsms were obtained using six other humanskin fibroblast
cell lines. Gel filtration
analysis of the unreduced labelled macromolecules in the
cell layer is presented in Fig. lb. not significantly Tfieae results relatively
It is noteworthy that this profile
altered by reduction
indicated
of the sample prior
was
to chromatography.
that the peak M component, if present, represented a
minor proportion
of the total
fucosylated
species in the cell
layer. Further experiments were conducted to determine whether the high molecular weight [%]f ucose-labelled represented a major constituent electrophoresis threitol
macromolecule present in the medium
released by the cells.
of the mediummacromolecules after
demonstrated that the major protein
molecular weight of approximately
Polyacrylamide
reduction vith
2).
material
200,000, stained strongly
with the PAS
It was also established that the [%]fucose-labelled recovered from the SDS-agarose column (Fig. la)
with the samemobility. conditions including
dithio-
present had an apparent
reagent and co-electrophoresed with the major [%]fucose-labelled (Fig.
gel
species Peak M
electrophoresed
Analysis of the cell layer proteins under reducing
revealed a complex series of CoomassieBlue-staining
bands
high molecular weight species which did not penetrate the sepsr-
381
VoL71,No.
BIOCHEMICAL
1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2: SDS-polyacrylamide gel electrophoresis of [3fI]fucose-labelled macromolecules released into the medium of skin fibroblast cultures. Proteins were detected by scanning a Coomassie Blue-stained gel at 620 nm, and the presence of [3H]glycoproteins detected in a duplicate gel by slicing and scintillation counting. The mobilities of the following standards are indicated: bovine serum albumin (A) trimer (204,000), (B) dimer (136,000), D) monomer (68,000); (C) phosphorylase (100,000). o-el ) [3B]fucose ; (- - - - ) E620.
ating from
gel.
Several
protein
iSO,OOO to 220,000
determine
whether
glycoprotein
medium
were
or not
I and Type III human skin
observed,
referred
whether
which (lo),
The macromolecules
the SDS-agarose
column
(fractions
graphed.
The results
proline-labelled labelled of tendon
22-23
without
the major
molecular work
is
weights
ranging
in progress
to
to the high
molecular
weight
presented
peak of MFGP, and the
@- and a-chains.
into
was reduced,
first
second
to the precursors
of Type
and secreted with
applied
eluting
alkylated
coincident
[3B]fucose-
in a position
A small
proportion
and to
and rechromatopeaks
with
by
at the void
3 show two included
eluting
382
[3B]fucose
the medium were
and the material
in Fig. the
in the
dual-labelled
released reduction
glycoprotein
to be synthesised
were
la)
fucosylated
was related
are known
in Fig.
polypeptides,
collagen
is related
cultures
[14C]proline.
volume
and further
to as MFGP)
collagens,
fibroblasts
apparent
in the medium.
to determine
(hereafter
having
any of these
identified
In order
bands
the
between of the
of [14C]-
standards
radioactivity
BIOCHEMICAL
Vol. 71, No. 1,1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3: Gel filtration on SDS-agarose (Bio-Bad A-5m) of reduced, high molecular weight mediummacromolecules labelled with [%l]fucose and [%Iproline. Dual-labelled proteins were initially chromatographed under nonreducing conditions, and the void volume fractions (see Fig. la and the text) concentrated and rechromatographed after reduction and alky were collected, aliquots (0.05 ml) assayed for pairs, hydrolysed and analysed for of standards are indicated aa in - o - 0 ) [14C]proline;
remained at the void volume, and is probably attributable tendency of MFGPto aggregate even in 0.1% SDS.
to the marked
Analyses of the fractions
for hydroxy[14C]p ro 1'ine demonstrated the collagenous nature of the second included [14C]proline
peak, which was thus identified
of procollagen molecules.
By comparison, the peak of [%]EFGP contained
only basal levels of hydroxy[14C]proline, collagenous glycoprotein.
Furthermore,
digestion with a highly purified hydroxy[14C]p ro 1'ine-containing level by this enzyme.
as reduced polypeptides
bacterial
suggesting it to be a nonthe [%l]MFGP peak was resistant collagenase (ll),
to
whereas the
procollagen peak was reduced to background
Pertinent
to these observations was the finding
that
the synthesis and secretion of procollagen peptides were much reduced when the cells were cultured
in ascorbate-deficient
mediumwhereas the amount of
radiolabelled-MFGP released into the mediumwas unaltered. DISCTJSSION: We believe that this study establishes for the first the major protein
released into the mediumby human skin fibroblaets
383
time that in
Vol. 71, No. 1,1976
culture
is
weight
BIOCHEMICAL
a non-collagenous
of MFGP has been
chromatography.
behave
quaternary
structure
During muscle
protein
which
found
in elastic
cells is
is unknown
molecule structural
raises
in culture
presumed
its
apparent
the possibility
glycoprotein
laid
Muir
that
protein et a1.(12)
is
complex. reported
out with
protein
by cultured
to the smooth also
that
glyco-
of the microfibrillar
a precursor
down in the extracellular
ACKNOWLEDGMENT: This work was carried Cystic Fibrosis Research Trust.
and after
weight
of MFGP synthesised
it
to
may possess
molecular
a subunit
similarity
gel
are known
glycoprotein
heterologous
a high
The function
filtration
of MFGP before
this
manuscript,
to represent
tissue. but
that
synthesise
by SDS-gel
as glycoproteins
of a larger
of&is
The molecular
by SDS-polyacrylemide
The behaviour
suggests
or be part
the preparation
smooth
obtained
systems.
(MFGP).
2 25,000
as 250,000
en underestimate,
bonds
RESEARCH COMMUNICATIONS
glycoprotein
of 200,000
in such
of disulphide
blasts
determined
is probably
anomalously
reduction
fucosylated
The value
electrophoresis
AND BIOPHYSICAL
muscle
fibro-
cell
or subunit
of a
matrix. the
aid
of a grant
from
the
-cEs I. 2.
3. 4.
5. 6. 7. ;: IO. 11. 12.
Anderson, J.C. (1976) Int. Rev. Connect. Tissue Res. 2, 251-322. Slavkin, H.C., Croissant&D. and Guenther,% (19751 Protides Biol. Fluids Proc. Co11 Sandberg, L.B. (197 Int. Rev. Connect. Tissue Res, 2, 159-210. Sear,C.H.J., Grant,M.E., Anderson,J.C. and Jackson,D.S. (1975) Biochem. Sot. Trans. f, 138-140. Gorham, L.W. aud Waymouth, C. (1965) Proc. Sot. Exp. Biol. Med. 119, 287-290. Harwood,R., Bhalla,A.K., Graut,M.E. and Jackson,D.S. (1974) Biochem. &3, 129-138. Juva, K. and Procko , D.J. (1966) Anal. Biochem. u, 77-83. Laemmli, U.K. (1970 P Nature 2, 680-685. Fairbauks,G., Steck,T.L. end Wallach,D.F.B. (1971) Biochemistry l0, 2606-2617. Lichtenstein,J.R., Byers,P.H., Smith,B.D. and Martin,G.R. (1975) Biochemistrv fk, 1589-1594. Lee-Own, V. and Anderson, J.C. (1975) Pre Muir,L.W., Bornstein,P. and Ross,R. (1976 105114.
384
J.
Vol. 71, No. 1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IDENTIFICATION OF A MAJOREXTRACELLULAR NON-COLLAGENOUS GLYCOPRGTEIN SYNTBFSISEDBYBCMANSKINFIBBCBLASTSINCULTURE Christopher H.J. Sear, Michael E. Grant & David S. Jackson Department of Medical Biochemistry,
University
of Manchester,
Medical School, Oxford Road, Manchester Ml3 gPT, U.K. Received
May 12,1976
-: The major protein released into the mediumby humanskin fibroblasts in culture has been shown to be a fucosylated glycoprotein (designated MFGP). Analysis by gel filtration chromatography and polyacrylsmide gel electrophoresis demonstrated that under reducing conditions MFGPhas a molecular weight of approx. 250,000, but occurs as a disulphide-linked aggregate in the medium. Three lines of evidence are presented to establish that MFGPis a non-collagenous molecule. INTRODUCTION: Studies with cultured
cells have contributed
to our uuder-
standing of the synthesis and secretion of both collagenous and proteoglycanrelated
components of the extracellular
matrix.
However, there is au
increasing amount of evidence to suggest that connective tissues also contain non-collagenous structural
glycoproteins
been very poorly characterised
(1).
which, with few exceptions,
In view of the possible importance of
such macromolecules in the development of connective tissues (2) disease processes (3) suitable
we have established optimal culture
for the study of glycoprotein
conditions
incorporate
into a high molecular weight non-collagenous glycoprotein, protein
and in
biosynthesis by human skin fibroblasts
In this paper we demonstrate that such cells
(4).
have
[%]fucose
which is the major
released into the culture medium.
were propagated as described previously cultures maintained in a ch ically defined medium, MAB 87/T were labelled for 12h or 24h with L-[lfucose (10 @X/ml). When fucose-labelled glycoproteins were to be compared with secreted collagenous molecules both L[l-%I]fucose and [U-14C]proline (I @X/ml) were
EXPERIMENTAL:
Abbreviation:
Copyright All rights
Human skin fibroblasts
SDS, sodium fiorlecyl sulfate,
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
379
Vol. 71, No. 1,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
added to the maintenance medium, which acid (50 &ml) aa P-aminopropionitrile
was further supplemented (50 (*g/ml).
with
ascorbic
Non-diffusible radiolabelled macromolecules in the medium and in the cell layer were analysed by SDS-agarose gel filtration chromatography either unreduced or after prior reduction aa alkylation (6). The elution positions of collagenous polypeptides were determined by a specific radiochemical assay c01ultms for hydroxy[%]proline following hydrolysis of the fractions (7). ovalbumin, bovine serum were calibrated using the following standards: albumin and rat tail tendon collagen OL- end P-chains. The %-macromolecules in the medium and cell layer were analysed by using a separating gel 10 cm 10% SDS-polyacrylamide gel electrophoresis (8) The proteins in the medium were aa containing 8$ (w/v) acrylamide. concentrated prior to analysis by precipitation with (NRk)2SOh at i'?$ saturation in the presence of 25 mM EIYTA, 10 mM N-ethylmaleimide and 2 mM Gels were stained with either Coomassie phenylmethylsulphonyl fluoride. Brilliant Blue B or by the periodic acid-Schiff procedure (!I), and the distribution of [%]f ucose-labelled molecules determined by slicing and assaying for radioactivity. RESULTS: our
stocks)
Glycoprotein was examined
non-diffusible proteins
biosynthesis by studying
the
After
macromolecules. present
by human skin
i? the medium were
incorporation
a 24h labelling
aualysed
fibroblasts
(line
BF3 of
of [%]fucose period
by SDS-agarose
into
the %-glycogel
filtration
a
20
40 FRACTION
60 NO.
Fig. 1: Gel filtration on SDS-agarose (Bio-Rad A-5m) of macromolecules labelled with [%I -fucose synthesised by cultured skin fibroblasts. Glycoproteins (a 3 released into the medium, (b) resent in the cell ES!; The elution ositions of collagen a- and P-chains 98,000 and 196,000 and the void volume of the column (57 ml) are amowed. nnreducea sample; (0 - 0 - 0 ) reduced sample.
380
.
BIOCHEMICAL
Vol. 71, No. 1,1976
chromatography (Fig.
la).
accounting for 253% of the column.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In the absence of reducing agents the major peak,
of the total
radioactivity,
eluted at the void volume
However, when the labelled mediumproteins were chromato-
graphed following
reduction
and alkylation
the radioactivity
at the void
volume was markedly diminished and a new major peak appeared (peak M of Fig. The molecular weight of this major species was estimated from six
la).
experiments as 250,000 t 25,000 by extrapolation based on the elution
positions
from a calibration
of standard proteins.
Similar results were
obtained when the cells were labelled with [i-i4C]fucose identical
curve
and essentially
chromatogrsms were obtained using six other humanskin fibroblast
cell lines. Gel filtration
analysis of the unreduced labelled macromolecules in the
cell layer is presented in Fig. lb. not significantly Tfieae results relatively
It is noteworthy that this profile
altered by reduction
indicated
of the sample prior
was
to chromatography.
that the peak M component, if present, represented a
minor proportion
of the total
fucosylated
species in the cell
layer. Further experiments were conducted to determine whether the high molecular weight [%]f ucose-labelled represented a major constituent electrophoresis threitol
macromolecule present in the medium
released by the cells.
of the mediummacromolecules after
demonstrated that the major protein
molecular weight of approximately
Polyacrylamide
reduction vith
2).
material
200,000, stained strongly
with the PAS
It was also established that the [%]fucose-labelled recovered from the SDS-agarose column (Fig. la)
with the samemobility. conditions including
dithio-
present had an apparent
reagent and co-electrophoresed with the major [%]fucose-labelled (Fig.
gel
species Peak M
electrophoresed
Analysis of the cell layer proteins under reducing
revealed a complex series of CoomassieBlue-staining
bands
high molecular weight species which did not penetrate the sepsr-
381
VoL71,No.
BIOCHEMICAL
1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2: SDS-polyacrylamide gel electrophoresis of [3fI]fucose-labelled macromolecules released into the medium of skin fibroblast cultures. Proteins were detected by scanning a Coomassie Blue-stained gel at 620 nm, and the presence of [3H]glycoproteins detected in a duplicate gel by slicing and scintillation counting. The mobilities of the following standards are indicated: bovine serum albumin (A) trimer (204,000), (B) dimer (136,000), D) monomer (68,000); (C) phosphorylase (100,000). o-el ) [3B]fucose ; (- - - - ) E620.
ating from
gel.
Several
protein
iSO,OOO to 220,000
determine
whether
glycoprotein
medium
were
or not
I and Type III human skin
observed,
referred
whether
which (lo),
The macromolecules
the SDS-agarose
column
(fractions
graphed.
The results
proline-labelled labelled of tendon
22-23
without
the major
molecular work
is
weights
ranging
in progress
to
to the high
molecular
weight
presented
peak of MFGP, and the
@- and a-chains.
into
was reduced,
first
second
to the precursors
of Type
and secreted with
applied
eluting
alkylated
coincident
[3B]fucose-
in a position
A small
proportion
and to
and rechromatopeaks
with
by
at the void
3 show two included
eluting
382
[3B]fucose
the medium were
and the material
in Fig. the
in the
dual-labelled
released reduction
glycoprotein
to be synthesised
were
la)
fucosylated
was related
are known
in Fig.
polypeptides,
collagen
is related
cultures
[14C]proline.
volume
and further
to as MFGP)
collagens,
fibroblasts
apparent
in the medium.
to determine
(hereafter
having
any of these
identified
In order
bands
the
between of the
of [14C]-
standards
radioactivity
BIOCHEMICAL
Vol. 71, No. 1,1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3: Gel filtration on SDS-agarose (Bio-Bad A-5m) of reduced, high molecular weight mediummacromolecules labelled with [%l]fucose and [%Iproline. Dual-labelled proteins were initially chromatographed under nonreducing conditions, and the void volume fractions (see Fig. la and the text) concentrated and rechromatographed after reduction and alky were collected, aliquots (0.05 ml) assayed for pairs, hydrolysed and analysed for of standards are indicated aa in - o - 0 ) [14C]proline;
remained at the void volume, and is probably attributable tendency of MFGPto aggregate even in 0.1% SDS.
to the marked
Analyses of the fractions
for hydroxy[14C]p ro 1'ine demonstrated the collagenous nature of the second included [14C]proline
peak, which was thus identified
of procollagen molecules.
By comparison, the peak of [%]EFGP contained
only basal levels of hydroxy[14C]proline, collagenous glycoprotein.
Furthermore,
digestion with a highly purified hydroxy[14C]p ro 1'ine-containing level by this enzyme.
as reduced polypeptides
bacterial
suggesting it to be a nonthe [%l]MFGP peak was resistant collagenase (ll),
to
whereas the
procollagen peak was reduced to background
Pertinent
to these observations was the finding
that
the synthesis and secretion of procollagen peptides were much reduced when the cells were cultured
in ascorbate-deficient
mediumwhereas the amount of
radiolabelled-MFGP released into the mediumwas unaltered. DISCTJSSION: We believe that this study establishes for the first the major protein
released into the mediumby human skin fibroblaets
383
time that in
Vol. 71, No. 1,1976
culture
is
weight
BIOCHEMICAL
a non-collagenous
of MFGP has been
chromatography.
behave
quaternary
structure
During muscle
protein
which
found
in elastic
cells is
is unknown
molecule structural
raises
in culture
presumed
its
apparent
the possibility
glycoprotein
laid
Muir
that
protein et a1.(12)
is
complex. reported
out with
protein
by cultured
to the smooth also
that
glyco-
of the microfibrillar
a precursor
down in the extracellular
ACKNOWLEDGMENT: This work was carried Cystic Fibrosis Research Trust.
and after
weight
of MFGP synthesised
it
to
may possess
molecular
a subunit
similarity
gel
are known
glycoprotein
heterologous
a high
The function
filtration
of MFGP before
this
manuscript,
to represent
tissue. but
that
synthesise
by SDS-gel
as glycoproteins
of a larger
of&is
The molecular
by SDS-polyacrylemide
The behaviour
suggests
or be part
the preparation
smooth
obtained
systems.
(MFGP).
2 25,000
as 250,000
en underestimate,
bonds
RESEARCH COMMUNICATIONS
glycoprotein
of 200,000
in such
of disulphide
blasts
determined
is probably
anomalously
reduction
fucosylated
The value
electrophoresis
AND BIOPHYSICAL
muscle
fibro-
cell
or subunit
of a
matrix. the
aid
of a grant
from
the
-cEs I. 2.
3. 4.
5. 6. 7. ;: IO. 11. 12.
Anderson, J.C. (1976) Int. Rev. Connect. Tissue Res. 2, 251-322. Slavkin, H.C., Croissant&D. and Guenther,% (19751 Protides Biol. Fluids Proc. Co11 Sandberg, L.B. (197 Int. Rev. Connect. Tissue Res, 2, 159-210. Sear,C.H.J., Grant,M.E., Anderson,J.C. and Jackson,D.S. (1975) Biochem. Sot. Trans. f, 138-140. Gorham, L.W. aud Waymouth, C. (1965) Proc. Sot. Exp. Biol. Med. 119, 287-290. Harwood,R., Bhalla,A.K., Graut,M.E. and Jackson,D.S. (1974) Biochem. &3, 129-138. Juva, K. and Procko , D.J. (1966) Anal. Biochem. u, 77-83. Laemmli, U.K. (1970 P Nature 2, 680-685. Fairbauks,G., Steck,T.L. end Wallach,D.F.B. (1971) Biochemistry l0, 2606-2617. Lichtenstein,J.R., Byers,P.H., Smith,B.D. and Martin,G.R. (1975) Biochemistrv fk, 1589-1594. Lee-Own, V. and Anderson, J.C. (1975) Pre Muir,L.W., Bornstein,P. and Ross,R. (1976 105114.
384
J.