Investigative Ophthalmology & Visual Science, Vol. 29, No. 7, July 1988
Copyright © Association for Research in Vision and Ophthalmology
Corneal Collagen Fibrils: Dissection With Specific
Collagenases and Monoclonal Antibodies
John M. Fitch,* David E. Dirk4 Anita Mentzer,* Karen A. Hasty, j- Carlo Mainardi.t and Thomas F. Linsenmayer"
To investigate the relationship of collagen types I and V within corneal fibrils, and the collagenolytic
mechanisms potentially involved in corneal development and remodeling, we have incubated cryostat
sections of avian corneas with collagenases that specifically degrade collagen types I or V to digest
selectively the collagen in situ. These preparations were then analyzed by immunofluorescence histochemistry and immunoelectron microscopy using anti-collagen type-specific monoclonal antibodies.
Digestion of corneal sections with the type I-specific collagenase ("I'ase") exposed antigenically
masked type V collagen, indicating that epitopes on type V collagen in heterotypic fibrils are inaccessible to the antibody due to their interaction with type I collagen. Sections digested with the type V
collagen-degrading enzyme ("Vase") showed no removal of type V collagen. However, after the
fibrillar structure was disrupted by acetic acid treatment before enzyme digestion, the type V collagen
was then degraded. Likewise, prior digestion of type I collagen by I'ase also rendered type V collagen
susceptible to digestion by Vase. These results suggest that the cleavage sites on type V collagen also
are buried within heterotypic fibrils and therefore inaccessible to the enzyme. They also document, for
the first time, Vase activity against type V collagen in situ. Electron microscopic observations of
sections partially digested with the I'ase revealed many short striated fibrillar segments from which
smaller filaments protrude. Both the striated regions and some of the filaments were labeled by an
antibody against type I collagen; anti-type V antibody reacted preferentially with the thin filaments.
Thus avian corneal fibrils contain type I collagen, in which filaments of type V collagen are embedded.
Complete removal of the fibrillar stromal matrix in the course of normal or pathological remodeling
requires at least two different collagenases acting in concert. Invest Ophthalmol Vis Sci 29:11251136,1988
type V.1 Since acetic acid interferes with acid-labile
intramolecular crosslinks and causes disaggregation
(swelling) of compact collagen fibrils (unpublished
observations), we proposed that type V collagen was
masked by its supramolecular fibrillar organization,
perhaps due to an association with type I collagen,
with which it might be coassembled into heterotypic
fibrils. This hypothesis was tested in the embryonic
corneal stroma using other methods to perturb fibrillar organization, such as: (1) experimental lathyrism,
in which collagenfibrilsdeficient in aldehyde-derived
crosslinks become disaggregated when cooled2"4; and
(2) digestion with a mammalian collagenase which
degrades type I collagen but not type V.5"8 As predicted by this hypothesis, both methods exposed the
epitopes, as determined by immunofluorescence histochemistry.910 More recently, immunoelectron microscopic analysis of corneal sections from lathyritic
embryos labeled with antibodies coupled to electrondense markers have provided direct evidence that
each 25 nm fibril in the avian corneal stroma is a
heterotypic structure composed of molecules of type I
and type V collagens.1112
In previous immunofluorescence histochemical
studies of connective tissues of embryonic chickens,
we observed that epitopes on type V collagen in stromal matrices from a wide variety of ocular and extraocular tissues are normally unavailable to monoclonal antibodies that specifically recognize that collagen type. Reactivity of such matrices within tissue
sections could be achieved by pretreatment of the
sections with dilute acetic acid; treatments with a variety of neutral proteases and glycosaminoglycan-degrading enzymes failed to unmask the epitopes on
From the *Department of Anatomy and Cellular Biology, Tufts
University School of Medicine, Boston, Massachusetts; the fDepartment of Medicine, University of Tennessee, Memphis, Tennessee; and the ^Department of Pathology, UMDNJ-Robert Wood
Johnson Medical School, Piscataway, New Jersey.
Supported by grants EY-05191, EY-05129, and AI-22603 from
the National Institutes of Health, Bethesda, Maryland.
Submitted for publication: October 26, 1987; accepted January
11, 1988.
Reprint requests: John M. Fitch, Department of Anatomy and
Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1988
The molecular arrangement of collagen types I and
V within these fibrils, the mechanism of their coassembly, and the means by which such fibrils are degraded are unclear. For the fibrillar structure, a variety of models can be posed. For example, such fibrils
in the corneal stroma might contain a central core of
type V collagen insulated by type I; alternating concentric layers of collagen types I and V; intermixed
molecules; or multiple microfibrillar units consisting
of homogeneous aggregates of type I or V molecules,
or of heterogeneous aggregates of both collagen types.
The possibility even exists that only a portion of each
type V collagen molecule is embedded within a fibril,
the remainder being exposed on the surface.
For degradation of heterotypic fibrils, one can envision the need for several different collagenases,
since it is known that type V collagen is refractory to
degradation with the enzyme that degrades type I
collagen.7-8
In the present study, we have investigated the relationship of these two collagens within fibrils, and the
degradation of such fibrils, by performing digestions
with two different collagen type-specific collagenases
and analyzing the results using a combination of immunofluorescence histochemistry and immunoelectron microscopy. The two enzymes we have employed are a rabbit macrophage collagenase (termed
"I'ase") which degrades type I collagen in solution
but not type V, 713 and a metalloprotease (termed
"Vase") produced by cultured MDCK cells which
we have recently characterized14 as degrading native
type V collagen molecules in solution but having no
detectable activity against native type I molecules.715"18 This latter enzyme is also a gelatinase
which digests the a-chains of denatured interstitial
collagens. We have purified these enzymes, and have
used them as tools to dissect the structure of collagen
fibrils in corneal tissue sections. By immunofluorescence analyses, we have found that type V collagen in
situ is not normally available for digestion by the
Vase, but can be degraded only after it has been
exposed by prior treatment with dilute acetic acid or
active I'ase. Our electron microscopic analyses revealed that, following partial digestion of corneal fibrils with Fase, antibodies against type V collagen
preferentially bind to thin filaments that appear to be
derived from more intact fibrillar regions. These results directly confirm the heterotypic nature of avian
corneal fibrils, suggest that type V collagen is largely
buried within the fibril, and indicate that there is no
subpopulation of type I collagen molecules that itself
is masked by type V. They also show that removal of
corneal fibrils must involve the concerted activities of
at least two collagenases.
Vol. 29
Materials and Methods
Tissue
White leghorn chicken embryos (Spafas, Norwich,
CT) were incubated at 37°C in a humid atmosphere.
Embryos were removed at 14 days of incubation, and
staged according to Hamburger and Hamilton.19
Corneas with adjacent sclera and lens were removed
in Hanks BSS, and immersed in phosphate buffered
saline (PBS) with 7% sucrose for 10 min. They were
then embedded in Tissue Tek OCT (Miles Laboratories, Elkhart, IN) and frozen in liquid nitrogen.
Cryostat sections (8 ixm) of such unfixed material
were mounted on 12-spot glass slides (Shandon Scientific, Sewickley, PA) coated with polylysine
(MW~350,000, Sigma Chemical Co., St. Louis,
MO), air-dried, and stored dessicated at -20°C until
used. The investigations using animals, as described
in this manuscript, conform to the ARVO Resolution
on the Use of Animals in Research.
Enzymes
Type I (interstitial) collagenase (I'ase) that specifically degrades collagen type I but not type V was
purified from serum-free supernatants of "in vivo"
activated rabbit alveolar macrophages. Cultures were
prepared as previously described,7 and the pooled supernatants concentrated 50-fold using a hollow-fiber
filter apparatus (Amicon). The concentrated supernatants were subjected to DEAE chromatography to
separate the collagenase from other metalloproteases
as previously described13 using conditions under
which the enzyme passes through the column unretarded. The unbound fraction which contained all of
the collagenase activity against type I collagen was
dialyzed against 0.05 M Tris-acetate, 5 mM CaCl2,
pH 6.8 containing 0.05% (v/v) Brij. The sample was
pumped onto a MonoS column (Pharmacia, Piscataway, NJ) which had been equilibrated with the same
buffer using a Hewlett-Packard 1090 HPLC apparatus equipped with a diode array detector. The column was washed with 20 volumes of the above buffer
and the bound proteins were eluted with a stepwise
linear gradient to 0.5 M NaCl in the same buffer. The
flow rate was 1 ml/min and the gradient was to 0.1 M
NaCl at 10 min, 0.4 M NaCl at 80 min, and 0.5 M
NaCl at 90 min. Under these conditions collagenase
was eluted as a single peak with a retention time of 52
min. The enzyme was further purified by gel filtration
using a Superose 12 column equilibrated with 0.05 M
Tris-HCl, 0.05 M NaCl, 5 mM CaCl2,0.05% Brij, pH
7.6 on the same apparatus. The enzyme activity
eluted as a single homogenous peak on this column.
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STRUCTURE OF HETEROTYPIC CORNEAL COLLAGEN FIBRILS / Firch er ol.
Activation of latent collagenase was not necessary as
the enzyme totally autoactivated with purification.
Type V collagenase (Vase) was purified from culture supernatants of canine kidney epithelial cells by
affinity chromatography on gelatin-Sepharose.14 The
bound enzyme was washed with 0.5 M NaCl in 0.05
M Tris-HCl pH 7.4 containing 5 mM CaCl2 and
eluted with 15% DMSO in the same buffer. The enzyme was characterized as a metalloprotease which
would degrade native type V collagen, but showed no
activity against native types I or IV collagen.
The enzymes were stored at -70°C until used. The
Vase was activated with 0.01 M 4-aminophenylmercuric acetate just before use. The I'ase was spontaneously active.
Samples of each enzyme were checked for typespecific collagenolytic activity in vitro by mixing 100
ng of chicken type I or type V collagen with 0, 2.5 n\,
5 (d, 10 n\ or 20 ix\ of enzyme in a total volume of 150
Ml of 0.05 M Tris-HCl buffer, pH 7.4, with 5 mM
CaCl2 and 0.02% NaN3. The reaction mixtures were
incubated at 36°C in a temperature-controlled heat
block for 18-20 hr. They were analyzed by SDS polyacrylamide (7%) gel electrophoresis by the method of
Laemmli.20
Antibodies
Monoclonal antibodies against collagen types I
(I-BA121), V (V-DH2 and V-AB121), and IV (IVIA822) were produced and characterized as described
previously. All antibodies recognize helical epitopes
that are type-specific; none of these antibodies crossreacts with any other known collagen. For immunofluorescence histochemistry, the antibodies were used
as hybridoma culture supernatants. For immunoelectron microscopy, monoclonal IgG was employed (see
below).
Enzyme Digestions
Slides containing unfixed 8 nm cryostat sections of
14-day embryonic anterior eyes were rinsed in Tris/
NaCl buffer (50 mM Tris, 150 mM NaCl, pH 7.4).
Unmasking of type V collagen in situ was then accomplished by pretreating sections in 0.2 M acetic
acid for 20 min at room temperature. Control sections, in which type V collagen remained masked
during subsequent enzyme digestions, were immersed in Tris/NaCl for 20 min. The slides were
rinsed in Tris/NaCl X3, and the sections were incubated for 20 min with a drop of 0.1% BSA in Tris/
NaCl containing 5 mM CaC^. This was removed by
aspiration, and replaced with a drop (30-35 n\) of
either I'ase or Vase. Control sections received either
1127
enzyme inhibited with 25 mM EDTA, or buffer
without enzyme. (Sections incubated with either
control solution were identical, showing no degradation of any collagen type, as measured by immunofluorescence histochemistry.) Incubations were carried out in humid chambers for 6-74 hr. In experiments involving long incubations, enzyme solutions
were replaced with fresh enzyme after about 48 hr of
incubation. The course of collagen degradation in
situ was monitored by indirect immunofluorescence
histochemistry with the monoclonal antibodies.
Indirect Immunofluorescence Histochemistry
Cryostat sections of unfixed anterior eyes treated in
various ways (±acetic acid; ±active collagenase(s))
were rinsed twice in Tris/NaCl and once in PBS. In
some experiments the sections were then fixed in 4%
paraformaldehyde in PBS for 15 min at 4°C, washed
in PBS, and treated with 0.05% NaBH4 in PBS for 1
hr at 4°C to quench free aldehydes. In other experiments, the fixation/quenching steps were omitted.
(Essentially the same results were obtained from both
thefixedand unfixed sections, except that the fluorescent signals observed in the unfixed sections were
noticeably greater. In the experiments pictured in
Figures 1, 4 and 5, fixation was not done.) Sections
were incubated with 1% BSA/PBS for 20 min, and
then reacted with a monoclonal anti-collagen antibody followed by a rhodamine-labeled goat antimouse second antibody (Cappel, Malvern, PA) as described previously.22 Sections were observed and
photographed using a Nikon Fluophot microscope
equipped for epifluorescence, as described previously.1-22
Pre-Embedding Immunoelectron Microscopy
Cryostat sections treated with active I'ase or I'ase
buffer for 6-49 hr were rinsed in Tris/NaCl and PBS,
and then fixed with 4% paraformaldehyde in PBS for
15 min at 4°C. The sections were quenched with
0.05% NaBH4 as described above, washed in PBS,
and incubated with 1% BSA in PBS for 90 min at
room temperature to block nonspecific binding of
colloidal gold particles. The sections were then incubated overnight at 4°C with colloidal gold particles
coated with monoclonal anti-collagen IgG. In some
cases sections were pretreated with testicular hyaluronidase (type IV-S, Sigma Chemical Co.) before
application of antibody-gold conjugates. Just before
use, the antibody-gold complexes were diluted with
PBS/1 % BSA (1 /20-1 /50) to give a pale pink color on
visual examination, and large aggregates were removed by centrifugation (9000g, 5 min). After reaction with antibody-gold, the sections were washed
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1988
with four changes of PBS containing 0.05% Tween20, once with PBS without Tween-20, and once with
0.1 M sodium cacodylate, pH 7.4. All washes were for
10-15 min, with gentle agitation. The sections were
fixed for 1 hr with 4% paraformaldehyde with 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, and postfixed in 1.25% osmium tetroxide in 0.1 M cacodylate.
Following dehydration in graded alcohols, the sections were infiltrated overnight with epon-araldite,
covered with BEEM capsules filled with epon-araldite, and polymerized. Embedded sections were
popped from the glass slide after briefly heating them
on a hot plate.
Sections with a pale gold interference color were
picked up onto copper grids and stained with 2%
aqueous uranyl acetate and either 1% lead stain or 1%
phosphotungstic acid, pH 3.2. Sections were observed
and photographed with either a Philips 200 or CM-10
transmission electron microscope.
Preparation of Colloidal Gold-Antibody Complexes
Monoclonal anti-collagen IgG was purified from
ascites fluid or concentrated hybridoma culture supernatant by affinity chromatography on Protein Aagarose columns using the Affi-Gel System (Bio-Rad,
Richmond, CA). Purified IgG was dialyzed into PBS
with 0.05% sodium azide, and stored at 4°C until
used.
Colloidal gold particles with diameters of 5 or 10
nm were obtained from Janssen Pharmaceuticals
(Piscataway, NJ). They were coated with purified
monoclonal IgG in 10 mM phosphate buffer at pH
8-9, as described.12 Concentrated gold-antibody
complexes were stored at 4°C until used. Just before
being applied to tissue sections, the antibody-gold
colloids were diluted with 1% BSA in PBS, and aggregates were removed by centrifugation (9000#,
5 min).
Results
Digestion With Type I-Specific Collagenase ("I'ase")
The specificity of the I'ase used in these experiments for type I collagen in solution is demonstrated
in Figure 1 A. Collagen types V or I were mixed with
increasing amounts of I'ase and incubated for 18 hr at
36°C. Visualization of the reaction mixtures after
polyacrylamide gel electrophoresis showed that the
characteristic al(V) and «2(V) bands migrated as expected for intact a-chains, indicating that no degradation of the native type V collagen molecules had occurred. The enzymatic digestion of native type I collagen is demonstrated by the cleavage of the a 1(1) and
«2(I) chains into smaller fragments. The extent of
Vol. 29
type I collagen degradation was proportional to the
amount of I'ase added.
This enzyme was applied to unfixed frozen sections
of 14d. embryonic corneas and incubated for 12-49
hr at 37°C. Controls received either buffer without
enzyme or enzyme inhibited with EDTA. Sections
were then reacted with anti-collagen monoclonal antibodies, and the bound antibody was visualized by
indirect immunofluorescence. A representative experiment is shown in Figure 1B-G. Corneal sections
incubated with buffer or inhibited enzyme exhibited
type I collagen-specific immunofluorescence throughout the corneal stroma and Bowman's membrane
(Fig. IB). Type V collagen-specific fluorescence was
largely confined to Bowman's membrane (arrows,
Fig. 1C), a site in which type V collagen is normally
available for antibody binding. Sections incubated
with active I'ase for various time intervals showed a
progressive loss of the type I collagen as demonstrated
by a decrease in the fluorescence signal for this collagen (Fig. IF, G). The disappearance of type I collagen-specific immunoreactivity always proceeded
more slowly in the interface between the stroma and
Descemet's membrane (asterisk, Fig. 1G). The decrease in type I collagen was mirrored by a corresponding increase throughout the cornea in detectable type V collagen immunoreactivity (Fig. 1D, E).
Thus, as previously reported,9 epitopes on type V
collagen in the corneal stroma become available for
antibody binding as type I collagen is enzymatically
removed. Treatment with I'ase did not affect the pattern of immunofluorescence elicited by antibodies
against collagen type IV (not shown).
Fine Structure of Sections Partially Digested With
I'ase
The structure of the corneal stromal type V collagen exposed by digestion with I'ase and identified by
anti-collagen antibody-coated gold particles was examined by electron microscopy. For analysis of the
structural relationship between collagen types I and V
in corneal fibrils, sections partially digested (for
12-24 hr) with I'ase, containing both essentially intact (striated) segments along with variably degraded
fibrillar regions, revealed considerably more information than did more completely digested preparations, which will not be described here.
The observations pictured in Figures 2 and 3 are of
sections digested for 24 hr, containing partially unmasked type V collagen along with abundant type I
(see Fig. ID, F). Control sections incubated with I'ase
buffer contained the small-diameter (25 nm) striated
fibrils characteristic of the avian corneal stroma.
These were well labeled by antibody against type I
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STRUCTURE OF HETEROTYPIC CORNEAL COLLAGEN FIBRILS / Firch er ol.
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Fig. 1. Digestion with macrophage collagenase (I'ase). (A) Activity against purified collagens in solution. Aliquots of type V collagen (the
five lanes on the left) or type I collagen (thefiveright-hand lanes) were mixed with increasing amounts of active I'ase, and incubated for 18 hr
at 36°C. Analysis of the reaction mixtures by SDS-PAGE showed persistent a 1(V) and a2(V) bands in all the left-hand lanes, indicating that
the enzyme did not degrade native type V collagen in solution. In contrast, increasing amounts of I'ase (lanes on the right) degraded
progressively more type I collagen into smaller fragments. The band at the bottom of each lane represents BSA, a component of the reaction
mixtures. (B-G) show unfixed sections of embryonic chicken cornea incubated with I'ase or buffer without enzyme at 37°C for 24 hr or 49 hr
and then reacted with an antibody against collagen type I or V. Bound antibody was visualized by indirect immunofluorescence. Bar = 100
/im. (B) and (C) show control sections incubated with buffer without enzyme for 49 hr. A section reacted with anti-type I collagen antibody (B)
is strongly fluorescent throughout the cornea! stroma (st) and in Bowman's membrane (arrows). In (C), a section reacted with an antibody
against type V collagen bound antibody almost exclusively within Bowman's membrane (arrows). (D-G) document the emergence of
anti-type V collagen immunoreactivity (D, E) and the concomitant loss of anti-type I immunoreactivity (F, G) with increasing time of
digestion with active I'ase. (D) and (F) show sections digested for 24 hr; the sections in (E) and (G) were digested for 49 hr.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / July 1988
Vol. 29
6
"• c .
•*
• • * • ••
#
••
• • • • • # •#.••• •
•
9
Fig. 2. Fine structure of corneal stromalfibrilspartially digested with I'ase. (A-E) show cryostat sections of comeal stroma incubated for 24
hr at 37°C with active I'ase or buffer without enzyme, and then reacted with 10 nm gold particles coupled to a monoclonal antibody against
collagen types I, V, or IV. The sections were then fixed and processed for electron microscopy. Bars = 100 nm. (A) and (B) show control
sections incubated with buffer before treatment with immunogold particles that recognize collagen type I (A) or type V (B). In (A), the
characteristic 25 nm corneal collagenfibrilsare decorated with type I collagen-specific antibody. In contrast, a similar section reacted for type
V collagen (B) contains very little gold label within the corneal stroma. (C-E) show enzyme-digested sections reacted with antibody against
collagen types I, IV, or V. These preparations contained a network of smallfilamentousstrands, with diameters of 10 nm or less, along with
thicker segments of more intact striated collagenfibrils.In some cases, the thinnerfilamentsappear to be derived from a contiguous striated
segment. (C) shows a partially-digested section labeled with an antibody against type I collagen. Gold particles are associated with both the
more intact fibrillar segments as well as some of the thinner filaments that surround them. In enzyme-treated sections reacted for type IV
collagen (D), the corneal stroma is devoid of gold particles. In (E), a section reacted with the type V collagen-specific antibody shows gold
particles preferentially bound to the thinner strands liberated by the enzyme treatment. The thickerfibrillersegments are only lightly labeled
by this antibody.
collagen (Fig. 2A) but not by antibody against type V
collagen (Fig. 2B). After digestion with the enzyme,
sections of corneal stroma showed a mixture of thin
filamentous strands and regions of relatively intact
striated fibrils, from which the thinner strands could
sometimes be seen to emerge (Figs. 2C-E, 3). Antibody against type V collagen preferentially bound to
the thin filaments which were only seen after hydrolysis with I'ase, and labeled striated regions only lightly
(Fig. 2E and lower-power overview in Fig. 3). The
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STRUCTURE OF HETEROTYPIC CORNEAL COLLAGEN FIDRILS / Firch er QI.
No. 7
.
.
.•
••
i
Fig, 3. Overview of a I'ase-digested section. This micrograph shows a wider field of a section incubated with I'ase for 24 hr, and then labeled
with an antibody against type V collagen. This view offers a variety of examples of thin filamentous strands appearing to emerge from thicker
segments of striated collagen fibrils. Also note the pattern of immunogold labeling which demonstrates antibody against type V collagen
bound preferentially to the thin filaments. Bar = 200 nm,
filaments in such preparations had diameters of 10
nm or less (Fig. 2C-E).
In contrast, type I collagen-specific antibodies labeled both the thicker striated regions and the filamentous strands (Fig. 2C). Antibody against type IV
collagen decorated neither striated nor filamentous
collagen in the stroma (Fig. 2D).
Digestion With Type V-Specific Collagenase (Vase)
We then determined the conditions under which
the Vase could degrade type V collagen. The activity
against purified type I and V collagen in solution is
shown in Figure 4A. After 19 hr at 36°C, increasing
amounts of enzyme degraded a progressively greater
portion of the type V collagen. This is seen as a decrease in the amount of al(V) and a2(V) chains, and
an increasing amount of a limited number of smaller
fragments. Type I collagen was unaffected by this enzyme.
Sections of 14-day corneas were incubated with
Vase at 37°C for various time intervals, reacted with
anticollagen antibodies, and examined by indirect
immunofluorescence histochemistry. A representative experiment is shown in Figure 4B-G. When sec-
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STRUCTURE OF HETEROTYPIC CORNEAL COLLAGEN FIDRILS / Firch er ol.
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Fig. 4. Digestion with MDCK collagenase/gelatinase (Vase). (A) Activity against purified collagens in solution. Aliquots of collagen type V
(thefivelanes on the left) or I (thefiveright-handlanes) were mixed with increasing amounts of active Vase and incubated for 19 hr at 36°C.
Analysis of the reaction mixtures by SDS-PAGE revealed cleavage of the native type V collagen molecules that was reflected in the
degradation of the al(V) and a2(V) bands into smaller polypeptide chains (lanes on the left). The lanes on the right reveal no proteolytic
activity against type I collagen. (B-G) show unfixed sections of embryonic chicken cornea treated with active or inactive Vase for 69 hr, and
then reacted with a monoclonal antibody against collagen type I or V and visualized by indirect immunofluorescence. Bar = 100 pm. In (B, C,
F), sections were treated with Vase without previous acetic acid treatment to disruptfibrils.(B) shows such a section reacted with an antibody
against type V collagen after Vase digestion. The normal pattern of type V-specific immunofluorescence, largely confined to Bowman's
membrane, is unchanged by exposure to Vase. In (F), a section treated with active Vase was then swollen in acetic acid (to expose masked
type V collagen) before reaction with an antibody against type V collagen. The continued presence of stromal type V collagen is revealed by
bright fluorescence throughout the cornea. In (C), a Vase-treated section was reacted with an antibody against type I collagen. This
demonstrates that prolonged exposure to Vase does not affect type I-specific immunofluorescence. (D) and (E) show sections that were first
treated with acetic acid to disruptfibrilsbefore treatment with active or inactive Vase and subsequent application of antibody against type V
collagen. The section in (D), treated with Vase inhibited by EDTA, shows abundant type V collagen that has remained available to antibody
(unmasked) throughout the course of the experiment. (E) demonstrates the removal of type V collagen-specific immunoreactivity from an
acid-treated section by digestion with active Vase. The asterisk identifies a dense band of persistent type V collagen immunoreactivity in the
interface between Descemet's membrane and the stroma. (G) shows a similar section that was acid-treated, digested with active Vase, and
then reacted with an antibody against type I collagen. Even after fibrillar disruption by acid, this collagen was unaffected by the enzyme
treatment.
tions of normal corneas were incubated with Vase
for as long as 74 hr, the fluorescent signal for type I
collagen appeared unchanged (Fig. 4C), and there
was only a slight diminution of the type V collagenspecific immunofluorescence in Bowman's membrane (Fig. 4B; see Discussion).
To determine whether, under these conditions, the
enzyme had in fact degraded the antigenically
masked type V collagen in the stroma, the Vasetreated sections were subsequently treated with acetic
acid before application of antibody. As can be seen in
Figure 4F, this treatment revealed that abundant type
V collagen remained. This indicated that most, if not
all, of the type V collagen was protected from digestion, presumably by its supramolecular fibrillar organization.
That type V collagen is indeed susceptible to in situ
degradation by Vase was demonstrated in corneal
sections that were pretreated with dilute acetic acid to
expose the type V collagen before being incubated
with the enzyme. Acid-treated sections incubated
with inhibited Vase for as long as 3 days retained
abundant unmasked type V collagen (Fig. 4D), while
similar sections incubated with the active enzyme lost
progressively more immunoreactive type V collagen
with increasing time of incubation (Fig. 4E). Type V
collagen was lost from both Bowman's membrane
and the stroma, at about the same rate. In these preparations, although most type V collagen-specific immunofluorescence could be removed given sufficient
incubation time, complete removal was never
achieved, even in sections exposed to fresh enzyme.
Particularly in the vicinity of the Descemet's membrane-stroma interface (asterisk, Fig. 4E), some type
V collagen remained (see Discussion). Vase digestion
of such acid-treated sections caused no detectable
change in type I collagen-specific immunoreactivity
(Fig. 4G) or in type IV collagen-specific immunoreactivity within the lens capsule, Descemet's membrane,
and iris (not shown).
Sequential Digestion With I'ase and Vase
We also examined whether type V collagen, when
exposed by digestion of type I collagen with the I'ase,
is degradable by the Vase. Corneal sections were incubated with active Fase until the immunoreactive
type I collagen was removed (24-48 hr), as determined by indirect immunofluorescence on parallel
sections. These then were incubated with Vase and
monitored at various time intervals by indirect immunofluorescence with the anticollagen antibodies.
Controls received buffer without enzyme. The endpoint of such an experiment is shown in Figure 5. In
sections incubated with the active enzymes, we observed a slow loss of type V collagen-specific immunoreactivity with time, until, in some sections, very
little type I (Fig. 5C) or type V (Fig. 5D) collagen
could be detected. Other sections retained somewhat
more residual type V collagen within the corneal
stroma even after three days exposure to Vase (data
not shown). Control sections showed the characteristic patterns of type I and V collagen immunoreactivity after more than five days in buffer (Fig. 5A, B).
Parallel sections sequentially digested with the enzymes until the endpoint was reached retained type
IV collagen associated with ocular basement membranes (not shown).
Discussion
We have used collagen type-specific collagenases
and monoclonal antibodies to probe the molecular
arrangement of corneal fibrils composed of collagen
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1988
Vol. 29
Fig. 5. Sequential digestion with I'ase and Vase. (A-D) show sections of unfixed embryonic cornea incubated with active I'ase, or buffer
without I'ase, for 48 hr and then active Vase, or buffer, for 74 hr. In this experiment, the active Vase was replenished with freshly activated
Vase after 48 hr of digestion. Bar = 100 ^m. (A) and (B) show sections incubated with the buffers without enzyme. Reaction with antibodies
against collagen types I (A) or V (B) reveal no change in the patterns of cornea] immunoreactivity characteristic of these collagens. In (C) and
(D), sections were sequentially digested with I'ase and Vase. Subsequent indirect immunofluorescence histochemistry with antibodies against
type I (C) or type V (D) collagen documents the virtually complete removal of immunoreactivity for these collagen types by this treatment.
types I and V. Using a collagenase that degrades type
I collagen but not type V, we have confirmed and
extended our earlier observation9 that concealed epitopes on type V collagen in situ can be unmasked by
enzymatic cleavage of type I collagen molecules. This
suggests that type I collagen in fibrils renders those
epitopes on type V unavailable to anti-type V collagen monoclonal antibodies. Electron microscopic
observations of corneal stromas partially digested
with I*ase showed type V collagen in thin filamentous
strands. These filaments frequently were observed to
emerge from regions of thicker striated collagen fibrils. While anti-type V collagen antibody preferentially labeled the filamentous strands in these preparations, antibody against type I collagen labeled both
the striated regions and many of the filaments. Since
the thin filamentous strands could be labeled by antibodies against both collagen types, we believe that at
least some of the filaments liberated by I'ase activity
may themselves be heterotypic, being composed of
collagen types I and V.
The differential pattern of collagen labeling reported here leads us to the tentative conclusion that
much of the type V collagen in stromalfibrilsis completely buried within thefibril.Otherwise, the thicker
regions of partially digestedfibrilsshould have bound
more type V antibody (also see below). In addition,
molecules of type V collagen appear to be diffusely
distributed throughout the fibrils, since one could
find examples of multiple, closely associated filaments containing type V collagen protruding from
the same contiguous segment of a striated fibril. Such
images are of partially digested structures, however,
and thus do not necessarily exclude the possibility
that type V collagen forms a central core in intact
fibrils. The elucidation of the precise molecular arrangement of collagen types I and V will require further ultrastructural studies on fibrils dissected in a
variety of ways.23"26
Whatever the configuration of collagen types I and
V in thesefibrils,any model that entails a population
of type I collagen molecules that is itself sequestered
by type V (for example, an arrangement of alternating layers of the two collagen types), is largely eliminated. Such a model would predict that the sequential
enzymatic digestion of the accessible molecules of
type I and type V collagen would then unmask an
inner population, possibly a core, of type I collagen.
This was not detected in any of the sections sequentially digested with the collagenases. In such sections,
regions of the corneal stroma devoid of type V collagen-specific immunofluorescence also lacked type I;
the small amount of residual immunoreactivity for
type I collagen (see below) also contained type V.
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No. 7
STRUCTURE OF HETEROTYPIC CORNEAL COLLAGEN FIDRILS / Firch er ol.
Type IV-specific immunofluorescence was essentially
unchanged.*
The assertion that type V collagen molecules are
largely buried beneath the fibril surface is strengthened by our experiments with the type V-specific collagenase purified from MDCK cell cultures. We
found that type V collagen in undisrupted corneal
fibrils is not degraded by this enzyme. This indicates
that the cleavage sites on the type V collagen molecule, of which there are probably two or more,16 are
also normally inaccessible to the enzyme in intact
fibrils. Thus, several loci along the length of the type
V molecule are hidden when the molecule is organized into heterotypic fibrils. These sites become
available to the enzyme or antibodies only after the
corneal fibrils are disrupted by treatment with acetic
acid or enzymatic removal of type I collagen. The
implication that type V collagen is largely buried beneath the fibril surface does not exclude the existence
of surface domains of type V collagen in heterotypic
fibrils. Indeed, in Bowman's membrane, epitopes on
at least some of the type V collagen molecules are
normally exposed in corneal sections, and yet this
collagen is also largely resistant to digestion with
Vase in sections of normal corneas and becomes
available for digestion after fibrillar disruption. Perhaps the organization of type V collagen in the
smaller diameter (18 nm) fibrils within Bowman's
membrane is such that the region containing the epitopes, which reside near one end of the molecule,10 is
relatively more exposed than those regions bearing
the collagenase cleavage sites.
In corneal sections in which type V collagen was
unmasked by treatment with acetic acid or I'ase, the
removal of type V collagen from the stroma was
sometimes complete, but usually some residual immunoreactivity persisted. In acid-treated sections,
this might have been due to insufficient swelling of
some fibrils, such that epitopes on type V collagen
were exposed but cleavage sites were not. Alternatively, some reformation of compact fibrils at 37°C
may have protected some of the type V collagen from
Vase, although wholesale fibril reformation in such
preparations certainly did not occur (see Fig. 4D). In
sections treated sequentially with I'ase and Vase,
most of the persistent type V (as well as some type I)
collagen was observed in a region of interfacial matrix
bordering Descemet's membrane that contains especially dense deposits of collagen types I, II, V, VI, and
* In addition, the persistence of type IV collagen in ocular basement membranes in these preparations suggests that this Vase,
unlike some other type V collagen-degrading enzymes,1718 does not
degrade type IV collagen in situ.
1135
perhaps IV, as well as proteoglycan (preliminary observations). Digestion of this region with type I collagen-degrading enzymes proceeds considerably more
slowly, and the collagen in this region has a higher
thermal stability,21 than elsewhere in the cornea.
These experiments show that removal of the fibrillar collagenous matrix of the cornea requires both
I'ase and Vase acting in concert. The production of
type I/II/III-specific animal collagenase by corneal
stromal cells has been documented;27"29 type V-specific collagenase activity has not yet been identified in
the cornea, but it is possible that corneal cells do
possess such an enzyme in their repertoire. In the
context of heterotypic fibrils composed of collagen
types sensitive to different degradative enzymes, the
differential control of enzyme activity (at the level of
production, activation, or inhibition) would produce
quite different extracellular matrices in the course of
normal or pathological processes of remodeling. In
the corneal stroma, for example, the coordinate expression of both I'ase and Vase would produce a
loose filamentous matrix of uncertain orientation
composed, perhaps, of type VI collagen, noncollagenous microfibrils, and proteoglycans. Conversely,
the differential expression of I'ase without Vase activity would also result in a loose filamentous matrix,
but one with a spatial organization (imposed by the
persistent type V collagen) that would precisely mirror that of its origin. This matrix might then serve as a
template for directing the assembly of a new connective tissue matrix with the appropriate organization.
Key words: cornea, collagen,fibrilstructure, collagenases,
immunocytochemistry
Acknowledgments
We wish to thank Dr. Richard Mayne for the purified
type V collagen used in the digestions.
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