Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
1048-1052
Pages
PREDOMINANCE OF TYPE III OVER TYPE I CELL SURFACE PROCOLLAGENS IN RAT LUNG FIBROBLASTS Yoshiki Department
Received
of
December
Biochemistry, of Vermont,
25,
Nagai*
College Burlington,
of Medicine, VT 05405
University
1990
125 Binding assays of I-labeled affinity-purified monospecific procollagen antibodies to fibroblasts have type 1 and III revealed the presence of type I and III procollagens on the cell surface, and that the amount of type III procollagen is 10 times greater than that of type I procollagen. Analyses by flow cytometry and fluorescent microscopy after treatment with an isothiocyanate (FITC)-conjugated second antibody supported this finding. 0 1991 Academic press, 1°C.
It the
widely
accepted
extracellular
They
are
N-and at
is
space
subsequently
C-terminals
which
Investigation
are
synthesized collagens
peptidases
are
binding
antibodies
indicated
the
surface in
being into
by specific
of
procollagen
exists
procollagens
to
larger
presence and,
of to of
monospecific cultured
collagens
type
quantitatively,
amounts
than
I
and that
type
type
rat
lung III
the
cell. of
exact and
the
sites further
been identified. a site for the assembly I
and
(2, type
fibroblasts
procollagens type
into
the
cleavage
But
(1).
converted
excreted
within
with
into collagen fibrils have not plasma membrane as proposed the of terminal peptides and fibrillar
Goldberg excision
cell
after
changed
procollagens
assembled
that
III
3). III has
on
the
procollagen
I procollagen.
MATERIALS AND METHODS Fetal rat lung fibroblasts grown on 75 cm2 flasks in minimal essential medium containing 10 i (v/v) fetal calf serum (Grand Island Biological co., and a mixture of NY) penicillin-streptomycin were trypsinized late in the log phase. Polyclonal type I or III procollagen antibody, prepared in *
To whom correspondence should be addressed at of Medicine, Faculty of Medicine, University Hongo, Bunkyo-ku, Tokyo 113, Japan. 0006-291X/91
$1.50
1048
First Department of Tokyo, 7-3-l
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rabbits and made monospecific by absorption to a type I or III collagen column followed by affinity chromatography on a,t2)5pe I or III procollagen-Sepharose column (4), was labeled with 1 by the chloramine-T method (specific activity, 0.069 and O.lOpCi/ug protein for types I and III procollagen antibodies, respectively) The type I and III procollagen antibodies were specific to (5). and their antibody properties were the respective antigens identical since the two antibodies, when used in equal almost amounts, made virtually the same quantities of antigen-antibody complex (4). Reactions were carried out in a total volume of 250 bovine serum ul of Dulbecco's phosphate buffer containing 2% albumin (generalll;n,~,.o~-ml polypropylene tubes. The,.$J,i;~;;~~~~~ ~~;il; cells) were incubated with or III procollagen antibody with continuous shaking at 37'C for Incubations were terminated by centrifugation of the 20 min. centrifugation, the supernatant reaction mixture. 12+fter containing unbound I-type I or III procollagen antibody was and the pellet was gently removed from the centrifuge tube, washed twice with Dulbecco's phosphate buffer solution. The tip of the tube containing the cell pellet was cut and the radioactivity was measured. The binding of both types of procollagen to fibroblasts was analyzed by flow cytometry after treatment with also an FITC-labeled second antibody. The dissociated cells were incubated with type I or III procollagen antibody for 1 h on and further incubated for 30 min on ice with ice, washed twice, dilutions of FITC-conjugated goat antirabbit IgG antiserum (IgG fraction) (Cooperbiomedical, PA). The cells were washed and analyzed by flow cytometry (2150 cytofluorograph; Ortho fluorescent cells Diagnostic, MA). In a different experiment, were fixed in 4% paraformaldehyde solution for 20 min at room temperature and mounted in 5% n-propyl gallate for analysis by fluorescent microscopy. Each experiment included controls was omitted. from which either primary or secondary antibody
RESULTS
AND DISCUSSION
The fibroblasts
binding was
of
either
dependent
type upon
of its
procollagen concentration
antibody (Figs.
1
to and
and the number of cells in the incubation medium. A 2) Scatchard plot of the binding data yielded a straight line for both III procollagen I and antibodies (Fig. 3) (6). type Dissociation constants for the two antibodies were very close, -7
2.0x10
antibodies.
M for
type
However,
and
the
type
III
type
I procollagen antibody, The dose-dependency of
fibroblasts both types capacity antibody assayed
procollagen
I
antibody
2.3~10~~ number
M for
of
was
10
2.5~10~
binding
was also demonstrated of procollagen (Fig.
III
sites
times greater and 2.1x105, of procollagen
procollagen per
cell
than that respectively. antibodies
for for to
flow cytometry Comparison of
analysis of the binding
of the two antibodies revealed that type III bound to fibroblasts in larger amounts than concentration in the primary at the same
procollagen type I when incubation
1049
by 4).
binding
type
Vol.
174,
01
No.
BIOCHEMICAL
2, 1991
1251-type
I procollagen
antibody
AND
BIOPHYSICAL
RESEARCH
(x10-8Mn)
Fig. 1. Concentration dependence of type antibody binding to ra$ lung fibroblasts. mixture contained 8.6x10 cells. Fig. 2. Concentration dependence procollagen antibody binding to ra$ The reaction mixture contained 2.5x10 medium, (Fig.
possibly
5). The
binding
fibroblasts
was
reflecting of
had
a patchy
some and of III
but results
fluorescent
larger
and
I
absent
of
the
microscopy
in
the the
binding analyses
both type of procollagen on the exists in larger amounts than
bound
Fig. type
points
3. If1
were
L-----‘25I-typz
III
procol;igen
of
binding
cell
the
strongly
cell
and surface, I. It
anfibb5dy
(x IO-%A)
surface.
It
and even
capped
flow
suggest
cell type
cytometry the
presence
and that should be
scatchard analysis of binding of 125 I-labeled procollagen antibody to fibroblasts. Data
derived from the binding curve in Fig. 2. constants were derived (Kd) from the negative reciprocal of the slope, and numbers of binding sites were derived from the intercept of the line with the abscissa.
Dissociation
1050
to
microscopy.
membrane
cytoplasm. assays
sites
antibodies
fluorescent on
III
type fibroblasts.
procollagen
only
along
was
of lung cells.
by
present
I procollagen The reaction
number
III
visualized
was distribution
cells, The
a
type
also
Immunofluorescence
COMMUNICATIONS
type noted
Vol.
174,
No.
2, 1991
200
j
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
200
l?.,ti,k,,;o
200
1,
fl"ore*ce"ce
,I
1
Imenslt"
r
200
400
600
800
200 -
c
~4~~,~,~o~~40~4~,6.,.~oo
1000
fluorescence
lntenslty
400
d
C
i
.iLLL 1
200
‘loo
800.3001000
1
@;L
2004006008001000
1
200400
600800
1000
Fig. 4. Flow cytometry analysis Of type 1 procollagen antibody binding to fibroblasts after treatment with a fluorescein-labeled second antibody. Dose-dependency fs striking. The reaction mixture contajned 6.25x10 cell -7 and 0 (a), I.~xIO-~ M (b), 4x10 M (c), or 8x10 M (d) of type I procollagen antibody. Fig. 5. Comparison of binding of types I and III procollagen antibody to fibroblasts analyzgd by flow cytomqtry. The reaction mixture contained 2x10 cells and 8x10M of type I (b) or III (c) procollagen antibody. Type III procollagen antibody has a higher a, control. affinity to fibroblasts than type I procollagen.
that
the
with
antibodies
antigenic
terminal
globular
react
by is
the
currently
were
is
not
of
with
intact
known in
both
the
the
chain
for
and,
procollagen
to
the
preparation
postulated fibrillar
that assembly
enzymatic could both
II),
membranes of of type collagen
cannot peptides
(7,
8).
No
antibodies
which
procollagens. procollagens
on the
and/or speculate
membrane may fibrils. collagen
into
Presence type III
of
plasma
processing
plasma
carboxyl
cell
surface
cytoplasm for excretion or attached the extracellular space. As to
further
the
and
react
therefore,
peptidases
significance of procollagens physiological the cell surface, it seems reasonable to of
amino-
study
and procollagen
procollagen
whether
the from
used in this
procollagen
available
synthesized surface cell
the
of
intact
action
exclusively It
in
regions
between
released
procollagen
determinants
distinguish method
against
processing occur while
fibroblasts I (11)
(2,
procollagen (9), and acid-soluble 1051
be
to the
collagens that
on
binding
necessary Goldberg
for has
of procollagens and precursors are bound to 3). type
I
collagen
collagen (12)
(10,
on
the
Vol.
174,
No.
BIOCHEMICAL
2, 1991
plasma reference
membrane
of
III
to
type
been III
the
type
of I
procollagen
is
cell 10
a difference
BIOPHYSICAL
type
III on
in
of suggests
greater the
rate
COMMUNICATIONS
previously, procollagen
analysis surface times
RESEARCH
reported
procollagen
Quantitative on the
reflecting
been
presence
to
made.
procollagen
have
AND
the
or
plasma
both that
than
that
of
cellular
but no the ratio
membrane
has
type 1 and the amount of
type
of
type
I,
III
possibly
production.
ACKNOWLEDGMENT I wish encouragement
to to
thank this
Dr. Kenneth work.
R.
Cutroneo
for
his
support
and
REFERENCES 1.
Fessler,J.H.,
2. 3. 4.
~~id~~~g~~!.(1979) Cell 16 265-275. Goldberg,B.D. (1982) J.Celi Biol. Shull,S., and Cutroneo,K.R. (1983) 3369. Bordin,S., Kolb,W.P., and Page,R.C.
5.
and
Fessler,L.I.
(1978)
Ann.Rev.Biochem. 95, 747-751. J.Biol.Chem. (1984)
258, J.Immunol.
3364130,
1871-1875. 6. 7. 8.
Scatchard,G. (1949) Nowack,H., Gay,S., J.Immunol.Methods Timpl,R., Wick,G., 18,
9. IO. 11. 12.
Ann.N.Y.Acad.Sci. Wick,G., Becker,U., 12, 117-124. and Gay,S. (1977)
51, 660-672. and Timpl,R.
(1976)
J.Immunol.Methods
165-182.
Phelps,R.G., Matinez-Hernandez,A., and Goldberg,B.D. (1985) Collagen Rel.Res. 5, 405-414. Lichtenstein,J.R., Bauer,E.A., Hoyt,R., and Wedner,H.J. (1976) J.Exp.Med. 144, 145-154. Breen,E., Falco,V.M., Absher,M., and Cutroneo,K.R. (1990) J.Biol.Chem. 265, 6286-6290. Faulk,W.P., Conochie,L.B., Temple,A., and Papamichail,M. (1975) Nature 256, 123-125.
1052
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
1048-1052
Pages
PREDOMINANCE OF TYPE III OVER TYPE I CELL SURFACE PROCOLLAGENS IN RAT LUNG FIBROBLASTS Yoshiki Department
Received
of
December
Biochemistry, of Vermont,
25,
Nagai*
College Burlington,
of Medicine, VT 05405
University
1990
125 Binding assays of I-labeled affinity-purified monospecific procollagen antibodies to fibroblasts have type 1 and III revealed the presence of type I and III procollagens on the cell surface, and that the amount of type III procollagen is 10 times greater than that of type I procollagen. Analyses by flow cytometry and fluorescent microscopy after treatment with an isothiocyanate (FITC)-conjugated second antibody supported this finding. 0 1991 Academic press, 1°C.
It the
widely
accepted
extracellular
They
are
N-and at
is
space
subsequently
C-terminals
which
Investigation
are
synthesized collagens
peptidases
are
binding
antibodies
indicated
the
surface in
being into
by specific
of
procollagen
exists
procollagens
to
larger
presence and,
of to of
monospecific cultured
collagens
type
quantitatively,
amounts
than
I
and that
type
type
rat
lung III
the
cell. of
exact and
the
sites further
been identified. a site for the assembly I
and
(2, type
fibroblasts
procollagens type
into
the
cleavage
But
(1).
converted
excreted
within
with
into collagen fibrils have not plasma membrane as proposed the of terminal peptides and fibrillar
Goldberg excision
cell
after
changed
procollagens
assembled
that
III
3). III has
on
the
procollagen
I procollagen.
MATERIALS AND METHODS Fetal rat lung fibroblasts grown on 75 cm2 flasks in minimal essential medium containing 10 i (v/v) fetal calf serum (Grand Island Biological co., and a mixture of NY) penicillin-streptomycin were trypsinized late in the log phase. Polyclonal type I or III procollagen antibody, prepared in *
To whom correspondence should be addressed at of Medicine, Faculty of Medicine, University Hongo, Bunkyo-ku, Tokyo 113, Japan. 0006-291X/91
$1.50
1048
First Department of Tokyo, 7-3-l
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rabbits and made monospecific by absorption to a type I or III collagen column followed by affinity chromatography on a,t2)5pe I or III procollagen-Sepharose column (4), was labeled with 1 by the chloramine-T method (specific activity, 0.069 and O.lOpCi/ug protein for types I and III procollagen antibodies, respectively) The type I and III procollagen antibodies were specific to (5). and their antibody properties were the respective antigens identical since the two antibodies, when used in equal almost amounts, made virtually the same quantities of antigen-antibody complex (4). Reactions were carried out in a total volume of 250 bovine serum ul of Dulbecco's phosphate buffer containing 2% albumin (generalll;n,~,.o~-ml polypropylene tubes. The,.$J,i;~;;~~~~~ ~~;il; cells) were incubated with or III procollagen antibody with continuous shaking at 37'C for Incubations were terminated by centrifugation of the 20 min. centrifugation, the supernatant reaction mixture. 12+fter containing unbound I-type I or III procollagen antibody was and the pellet was gently removed from the centrifuge tube, washed twice with Dulbecco's phosphate buffer solution. The tip of the tube containing the cell pellet was cut and the radioactivity was measured. The binding of both types of procollagen to fibroblasts was analyzed by flow cytometry after treatment with also an FITC-labeled second antibody. The dissociated cells were incubated with type I or III procollagen antibody for 1 h on and further incubated for 30 min on ice with ice, washed twice, dilutions of FITC-conjugated goat antirabbit IgG antiserum (IgG fraction) (Cooperbiomedical, PA). The cells were washed and analyzed by flow cytometry (2150 cytofluorograph; Ortho fluorescent cells Diagnostic, MA). In a different experiment, were fixed in 4% paraformaldehyde solution for 20 min at room temperature and mounted in 5% n-propyl gallate for analysis by fluorescent microscopy. Each experiment included controls was omitted. from which either primary or secondary antibody
RESULTS
AND DISCUSSION
The fibroblasts
binding was
of
either
dependent
type upon
of its
procollagen concentration
antibody (Figs.
1
to and
and the number of cells in the incubation medium. A 2) Scatchard plot of the binding data yielded a straight line for both III procollagen I and antibodies (Fig. 3) (6). type Dissociation constants for the two antibodies were very close, -7
2.0x10
antibodies.
M for
type
However,
and
the
type
III
type
I procollagen antibody, The dose-dependency of
fibroblasts both types capacity antibody assayed
procollagen
I
antibody
2.3~10~~ number
M for
of
was
10
2.5~10~
binding
was also demonstrated of procollagen (Fig.
III
sites
times greater and 2.1x105, of procollagen
procollagen per
cell
than that respectively. antibodies
for for to
flow cytometry Comparison of
analysis of the binding
of the two antibodies revealed that type III bound to fibroblasts in larger amounts than concentration in the primary at the same
procollagen type I when incubation
1049
by 4).
binding
type
Vol.
174,
01
No.
BIOCHEMICAL
2, 1991
1251-type
I procollagen
antibody
AND
BIOPHYSICAL
RESEARCH
(x10-8Mn)
Fig. 1. Concentration dependence of type antibody binding to ra$ lung fibroblasts. mixture contained 8.6x10 cells. Fig. 2. Concentration dependence procollagen antibody binding to ra$ The reaction mixture contained 2.5x10 medium, (Fig.
possibly
5). The
binding
fibroblasts
was
reflecting of
had
a patchy
some and of III
but results
fluorescent
larger
and
I
absent
of
the
microscopy
in
the the
binding analyses
both type of procollagen on the exists in larger amounts than
bound
Fig. type
points
3. If1
were
L-----‘25I-typz
III
procol;igen
of
binding
cell
the
strongly
cell
and surface, I. It
anfibb5dy
(x IO-%A)
surface.
It
and even
capped
flow
suggest
cell type
cytometry the
presence
and that should be
scatchard analysis of binding of 125 I-labeled procollagen antibody to fibroblasts. Data
derived from the binding curve in Fig. 2. constants were derived (Kd) from the negative reciprocal of the slope, and numbers of binding sites were derived from the intercept of the line with the abscissa.
Dissociation
1050
to
microscopy.
membrane
cytoplasm. assays
sites
antibodies
fluorescent on
III
type fibroblasts.
procollagen
only
along
was
of lung cells.
by
present
I procollagen The reaction
number
III
visualized
was distribution
cells, The
a
type
also
Immunofluorescence
COMMUNICATIONS
type noted
Vol.
174,
No.
2, 1991
200
j
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
200
l?.,ti,k,,;o
200
1,
fl"ore*ce"ce
,I
1
Imenslt"
r
200
400
600
800
200 -
c
~4~~,~,~o~~40~4~,6.,.~oo
1000
fluorescence
lntenslty
400
d
C
i
.iLLL 1
200
‘loo
800.3001000
1
@;L
2004006008001000
1
200400
600800
1000
Fig. 4. Flow cytometry analysis Of type 1 procollagen antibody binding to fibroblasts after treatment with a fluorescein-labeled second antibody. Dose-dependency fs striking. The reaction mixture contajned 6.25x10 cell -7 and 0 (a), I.~xIO-~ M (b), 4x10 M (c), or 8x10 M (d) of type I procollagen antibody. Fig. 5. Comparison of binding of types I and III procollagen antibody to fibroblasts analyzgd by flow cytomqtry. The reaction mixture contained 2x10 cells and 8x10M of type I (b) or III (c) procollagen antibody. Type III procollagen antibody has a higher a, control. affinity to fibroblasts than type I procollagen.
that
the
with
antibodies
antigenic
terminal
globular
react
by is
the
currently
were
is
not
of
with
intact
known in
both
the
the
chain
for
and,
procollagen
to
the
preparation
postulated fibrillar
that assembly
enzymatic could both
II),
membranes of of type collagen
cannot peptides
(7,
8).
No
antibodies
which
procollagens. procollagens
on the
and/or speculate
membrane may fibrils. collagen
into
Presence type III
of
plasma
processing
plasma
carboxyl
cell
surface
cytoplasm for excretion or attached the extracellular space. As to
further
the
and
react
therefore,
peptidases
significance of procollagens physiological the cell surface, it seems reasonable to of
amino-
study
and procollagen
procollagen
whether
the from
used in this
procollagen
available
synthesized surface cell
the
of
intact
action
exclusively It
in
regions
between
released
procollagen
determinants
distinguish method
against
processing occur while
fibroblasts I (11)
(2,
procollagen (9), and acid-soluble 1051
be
to the
collagens that
on
binding
necessary Goldberg
for has
of procollagens and precursors are bound to 3). type
I
collagen
collagen (12)
(10,
on
the
Vol.
174,
No.
BIOCHEMICAL
2, 1991
plasma reference
membrane
of
III
to
type
been III
the
type
of I
procollagen
is
cell 10
a difference
BIOPHYSICAL
type
III on
in
of suggests
greater the
rate
COMMUNICATIONS
previously, procollagen
analysis surface times
RESEARCH
reported
procollagen
Quantitative on the
reflecting
been
presence
to
made.
procollagen
have
AND
the
or
plasma
both that
than
that
of
cellular
but no the ratio
membrane
has
type 1 and the amount of
type
of
type
I,
III
possibly
production.
ACKNOWLEDGMENT I wish encouragement
to to
thank this
Dr. Kenneth work.
R.
Cutroneo
for
his
support
and
REFERENCES 1.
Fessler,J.H.,
2. 3. 4.
~~id~~~g~~!.(1979) Cell 16 265-275. Goldberg,B.D. (1982) J.Celi Biol. Shull,S., and Cutroneo,K.R. (1983) 3369. Bordin,S., Kolb,W.P., and Page,R.C.
5.
and
Fessler,L.I.
(1978)
Ann.Rev.Biochem. 95, 747-751. J.Biol.Chem. (1984)
258, J.Immunol.
3364130,
1871-1875. 6. 7. 8.
Scatchard,G. (1949) Nowack,H., Gay,S., J.Immunol.Methods Timpl,R., Wick,G., 18,
9. IO. 11. 12.
Ann.N.Y.Acad.Sci. Wick,G., Becker,U., 12, 117-124. and Gay,S. (1977)
51, 660-672. and Timpl,R.
(1976)
J.Immunol.Methods
165-182.
Phelps,R.G., Matinez-Hernandez,A., and Goldberg,B.D. (1985) Collagen Rel.Res. 5, 405-414. Lichtenstein,J.R., Bauer,E.A., Hoyt,R., and Wedner,H.J. (1976) J.Exp.Med. 144, 145-154. Breen,E., Falco,V.M., Absher,M., and Cutroneo,K.R. (1990) J.Biol.Chem. 265, 6286-6290. Faulk,W.P., Conochie,L.B., Temple,A., and Papamichail,M. (1975) Nature 256, 123-125.
1052
