Vol. 86, No. 3, 1979 February
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 465-472
14, 1979
COLLAGEN SYNTHESIS BY CULTURES OF STROMAL CELLS FROM NORMAL HUMAN AND KERATOCONUS CORNEAS* Beatrice
Y.J.T.
Yue,
Jules
L, Baumt and Barbara
D. Smith
Department of Ophthalmology New England Medical Center Hospital Tufts University School of Medicine and Connective Tissue-Aging Research Laboratory, Veterans Administration Outpatient Clinic Received
November
28,
1978
Summary Keratoconus is a disease which thins and scars the central cornea. Confluent cultures of cornea1 stromal cells were derived from patients with keratoconus. The collagen synthesized by these cultures was compared to the collagen synthesized by age-matched normal human cornea1 stromal cultures, Although the amount of collagen and the types of collagen synthesized were similar, relative proportion of type I collagen and A, B chains produced was significantly altered in keratoconus cultures. The DEAE-cellulose chromatograms of procollagen in the medium fraction were different, not only between normal control and keratoconus cultures but also among keratoconus patients. Introduction Keratoconus tion
is
a disease
of the central
cribed
(l-4),
noted
that
joints, tive
It
(5)
(8,9)
are distributed
(7)
is
of this
sometimes
suggesting
established
and possibly
exist
condition
tissues
It
has been
hypermobility disorder
distinct
of collagen,
and cell manner.
have been des-
of the of connec-
(6),
genetically types
and distor-
unclear.
with
heritable
syndrome
additional
in a tissue-specific
remains
a generalized
four
thinning changes
seen in patients
that
in mammalian
in a gradual
ultrastructural
such as Ehlers-Danlos
is now well
of collagen chains
keratoconus
results
Although
the pathogenesis
an association tissue
cornea.
which
cultures.
In cornea1
types
(I-IV)
such as A and B These strona,
collagens the collagen
*
This investigation was supported in part by research the National Eye Institute and in part by the Medical the Veterans Administration,
grant EY 01793 from Research Service of
t
Requests for reprints should be addressed to Jules L, Baum, MI,D, Department of Ophthalmology, New England Medical Center Hospital, 171 Harrison Avenue, Boston, MA 02111, 0006-291X/79/030465-08$01,00/0 465
Copyright All rights
@ 1979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 86, No. 3, 1979
is highly
glycosylated
Type III
(12)
stroma.
The collagen
studied
(15)
have been
mainly
increased
biochemical reason
in the
(13)
cornea
suggested.
have also
abnormalities
It
of type
of patients
of lysine,
I collagen been
with
has also
been reported
12), in cornea1
keratoconus
in the collagen
of structural
(11,
identified
and the glycosylation
and the amount
has not been
cross-linking of hydroxylsine
that
the total
glycoprotein
is relati-
(15).
grown
in tissue
analyses
keratoconus were
culture
have
in relation
we have established
experiments
chains
although
is decreased
Cells
with
and consists
the hydroxylation
collagen vely
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and A, B-like
extensively,
(14),
BIOCHEMICAL
to examine performed
to various
cultures
normal
a good system
diseases
of cornea1
the synthesis with
provided
stromal
of collagen. human cornea1
(16,
for
17).
cells
detailed
For this from patients
Parallel stromal
cell
cultures
as controls. Materials
and Methods
Age-matched normal human corneas were obtained from the New England Eye Bank. Cornea1 buttons from patients with keratoconus were placed into McCarey-Kaufman medium (18) and sent to our laboratory, The middle portion of the cornea1 stromal layer was explanted and cultured as described previously (19). After 3-4 weeks, when cultures cells were trypsinized and passed. Normal control and were confluent, keratoconus cells appeared similar under phase contrast microscopy. Collagen studies were performed between the 2nd and the 7th passages. Four flasks (75 cm2) of confluent cornea1 cultures were incubated for 24 hours with Eagles' minimum essential medium (Cat.{/ 12-1258, Microbiological Co.) containing 5 pCi/ml each of (3H)glycine and (3H)proline in the presence of @aminopropionitrile and ascorbic acid (100 pg/ml). After labeling, the medium was removed from each flask and pooled, The cultures were washed six times with 8 ml of Dulbecco's phosphate buffered saline (1 x, pH 7.0, Cat.# 17-5138, Microbiological Co,), and then combined with the medium fraction. The cells were harvested in 0.5 M acetic acid and broken with a Polytron homogenizer as the cell layer fraction, Aliquots of the cell layer and the medium fraction were dialyzed extensively against distilled water, lyophilized, and the amount of collagenous and non-collagenous proteins were determined with a purified bacterial collagenase (Advance Biofactures, Form IV) as described previously (20). All experiments were performed in duplicate. The cell layer together with 5 mg of carrier type I collagen [prepared from lathyritic rat skin (21)] was extracted with 0.5 M acetic acid at 4OC overnight. After centrifugation at 15,000 x g for 40 minutes, the supernatant was dialyzed against 0.05 M sodium acetate buffer, pH 4.8, heated and placed on a column of carboxylmethyl (CM)-cellulose as previously described (22), Certain cell homogenates were suspended in
466
Vol.
86,
No.
BIOCHEMICAL
3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.5 M acetic acid and treated with pepsin (1 mg/lO ml) for 16 hours at 4'C before analyses by CM-cellulose column chromatography. The individual c1 chains were obtained from the CM-cellulose column, for slab gel pooled, desalted by a Biorad P-2 column, and lyophilized electrophoresis. Samples were treated with 1% sodium dodecyl sulfate (SDS) at 80°C for 5 min, and certain samples were reduced with 10 mM of dithiothreitol prior to layering onto 5% SDS-polyacrylamide slab gels (23). After the completion of electrophoresis, the gels were fixed and stained in Coomassie Blue. The radioactively labeled bands were detected by fluorography (24), The medium fraction was dialyzed against solutions of 0,15 M NaCl, 0,05 M Tris and 0.02 M EDTA, pH 7.4, containing the protease inhibitors phenylmethyl sulfonyl fluoride (10 mM) and p-chloromercuribenzoate (1 mM). Samples were prepared and chromatographed on DEAE-cellulose columns as previously described (25) to separate collagen, procollagen Type I, and procollagen type III. Results
and Discussion
Collagen
synthesis
human stroma synthesized products
is
shown in Table
by these were
The labeled cultures
together
with
carrier
samples.
samples
(open
elution
patterns of a,(I)
material
(Fig, various crete
1, closed peaks
found
between
The CM-cellulose circles) compared
a 1 and a2 peaks.
and percentage
of collagen
More labelled than
layer
type
of cell
in all
it
chromatogram
normal
samples
of the
and five
labeled
from normal
between
the
are shown in Fig.
cultures
1,
kera-
normal
control
Similar corneas,
2,O and 2.3.
with
the
The labeled
and the a2 peaks was collagenase
was collagen. of samples
showed marked to normal
samples
standard
preceeding
and an a2 peak,
the al(I)
that
on CM-cellulose
peak,
slightly
and normal
The carrier
layer
profile
layer.
of keratoconus
S12 component
from four
collagenous
in the cell
I collagen.
and a2 peaks with
to a2 varying
demonstrating
The amount
fraction
had an al(I)
were
and normal
similar.
The radioactivity
appearing
sensitive
skin
profiles
circles)
of keratoconus
and chromatographed
patterns
typical
cultures
from the cell
rat
al(I),
toconus
ratio
were
was extracted
The elution
representing
I.
in the medium
protein
had the typical a2 peak,
cultures
found
control
by confluent
control
The radioactivity
467
from keratoconus
differences samples.
cultures
in the proportions There
in the region
was no disbetween
al(I)
of
Vol. 86, No. 3, 1979
BIOCHEMICAL
Table synthesis by and keratoconus
Collagen human
Cell
Age of Donor (years)
Type
(3H)proline incorporated proteins(cpm/mg Cell
Normal human
Keratoconus
Each essential 24 hours and
flask medium in
the
percentage
as described
17
3164
26 40 51
2255
23 31 44 48 68
of
in
I 50
( 3 H)glycine collagenous of dry weight) Cell
f 3 H)collagenous as % of total
+ Medium
2.9
1177 2130
2.0 2.4
3234 2556 2745 6530 3079
22937 13677 11040 8465 12347
26171 16233 13785 14995 15426
2.6 3.0 2.3 3.8 3.0
of
cultures
was
5 uCi/ml
each
incubated of
with
and
proteins
were
Medium 8.9 11.0 16.9 19.6
3.0
(3H)proline
B-aminopropionitrile
proteins (3H)proteins
Cell
11156 14602 10084
(3H)collagenous
Material
Medium
of normal cells
15718
containing
of
and into
I cultures stromal
12617 8901 13425 7954
confluent
presence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3 ml and
ascorbic
determined
of
18.9 9.2 9.8
Eagle's
(3H)glycine acid.
by
18.5 17.7
minimum for
The amount
collagenase
assays
and Methods.
I 100
I 150
EFFLUENT
I 200
I 250
VOLUME, m/
468
! 300
I 350
400
BIOCHEMICAL
Vol. 86, No. 3, 1979
and CL chains 2 cultures
was increased
as opposed
AND BIOPHYSICAL RESEARCH COMMJNICATIONS
to 40-50%
to 20-25%
in normal
could
be procollagen
(26),
based
on its
from CM-cellulose
elution
To further keratoconus
samples
extension
peptides,
of these
proteins
migrating
slower
type
eluting
than
of type
This
I collagen
stromal
III
differs
treated
still
with
contained the a
1
of
pepsin
to re-
by CM-cellulose proportions
and u 2 chains,
region
(8).
is
in keratoconus (27)
product
consistent
and normal
which
showed
in normal
column
cultures
cultures
(closed
(open
II,
approximately
of cornea1
chromatography, procollagens
by SDS-gel at peak
fraction
circles)
circles), electrophoresis which
Fig,
cultures
2 shows the
from the most
severely
and the representative The product as type
was completely
appearing
control
that
human cornea1
were
collagenase
6-15% of the radioactivity
analyzed
elution affected normal
at peak
I procollagen,
I was
The
digestable, in four
normal
chromatogram of labeled collagen isolated Figure 1. CM-cellulose from the cell layer of keratoconus (o---o) and normal control cultures (o---o). The column (1.5 x 10 cm) was equilibrated with 0.05 M sodium acetate buffer pH 4.8 and elution was performed with a linear gradient from 0 to 0.1 M NaCl over a total volume of 400 ml. Fractions Carrier collagen of 5 ml were collected and assayed for radioactivity. was type I collagen isolated from lathyritic rat skin,
469
bands
No radioactive
indicating
therefore,
report
Analysis
in two radioactive
dithiothreitol
The data, chains
set
similar
resulted
with
collagenous
in the medium
of radioactive
keratoconus
represented
(8)
cultures,
patterns
component
material
A, B chains
another
When examined
from a previous
was the only
by DEAE-cellulose
identified
were
reduction
collagen,
This
and/or
material,
between
of A, B-like
Procollagens
control
(16),
electrophoresis
even after
keratoconus
cultures.
the a1 band in the A, B chain
the idenfication
cultures.
samples
by SDS-gel
band was found
with
collagenous
these
in all
columns.
control
proteins
control
collagen
and normal
chromatography,
the absence
III
this
of collagenous
cl
type
identify
move the procollagen column
of the total
Vol. 86, No. 3, 1979
BIOCHEMICAL
0
I 0
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I 500
100
Figure 2, DEAE-cellulose from the medium fraction cultures (o---o).
chromatogram of keratoconus
I 600
of the procollagen purified (e---e) and normal control
Column was equilibrated with 0.05 M Tris pH 8,0, 2 M urea. Chromatography was performed at 4OC with an elution volume of 600 ml and the gradient from 0 to 0.2 M NaCl. Fractions of 11 ml were collected and assayed for radioactivity.
control
cultures,
approximately
but was increased 30-70%
of the
total
in five procollagen
keratoconus
cultures
depending
to
on the patient
analyzed. Variation with
keratoconus
This
has also
with
osteogenesis
found
in the DEAE-cellulose
suggests occurred
that in studies
imperfecta
cornea1
buttons,
a scarred
present
whether
the defects
diseased
cells
themselves
this
area
cornea1 with
(16),
chromatograms
skin
disease
among patients
is heterogeneous.
fibroblasts
from patients
In some of the keratoconus
was observed,
in the collagen or are associated
repair.
470
It
is not
synthesis with
clear
at
are due to the
the process
of
Vol.
86,
No.
3, 1979
BIOCHEMICAL
We have also glycosaminoglycans synthesis
matrix
produced
of cell
reduced
(28), which
demonstrated
This
AND
BIOPHYSICAL
abnormalities by these
layer-related suggests
shows modifications
sulfate
keratoconus in both
COMMUNICATIONS
in the metabolism
same keratoconus
heparan that
RESEARCH
is
cultures.
The
significantly
may be a disease
collagen
of
of
and glycosaminoglycan
syntheses. Acknowledgements We thank for
their
and Dr,
MS, Rasma Niedra,
technical A. Boruchoff
assistance. for
supplying
Mr,
John Salem,
We also
wish
and Mr, William to thank
Dr.
some of the keratoconus
P,R.
Lavin Laibson
cornea1
buttons,
References 1. 2. 3. 4. 5. 6, 7. 8. 9. 10. 11. 12. 13.. 14, 15, 160 17. 18, 19, 20. 21, 22. 23, 24,
Teng, C.C. (1963) Amer. J. Ophthalmol. 55, 18-47. (1970) Arch, Ophthalmol, Patta, CL,, Loyon, L,, and Roucher, F. (Paris) 30, 403-417. Kim, J.O., and Hassard, D,T,R. (1972) Canad. 9. Ophthalmol, 7, 176-180. Iwamoto, T., and Devoe, A.G. (1975) Arch. Ophthalmol. (Paris) 35, 65-72. Robertson, I. (1974) Aust, J. Ophthalmol. 2, 144-147. Uitto, J., and Prockop, D.J, (1974) Molecular Pathology, pp. 670-688, Thomas, Springfield, Illinois. Miller, E.J. (1976) Molecular and Cellular Biochemistry 13, 165-192. Burgeson, R.E., El Adli, F.A., Kaitila, I-I,, and Hollister, D.W, (1976) Proc. Natl. Acad, Sci. USA 12, 2579-2583, Chung, E., Rhodes, R.K., and Miller, E.J. (1976) Biochem. Biophys, Res, comm, 71, 1167-1169, Katzman, R.L., Kang, A.H,, and Dixit, S.N, (1974) Biochim. Biophys. Acta, 336, 367-369. Freeman, I.L, (1978) Invest. Ophthalmol. and Vis. Sci. 17, 171-177, Schmut, 0. (1977) Exp. Eye Res. 25, 505-509, Hong, B,S., Cannon, D.J. and Davison, P.F. (1978) Fed. Proc, 37, 1528, Cannon, J. and Foster, C.S. (1978) Invest. Ophthalmol. and Vis. Sci. l7-, 63-65. Robert, L,, Schillinger, G,, Moczar, M., Junqua, S., and Moczar, E. (1970) Arch. Ophthalmol. (Paris) 30, 590-607. Penttinen, R.P,, Lichtenstein, J,R., Martin, G.R,, and McKusick, V.A, (1975) Proc. Natl, Acad. Sci. USA 72, 586-589. Dorfman, A., and Matalon, R. (1976) Proc, Natl. Acad. Sci. USA 73, 630-637. McCarey, B.E., and Kaufman, H.E. (1974) Invest., Ophthalmol, 2, 165-173. Baum, J,L., Niedra, R,, Davis, C., and Yue, B,Y,J,T. Arch. Ophthalmol. (in press). Peterkofsky, B., and Diegelmann, R, (1971) Biochemistry I& 988-994, Piez, K.A. (1967) Treatise on collagen, Vol, 1, Chemistry of Collagen, ppO 201-248, Academic Press, New York. Miller, E.J., Martin, G,R,, Piez, K.A., and Powers, M,J. (1967) J. Biol, Chem, 242, 5481-5489. Laemmli, U,K. (1970) Nature 227, 680-685, Laskey, R.A., and Mills, A.D. (1975) Eur, J, Biochem. 56, 335-341.
471
Vol. 86, No. 3, 1979
25, 26, 27, 28.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(1972) Proc, Natl. Acad, Sci. Smith, B.D., Byers, P.H,, and Martin, G.R, USA 69, 3260-3262. (1977) Arch. Biochem, Biophys. 182, 33-41. Moro, L., and Smith, B.D, Stoesser, T.R., Church, R.L., and Brown, S.I. (1978) Invest. Opthalmol, and Vis. Sci. l7, 264-271, Yue, B.Y.J.T,, Baum, J.L,, and Silbert, J.E. Manuscript submitted for publication,
472
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 465-472
14, 1979
COLLAGEN SYNTHESIS BY CULTURES OF STROMAL CELLS FROM NORMAL HUMAN AND KERATOCONUS CORNEAS* Beatrice
Y.J.T.
Yue,
Jules
L, Baumt and Barbara
D. Smith
Department of Ophthalmology New England Medical Center Hospital Tufts University School of Medicine and Connective Tissue-Aging Research Laboratory, Veterans Administration Outpatient Clinic Received
November
28,
1978
Summary Keratoconus is a disease which thins and scars the central cornea. Confluent cultures of cornea1 stromal cells were derived from patients with keratoconus. The collagen synthesized by these cultures was compared to the collagen synthesized by age-matched normal human cornea1 stromal cultures, Although the amount of collagen and the types of collagen synthesized were similar, relative proportion of type I collagen and A, B chains produced was significantly altered in keratoconus cultures. The DEAE-cellulose chromatograms of procollagen in the medium fraction were different, not only between normal control and keratoconus cultures but also among keratoconus patients. Introduction Keratoconus tion
is
a disease
of the central
cribed
(l-4),
noted
that
joints, tive
It
(5)
(8,9)
are distributed
(7)
is
of this
sometimes
suggesting
established
and possibly
exist
condition
tissues
It
has been
hypermobility disorder
distinct
of collagen,
and cell manner.
have been des-
of the of connec-
(6),
genetically types
and distor-
unclear.
with
heritable
syndrome
additional
in a tissue-specific
remains
a generalized
four
thinning changes
seen in patients
that
in mammalian
in a gradual
ultrastructural
such as Ehlers-Danlos
is now well
of collagen chains
keratoconus
results
Although
the pathogenesis
an association tissue
cornea.
which
cultures.
In cornea1
types
(I-IV)
such as A and B These strona,
collagens the collagen
*
This investigation was supported in part by research the National Eye Institute and in part by the Medical the Veterans Administration,
grant EY 01793 from Research Service of
t
Requests for reprints should be addressed to Jules L, Baum, MI,D, Department of Ophthalmology, New England Medical Center Hospital, 171 Harrison Avenue, Boston, MA 02111, 0006-291X/79/030465-08$01,00/0 465
Copyright All rights
@ 1979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 86, No. 3, 1979
is highly
glycosylated
Type III
(12)
stroma.
The collagen
studied
(15)
have been
mainly
increased
biochemical reason
in the
(13)
cornea
suggested.
have also
abnormalities
It
of type
of patients
of lysine,
I collagen been
with
has also
been reported
12), in cornea1
keratoconus
in the collagen
of structural
(11,
identified
and the glycosylation
and the amount
has not been
cross-linking of hydroxylsine
that
the total
glycoprotein
is relati-
(15).
grown
in tissue
analyses
keratoconus were
culture
have
in relation
we have established
experiments
chains
although
is decreased
Cells
with
and consists
the hydroxylation
collagen vely
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and A, B-like
extensively,
(14),
BIOCHEMICAL
to examine performed
to various
cultures
normal
a good system
diseases
of cornea1
the synthesis with
provided
stromal
of collagen. human cornea1
(16,
for
17).
cells
detailed
For this from patients
Parallel stromal
cell
cultures
as controls. Materials
and Methods
Age-matched normal human corneas were obtained from the New England Eye Bank. Cornea1 buttons from patients with keratoconus were placed into McCarey-Kaufman medium (18) and sent to our laboratory, The middle portion of the cornea1 stromal layer was explanted and cultured as described previously (19). After 3-4 weeks, when cultures cells were trypsinized and passed. Normal control and were confluent, keratoconus cells appeared similar under phase contrast microscopy. Collagen studies were performed between the 2nd and the 7th passages. Four flasks (75 cm2) of confluent cornea1 cultures were incubated for 24 hours with Eagles' minimum essential medium (Cat.{/ 12-1258, Microbiological Co.) containing 5 pCi/ml each of (3H)glycine and (3H)proline in the presence of @aminopropionitrile and ascorbic acid (100 pg/ml). After labeling, the medium was removed from each flask and pooled, The cultures were washed six times with 8 ml of Dulbecco's phosphate buffered saline (1 x, pH 7.0, Cat.# 17-5138, Microbiological Co,), and then combined with the medium fraction. The cells were harvested in 0.5 M acetic acid and broken with a Polytron homogenizer as the cell layer fraction, Aliquots of the cell layer and the medium fraction were dialyzed extensively against distilled water, lyophilized, and the amount of collagenous and non-collagenous proteins were determined with a purified bacterial collagenase (Advance Biofactures, Form IV) as described previously (20). All experiments were performed in duplicate. The cell layer together with 5 mg of carrier type I collagen [prepared from lathyritic rat skin (21)] was extracted with 0.5 M acetic acid at 4OC overnight. After centrifugation at 15,000 x g for 40 minutes, the supernatant was dialyzed against 0.05 M sodium acetate buffer, pH 4.8, heated and placed on a column of carboxylmethyl (CM)-cellulose as previously described (22), Certain cell homogenates were suspended in
466
Vol.
86,
No.
BIOCHEMICAL
3, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.5 M acetic acid and treated with pepsin (1 mg/lO ml) for 16 hours at 4'C before analyses by CM-cellulose column chromatography. The individual c1 chains were obtained from the CM-cellulose column, for slab gel pooled, desalted by a Biorad P-2 column, and lyophilized electrophoresis. Samples were treated with 1% sodium dodecyl sulfate (SDS) at 80°C for 5 min, and certain samples were reduced with 10 mM of dithiothreitol prior to layering onto 5% SDS-polyacrylamide slab gels (23). After the completion of electrophoresis, the gels were fixed and stained in Coomassie Blue. The radioactively labeled bands were detected by fluorography (24), The medium fraction was dialyzed against solutions of 0,15 M NaCl, 0,05 M Tris and 0.02 M EDTA, pH 7.4, containing the protease inhibitors phenylmethyl sulfonyl fluoride (10 mM) and p-chloromercuribenzoate (1 mM). Samples were prepared and chromatographed on DEAE-cellulose columns as previously described (25) to separate collagen, procollagen Type I, and procollagen type III. Results
and Discussion
Collagen
synthesis
human stroma synthesized products
is
shown in Table
by these were
The labeled cultures
together
with
carrier
samples.
samples
(open
elution
patterns of a,(I)
material
(Fig, various crete
1, closed peaks
found
between
The CM-cellulose circles) compared
a 1 and a2 peaks.
and percentage
of collagen
More labelled than
layer
type
of cell
in all
it
chromatogram
normal
samples
of the
and five
labeled
from normal
between
the
are shown in Fig.
cultures
1,
kera-
normal
control
Similar corneas,
2,O and 2.3.
with
the
The labeled
and the a2 peaks was collagenase
was collagen. of samples
showed marked to normal
samples
standard
preceeding
and an a2 peak,
the al(I)
that
on CM-cellulose
peak,
slightly
and normal
The carrier
layer
profile
layer.
of keratoconus
S12 component
from four
collagenous
in the cell
I collagen.
and a2 peaks with
to a2 varying
demonstrating
The amount
fraction
had an al(I)
were
and normal
similar.
The radioactivity
appearing
sensitive
skin
profiles
circles)
of keratoconus
and chromatographed
patterns
typical
cultures
from the cell
rat
al(I),
toconus
ratio
were
was extracted
The elution
representing
I.
in the medium
protein
had the typical a2 peak,
cultures
found
control
by confluent
control
The radioactivity
467
from keratoconus
differences samples.
cultures
in the proportions There
in the region
was no disbetween
al(I)
of
Vol. 86, No. 3, 1979
BIOCHEMICAL
Table synthesis by and keratoconus
Collagen human
Cell
Age of Donor (years)
Type
(3H)proline incorporated proteins(cpm/mg Cell
Normal human
Keratoconus
Each essential 24 hours and
flask medium in
the
percentage
as described
17
3164
26 40 51
2255
23 31 44 48 68
of
in
I 50
( 3 H)glycine collagenous of dry weight) Cell
f 3 H)collagenous as % of total
+ Medium
2.9
1177 2130
2.0 2.4
3234 2556 2745 6530 3079
22937 13677 11040 8465 12347
26171 16233 13785 14995 15426
2.6 3.0 2.3 3.8 3.0
of
cultures
was
5 uCi/ml
each
incubated of
with
and
proteins
were
Medium 8.9 11.0 16.9 19.6
3.0
(3H)proline
B-aminopropionitrile
proteins (3H)proteins
Cell
11156 14602 10084
(3H)collagenous
Material
Medium
of normal cells
15718
containing
of
and into
I cultures stromal
12617 8901 13425 7954
confluent
presence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3 ml and
ascorbic
determined
of
18.9 9.2 9.8
Eagle's
(3H)glycine acid.
by
18.5 17.7
minimum for
The amount
collagenase
assays
and Methods.
I 100
I 150
EFFLUENT
I 200
I 250
VOLUME, m/
468
! 300
I 350
400
BIOCHEMICAL
Vol. 86, No. 3, 1979
and CL chains 2 cultures
was increased
as opposed
AND BIOPHYSICAL RESEARCH COMMJNICATIONS
to 40-50%
to 20-25%
in normal
could
be procollagen
(26),
based
on its
from CM-cellulose
elution
To further keratoconus
samples
extension
peptides,
of these
proteins
migrating
slower
type
eluting
than
of type
This
I collagen
stromal
III
differs
treated
still
with
contained the a
1
of
pepsin
to re-
by CM-cellulose proportions
and u 2 chains,
region
(8).
is
in keratoconus (27)
product
consistent
and normal
which
showed
in normal
column
cultures
cultures
(closed
(open
II,
approximately
of cornea1
chromatography, procollagens
by SDS-gel at peak
fraction
circles)
circles), electrophoresis which
Fig,
cultures
2 shows the
from the most
severely
and the representative The product as type
was completely
appearing
control
that
human cornea1
were
collagenase
6-15% of the radioactivity
analyzed
elution affected normal
at peak
I procollagen,
I was
The
digestable, in four
normal
chromatogram of labeled collagen isolated Figure 1. CM-cellulose from the cell layer of keratoconus (o---o) and normal control cultures (o---o). The column (1.5 x 10 cm) was equilibrated with 0.05 M sodium acetate buffer pH 4.8 and elution was performed with a linear gradient from 0 to 0.1 M NaCl over a total volume of 400 ml. Fractions Carrier collagen of 5 ml were collected and assayed for radioactivity. was type I collagen isolated from lathyritic rat skin,
469
bands
No radioactive
indicating
therefore,
report
Analysis
in two radioactive
dithiothreitol
The data, chains
set
similar
resulted
with
collagenous
in the medium
of radioactive
keratoconus
represented
(8)
cultures,
patterns
component
material
A, B chains
another
When examined
from a previous
was the only
by DEAE-cellulose
identified
were
reduction
collagen,
This
and/or
material,
between
of A, B-like
Procollagens
control
(16),
electrophoresis
even after
keratoconus
cultures.
the a1 band in the A, B chain
the idenfication
cultures.
samples
by SDS-gel
band was found
with
collagenous
these
in all
columns.
control
proteins
control
collagen
and normal
chromatography,
the absence
III
this
of collagenous
cl
type
identify
move the procollagen column
of the total
Vol. 86, No. 3, 1979
BIOCHEMICAL
0
I 0
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I 500
100
Figure 2, DEAE-cellulose from the medium fraction cultures (o---o).
chromatogram of keratoconus
I 600
of the procollagen purified (e---e) and normal control
Column was equilibrated with 0.05 M Tris pH 8,0, 2 M urea. Chromatography was performed at 4OC with an elution volume of 600 ml and the gradient from 0 to 0.2 M NaCl. Fractions of 11 ml were collected and assayed for radioactivity.
control
cultures,
approximately
but was increased 30-70%
of the
total
in five procollagen
keratoconus
cultures
depending
to
on the patient
analyzed. Variation with
keratoconus
This
has also
with
osteogenesis
found
in the DEAE-cellulose
suggests occurred
that in studies
imperfecta
cornea1
buttons,
a scarred
present
whether
the defects
diseased
cells
themselves
this
area
cornea1 with
(16),
chromatograms
skin
disease
among patients
is heterogeneous.
fibroblasts
from patients
In some of the keratoconus
was observed,
in the collagen or are associated
repair.
470
It
is not
synthesis with
clear
at
are due to the
the process
of
Vol.
86,
No.
3, 1979
BIOCHEMICAL
We have also glycosaminoglycans synthesis
matrix
produced
of cell
reduced
(28), which
demonstrated
This
AND
BIOPHYSICAL
abnormalities by these
layer-related suggests
shows modifications
sulfate
keratoconus in both
COMMUNICATIONS
in the metabolism
same keratoconus
heparan that
RESEARCH
is
cultures.
The
significantly
may be a disease
collagen
of
of
and glycosaminoglycan
syntheses. Acknowledgements We thank for
their
and Dr,
MS, Rasma Niedra,
technical A. Boruchoff
assistance. for
supplying
Mr,
John Salem,
We also
wish
and Mr, William to thank
Dr.
some of the keratoconus
P,R.
Lavin Laibson
cornea1
buttons,
References 1. 2. 3. 4. 5. 6, 7. 8. 9. 10. 11. 12. 13.. 14, 15, 160 17. 18, 19, 20. 21, 22. 23, 24,
Teng, C.C. (1963) Amer. J. Ophthalmol. 55, 18-47. (1970) Arch, Ophthalmol, Patta, CL,, Loyon, L,, and Roucher, F. (Paris) 30, 403-417. Kim, J.O., and Hassard, D,T,R. (1972) Canad. 9. Ophthalmol, 7, 176-180. Iwamoto, T., and Devoe, A.G. (1975) Arch. Ophthalmol. (Paris) 35, 65-72. Robertson, I. (1974) Aust, J. Ophthalmol. 2, 144-147. Uitto, J., and Prockop, D.J, (1974) Molecular Pathology, pp. 670-688, Thomas, Springfield, Illinois. Miller, E.J. (1976) Molecular and Cellular Biochemistry 13, 165-192. Burgeson, R.E., El Adli, F.A., Kaitila, I-I,, and Hollister, D.W, (1976) Proc. Natl. Acad, Sci. USA 12, 2579-2583, Chung, E., Rhodes, R.K., and Miller, E.J. (1976) Biochem. Biophys, Res, comm, 71, 1167-1169, Katzman, R.L., Kang, A.H,, and Dixit, S.N, (1974) Biochim. Biophys. Acta, 336, 367-369. Freeman, I.L, (1978) Invest. Ophthalmol. and Vis. Sci. 17, 171-177, Schmut, 0. (1977) Exp. Eye Res. 25, 505-509, Hong, B,S., Cannon, D.J. and Davison, P.F. (1978) Fed. Proc, 37, 1528, Cannon, J. and Foster, C.S. (1978) Invest. Ophthalmol. and Vis. Sci. l7-, 63-65. Robert, L,, Schillinger, G,, Moczar, M., Junqua, S., and Moczar, E. (1970) Arch. Ophthalmol. (Paris) 30, 590-607. Penttinen, R.P,, Lichtenstein, J,R., Martin, G.R,, and McKusick, V.A, (1975) Proc. Natl, Acad. Sci. USA 72, 586-589. Dorfman, A., and Matalon, R. (1976) Proc, Natl. Acad. Sci. USA 73, 630-637. McCarey, B.E., and Kaufman, H.E. (1974) Invest., Ophthalmol, 2, 165-173. Baum, J,L., Niedra, R,, Davis, C., and Yue, B,Y,J,T. Arch. Ophthalmol. (in press). Peterkofsky, B., and Diegelmann, R, (1971) Biochemistry I& 988-994, Piez, K.A. (1967) Treatise on collagen, Vol, 1, Chemistry of Collagen, ppO 201-248, Academic Press, New York. Miller, E.J., Martin, G,R,, Piez, K.A., and Powers, M,J. (1967) J. Biol, Chem, 242, 5481-5489. Laemmli, U,K. (1970) Nature 227, 680-685, Laskey, R.A., and Mills, A.D. (1975) Eur, J, Biochem. 56, 335-341.
471
Vol. 86, No. 3, 1979
25, 26, 27, 28.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(1972) Proc, Natl. Acad, Sci. Smith, B.D., Byers, P.H,, and Martin, G.R, USA 69, 3260-3262. (1977) Arch. Biochem, Biophys. 182, 33-41. Moro, L., and Smith, B.D, Stoesser, T.R., Church, R.L., and Brown, S.I. (1978) Invest. Opthalmol, and Vis. Sci. l7, 264-271, Yue, B.Y.J.T,, Baum, J.L,, and Silbert, J.E. Manuscript submitted for publication,
472
