Download Heterogeneity of collagens in rabbit cornea: type VI collagen.

Investigative Ophthalmology & Visual Science, Vol. 29, No. 5, May 1988
Copyright © Association for Research in Vision and Ophthalmology
Heterogeneity of Collagens in Rabbit Cornea:
Type VI Collagen
Charles Cinrron* and Bor-Shyue Hongf
Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic
acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction
was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a
high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was
converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These
physical characteristics, together with the susceptibility of these polypeptides to collagenase and their
amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas
from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [NC) glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin
and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue
residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed
for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is
synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either
corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI
collagen deposited in scar tissue is markedly lower than that found in neonate corneas. Invest Ophthalmol Vis Sci 29:760-766, 1988
The predominant banded collagen fibrils of mammalian corneal stroma are composed of type I collagen.1"4 Other than a single report, there is no evidence
indicating the presence of type II collagen in mammalian corneas.5 Although there is as yet some controversy as to the distribution and quantity of type III
collagen within the cornea, it appears to be associated
with neonate and healing adult tissues.346"10 Type IV
collagen seems to be restricted to the basement membrane of the corneal epithelium and the Descemet's
membrane of normal corneas."12 Type V collagen
constitutes about 10% of the normal stromal collagen.1314 The relative quantity of type V collagen
within the stroma of fetal and healing cornea is markedly different from the adult, suggesting that this collagen may play a role in the morphogenesis of the
corneal stroma. Although we do not know the ultrastructural distribution of type V collagen in mamma-
lian corneas, recent reports have indicated that chick
stromal collagen fibrils are copolymers of type I and
V collagens.15 Type VI collagen is also located in the
mammalian stroma and because of its unusual structure, it may be associated with fine filaments between
the banded collagen fibrils.16"19 Anchoring fibrils associated with the basement membrane of the corneal
epithelium and extending into the stroma have been
identified with type VII collagen.20-21 Finally, type
VIII collagen, originally designated "endothelial collagen,"22 has been described as a prominent collagen
in bovine and rabbit Descemet's membranes.2324 Because each corneal collagen has a unique structure,
distinctive chemical properties, a specific ultrastructural distribution within the tissue, and different and
varying abundance during morphogenesis, these
macromolecules must play an important role in normal corneal development and healing.
Our studies have shown that developing neonate
corneas and corneal scar tissue from adult corneas
synthesize and deposit mostly types I and V collagens.13 The limitations in our analyses were partially
due to the paucity of tissue and the low specific activity of radiolabeled collagen synthesized after in vivo
labeling of the corneal proteins. To increase the specific activity, we developed an organ culture method
that maintains the whole cornea deturgescent and ultrastructurally normal during incubation of the tis-
From the *Eye Research Institute, Boston, Massachusetts, and
fDepartment of Ophthalmology and Visual Sciences, Texas Tech
University Health Sciences Center, Lubbock, Texas.
Supported by grant EY-01199 from the National Institutes of
Health, Bethesda, Maryland, and a grant from Research to Prevent
Blindness.
Submitted for publication: April 14, 1987; accepted December
14, 1987.
Reprint requests: Charles Cintron, PhD, Eye Research Institute,
20 Staniford Street, Boston, MA 02114.
760
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No. 5
TYPE VI COLLAGEN IN RADBIT CORNEA / Cinrron ond Hong
sues with radiolabeled precursors.25 This we believe
also maintains a pattern of macromolecular synthesis
similar to that in vivo.
In the present study, we show that normal adult
rabbit corneas contain type VI collagen. Moreover,
rabbit neonate corneas and scar tissue from adult
corneas deposit types I, V and VI collagen and synthesize these collagens during organ culture. Semiquantitative analyses of the collagens deposited and
synthesized in these growing corneal tissues indicate
that the relative quantity and solubility of collagens
differ among different types of collagens as well as
between tissues. These differences have significance
to our understanding of scar formation.
Material and Methods
Type VI Collagen
Adult rabbit corneas, freed of Descemefs membrane, endothelium and epithelium were homogenized in 0.1 M acetic acid with a blender at high speed
for 1 min followed by limited pepsin digestion at 4°C
for 24 hr with a 5% pepsin/tissue wet weight ratio in
0.1 M acetic acid. The digest was centrifuged at
60,000 rpm for 10 min and the supernate directly
fractionated by differential salt precipitation at 0.7
M, 1.2 M and 2.5 M NaCl concentration in acid. The
2.5 M precipitate fraction was redissolved in 0.05 M
tris-HCl buffer, pH 7.5, containing 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM N-ethyl maleimide, 1 mM benzamidine, 25 mM ethylene diamine
tetra-acetic acid, and 0.5 M NaCl at 4°C for 2 hr. The
solution was centrifuged and the supernate dialyzed
against 0.05 M sodium acetate, pH 4.8, to precipitate
the proteins. These precipitated proteins could not
penetrate an SDS-8% polyacrylamide slab gel during
electrophoresis.26 Disulfide reduction with 5% 2mercaptoethanol dissociated the large protein aggregate, tentatively called high-molecular-weight disulfide-bonded aggregate (HMWDA), into small, Coomassie blue-stained proteins, which penetrated the
polyacrylamide gel.
The HMWDA was hydrolyzed with 6 N HC1 under
nitrogen at 105°C for 24 hr to determine the amino
acid composition with a Beckman amino acid analyzer, model 121 (Fullerton, CA).
Susceptibility of HMWDA to bacterial collagenase
was determined by incubating 100 yug of the
HMWDA with 50 /zg of Closlridium hislolyticum
collagenase, protease free (Cal Biochem, San Diego,
CA), in 200 ^1 of 0.05 M Tris-HCl buffer, pH 7.5,
containing 10 mM CaCl2 at 37°C for 2 hr. Controls
contained HMWDA in appropriate buffer and incubated at the same temperature for the same amount
of time. The hydrolysates and controls were then re-
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761
duced with mercaptoethanol and electrophoresed in
an SDS-8% polyacrylamide slab gel.
Corneal Organ Culture, Collagen Extraction,
and Analysis
Rabbits weighing about 2.5 kg were anesthetized
with intravenous injections of sodium pentobarbital
and topical application of proparacaine drops to each
eye before wounding the corneas. A 2 mm diameter,
full-thickness wound was made in each eye of six
rabbits and allowed to heal for 2 weeks.27 The
wounded rabbits and three newborn rabbits, approximately 3 days old, were killed with an overdose of
sodium pentobarbital. The whole cornea of each eye,
with an attached 1-2 mm scleral rim, was removed
and placed in ice-cold culture medium. One scarred
cornea was not used due to complications during
healing. Our culture medium contained Hank's basal
medium. Coon's concentrate, newborn calf serum
and the antibiotics, penicillin and streptomycin.28
The medium was modified by the addition of 2%
(w/v) chondroitin sulfate (Sigma, St. Louis, MO).
Corneas were washed with 4 ml of fresh medium
before incubation in Linbro tissue culture multi-well
plates with a well capacity of 7.5 ml (Flow Labs,
McLean, VA). Enough fresh medium was used to
cover the explant with the corneal endolhelium up.
The medium contained 75 nC\ of [I4C] glycine per ml
(New England Nuclear, Boston, MA) and the tissues
were incubated at 37°C in a 5% CO2/air mixture for
18 hr. These procedures have been shown to maintain the rabbit cornea deturgescent and ultrastructurally normal for 48 hr.25
After incubation, explants were washed in fresh
nonradioactive medium, and the scleral rims removed from the corneas. The total nconate cornea
and only the scar tissue of the adult cornea were
weighed, frozen in liquid nitrogen and pulverized.
Powdered corneas were digested with 0.02% (w/w)
pepsin/tissue weight in 0.5 M formic acid at 4°C for
18 hr. Digests were centrifuged for 15 min at 12,000
rpm and the supernate decanted. The tissue residues
were again treated with pepsin and centrifuged, and
the supernate eventually pooled with the first extraction. Collagens solubilized after the first and second
pepsin treatments were termed first and second pepsin-soluble fractions, respectively. Similarly, the undigested residues after each enzyme treatment were
referred to as the first and second pepsin-insoluble
fractions. The first pepsin-soluble fractions, containing collagens, were precipitated with 1.2 M NaCl
(pepsin-soluble, 1.2 M NaCl precipitate fraction).
The second pepsin-soluble fraction was too scant to
precipitate with salt. Precipitated collagens were re-
762
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1988
Vol. 29
Results
Type VI Collagen
1 2345
«» <200Kd
^
«116 Kd
m « 93Kd
•
SC3
« 66Kd
The HMWDA obtained in the 2.5 M precipitate
fraction from pepsin-solubilized proteins of adult
rabbit cornea were electrophoresed on an SDS-polyacrylamide gel with and without mercaptoethanol reduction (Fig. 1). Other than the intense Coomassie
blue-stained material in the stacking gel, containing
unreduced protein, no proteins were detected in the
running gel when mercaptoethanol was not present.
Upon reduction, three distinct bands, SCI, SC2 and
SC3, migrated between 45 and 66 Kd. The predominant corneal collagens, types I and V, migrated much
more slowly than the short-chain polypeptides.
Susceptibility of these polypeptides to bacterial
collagenase indicated the presence of collagen-like
proteins in the HMWDA (Fig. 2). Moreover, the
«45Kd
^
<31Kd
Fig. 1. Reduced and unreduced SDS-8% polyacrylamide slab gel
electrophoresis of rabbit cornea type VI collagen stained with Coomassie blue. Pepsin-digested, acetic acid-extracted collagens from
adult rabbit corneas were subjected to salt fractionation to obtain a
2.5 M NaCI-precipitated material which was then dissolved and
reprecipitated at pH 4.8. The precipitate was called high-molecular-weight disulfide-bonded aggregate (HMWDA). Lane 1,
HMWDA proteins remain in the stacking gel. Lane 2, HMWDA
proteins, reduced with 2-mercaptoethanol, migrate into the gel.
Three bands are identified as SCI, SC2 and SC3. Lane 3, type V
collagen. Lane 4, type I collagen. Lane 5, molecular weight markers
(myosin, 200 Kd; B-galactosidasc, 1 16 Kd; phosphorylase b, 93 Kd;
bovine serum albumin, 66 Kd; ovalbumin, 45 Kd; carbonic anhydrase, 31 Kd).
dissolved in 0.5 M formic acid, dialyzed against the
solvent and lyophilized. Collagen extracts and insoluble tissues were reduced with 5% 2-mercaptoethanol
and run on a 6% SDS-polyacrylamide slab gel. Proteins were stained with Coomassie blue and the relative quantity of collagen protein bands was measured
by densitometry. Gels were then treated with
En3hance (New England Nuclear, Boston, MA) and
exposed to X-ray film for fluorography. Developed
film was also subjected to densitometry to determine
the relative quantity of [14C] incorporation into collagens. All procedures adhered to the ARVO Resolution on the Use of Animals in Research.
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<200Kd
<116Kd
«93Kd
<66Kd
«45Kd
«31 Kd
i4Kd
Fig. 2. SDS-8% polyacrylamide slab gel electrophoresis of mercaptoethanol-reduced bacterial collagenase-digested rabbit cornea
type VI collagen stained with Coomassie blue. Lane I, bacterial
collagenase. Lane 2, HMWDA (see Fig. I) after digestion with
bacterial collagenase. Lane 3, Control HMWDA incubated with
collagenase buffer. Lane 4, Molecular weight markers, as in Figure
1, with addition of lysosome, 14 Kd.
No. 5
760
TYPE VI COLLAGEN IN RABBIT CORNEA / Cinrron and Hong
amino acid composition of the HMWDA indicated
the presence of amino acids common to collagen,
such as hydroxyproline, hydroxylysine and high
levels of glycine and proline (Table 1). The high content of hydroxylysine, leucine, valine and isoleucine,
and low content of alanine were similar to those in
types IV and V collagen. However, a high cysteine
content provided the most characteristic property of
type VI collagen.
Corneal Organ Culture
Radiolabeled glycine incorporated into fractions of
collagen extracts and residues from corneal tissue
were measured to compare collagen synthesis in neonate and scar tissue. Although total incorporation of
label was greater in neonate tissue, the proportions of
label distributed among the soluble and insoluble
fractions of neonate and scar tissue were similar
(Table 2). No marked changes in the counts of the
pepsin-insoluble fraction were noted between the first
and second pepsin digestion. Conversely, the number
of counts recovered from the tissue after the second
pepsin digestion was only 10% of the first pepsin-insoluble tissue fraction. These observations indicate
that most of the soluble collagens are extracted with
two digestions of pepsin. No further digestions or extractions were performed on these tissues. The
weights of the tissues before and after collagen extraction are consistent with our published findings,
indicating that scar tissue collagen is less soluble than
neonate corneal collagen.13
Polyacrylamide slab gel electrophoresis of mercaptoethanol-reduced pepsin-soluble and -insoluble collagen fractions from neonate corneas and 2-week-old
scars shows the presence of types I, V and VI collagen
polypeptides (Fig. 3). In neonate corneas, type I collagen was the predominant protein extracted with
pepsin treatment, leaving most of the types V and VI
in the insoluble fraction (Fig. 3, lanes 3 and 6). Very
little collagen could be detected in the first pepsin-soluble, 1.2 M NaCl supernate, and in second pepsinsoluble fractions (lanes 4 and 5). In scar, types I and V
collagens were present primarily in the pepsin-soluble, 1.2 M NaCl precipitate fraction (Fig. 3, lane 7).
As in the neonate cornea, type VI collagen in scar
remained in the insoluble fraction (lane 9). Delayed
mercaptoethanol reduction of these collagen fractions indicated the presence of type III collagen in
these tissues (not shown; see following paper10).
The relative optical densities of collagen polypeptides in Coomassie blue-stained SDS-PAGE are indicative of the relative quantities of different collagens present in the tissues (Table 3). The al:a2 ratios
greater than 2.00 indicated contamination of type I
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Table 1. Amino acid composition of the highmolecular-weight disulfide-bonded aggregate
(HMWDA) from rabbit corneal stroma
Amino Acid
Residues/1000 residues
4-Hydroxyproline
Aspartic acid
Threonine
65
65
24
35
104
109
309
38
30
19
8
20
29
17
16
43
14
5
61
Serine
Glutamic acid
Proline
Glycine
Alanine
Cysteine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Hydroxylysine
Lysine
Histidine
Arginine
High levels of hydroxyproline, hydroxylysine. glycine and proline are indicative of collagen. The high contents of hydroxylysine. leucine. valine and
isoleucine and low content of alanine are indicative of type IV. V. and VI
collagens. Finally, the high cysteine content indicates that the HMWDA
fraction is type VI collagen.
collagen polypeptides with types II, III, V or type I
trimer polypeptides. This was particularly evident in
the insoluble fractions. The quantity of type V collagen relative to the type I collagen in the soluble and
insoluble fractions, and the high ratio of type VI collagen in scar in comparison to neonate cornea, suggested marked differences in the molecular organization of these collagens in these tissues (Table 3).
To determine the relative proportion of collagens
in the tissues, we added the densities of certain polyTable 2. Summary of total [14C] glycine
incorporation into collagen from neonate rabbit
corneas and adult scar tissue in organ culture
cpm (% of total)
Fraction
Total cpm
First pepsin-soluble
1.2 M NaCl
precipitate
supernate
First pepsin-insoluble
Second pepsin-soluble
Second pepsin-insoluble
Number of corneas
Total wet weight (mg)
Total cpm per mg wet weight
Second pepsin-insoluble,
mg wet weight (% of
tissue)
Neonate
Scar
723.360(100)
138.400(100)
237.200(32.8)
39.750(5.5)
88.308(12.2)
386.160(53.4)
40.560(5.6)
345.546(47.8)
65.600 (47.4)
10.380(7.4)
13.680(9.9)
72.800 (52.6)
7.380(5.3)
65.420(47.3)
5
150
4822
30 (20)
11
33
4194
16(49)
764
Vol. 29
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Moy 1988
1
2
3
4
5
6
7
8
9
• * • •
•
Fig. 3. Type VI collagen from 3-day-old rabbit neonate corneas
and 2-wcek-old scars in adult corneas. Pepsin-digested, formic
acid-extracted collagens were precipitated with 1.2 M NaCl, reduced with mcrcaptoethanol, and subjected to SDS-6% polyacrylamide slab gel electrophoresis. Proteins were stained with Coomassie blue. Lane I, type V collagen standard. Lane 2, type VI collagen
standard. Lanes 3-6, neonate fractions. Lane 3, first pepsin-soluble, 1.2 M NaCl precipitated fraction. Lane 4, first pepsin-soluble,
1.2 M NaCl supernate fraction. Lane 5, second pepsin-soluble
fraction. Lane 6, pepsin-insoluble fraction. Lanes 7-9, scar fractions. Lane 7, first pepsin-soluble, 1.2 M NaCI-precipitated fraction. Lane 8, second pepsin-soluble fraction. Lane 9, pepsin-insoluble fraction. Dotted lines denote positions of SCI, SC2 and SC3.
These proteins are present only in the pepsin-insoluble fractions
from neonate and scar tissue. The low-molecular-weight double
band in lane 5 is pepsin, a-e: Migration of: a. aJV], b. a 2 [I], c. SCI,
d. SC2, and e. SC3.
peptide bands of each collagen type from each fraction. For type I collagen we added a2[l], and 1/4 Beta
(contributions by Gamma bands were insignificant).
For Type V collagen, al[V] polypeptide was measured. For type VI, we added SCI, 2, and 3 polypeptides. The results indicate that the relative amount of
type VI collagen to type I collagen in the insoluble
fraction from scar tissue is three times greater than
that in neonate corneas. However, the ratio of type VI
to total type I collagen in scar and neonate tissues is
0.131 and 0.372 respectively.
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The relative densities of silver deposits on autoradiographs of SDS PAGE slabs containing radiolabeled collagen polypeptides are suggestive of the relative rates of collagen synthesis, the processing of the
various collagens, or both in neonate and scar tissue
(Table 4). As in the Coomassie blue-stained gel, the
high ratio of a I :a2 in scar tissue indicated synthesis of
other types of collagen such as types II, III and I
trimer, or contamination with a2(V) polypeptide.
The ratio of al(V):a2(I) pepsin-soluble collagen polypeptides from scar is markedly different from that in
neonate corneas. In contrast to deposition no
marked difference was found in the incorporation of
label into type VI collagen in the insoluble collagen
fractions (Tables 3, 4). However, the ratio of the autoradiographic band densities of type VI polypeptides
to the sum of the band densities of a2(I) from soluble
and insoluble fractions of scar and neonate tissues are
0.376 and 0.606, respectively.
Discussion
Our studies confirm previous observations showing that rabbit corneal collagen is readily solubilized
from normal tissue after pepsin digestion in comparison to scar tissue. 1329 Moreover, the results are consistent with the presence of marked amounts of type I
trimer in scar tissue.29 That our corneal organ culture
method maintains the pattern of collagen synthesis
previously shown by in vivo labeling is evidenced by
the higher proportion of type V collagen synthesized
in scar tissue than in normal neonate corneas regardless of labeling method. 13 Radiolabeling of collagen
during organ culture has made it possible to see evidence of type VI and, as we show in the following
paper,10 type III collagen deposition in corneal tissues, which has not previously been detected by the in
vivo labeling method.
Our results show that the incorporation of [I4C]
glycine into collagen fractions from neonate corneas
Table 3. Semiquantitative analysis of collagen
present in neonate cornea and corneal scar*
Collagen polypeptide ratios
Tissues and
fractions
Corneal scar
Pepsin-soluble}:
Pepsin-insoluble
Neonate
Pepsin-soluble
Pepsin-insoluble
a 1(0
al(V)
K/f
a2(0
a2(I)
a2(i)
2.54
3.35
0.70
1.00
6.35
2.03
3.62
0.25
3.60
1.69
* Based on the relative band densities in Coomassie blue-stained SDSPAGE of collagen polypeptides from 3-day neonate corneas and 2-week-old
corneal scar tissue. Type I collagen polypeptides are calculated from a 1(1),
a2(I). and 1/4/3(1).
t The sum of SCI, SC2 and SC3 polypeptides of type VI collagen.
$ Pepsin-soluble, NaCl-precipitable collagen.
No. 5
765
TYPE VI COLLAGEN IN RADDIT CORNEA / Cinrron ond Hong
and scar tissue is about the same when expressed as
cpm per mg wet weight of tissue. The tissues in these
experiments, however, contain large quantities of
water and extracellular matrix. A more meaningful
way of expressing the data is to relate them to the
number of cells. The DNA values from similar tissues
in previous studies in our laboratory were 50 and 19
ixg DNA for the neonate and scar tissues, respectively.2730 Our calculations show that the incorporation of label per ng DNA into collagen fractions from
scar tissue is about half of that from neonate corneas,
suggesting that the rate of collagen synthesis is markedly lower in the scar.
Previous studies from our laboratory have indicated many morphological and biochemical similarities between healing adult cornea and the normal
development of fetal and neonate cornea.2730 If we
consider a 2-week-old corneal scar as temporally
equivalent to the first 14 days of fetal corneal development (27 days of gestation), scar tissue has deposited a greater amount of collagen per cell than has the
27-day fetal cornea.30 It is not until the first week of
birth that the neonate cornea accumulates collagen at
a rate comparable to the 2-week-old scar. These neonate corneal cells are morphologically well differentiated into keratocytes, albeit they contain enormous
quantities of rough endoplasmic reticulum. Thus, the
deposition of collagen in healing wounds is high during early stages of healing, whereas normal developing cornea accumulates collagen at a high rate only
after cell morphological differentiation is almost
complete. Since the present data suggest that scar tissue synthesizes collagen at a lower rate than that of
the neonate cornea, assuming the specific activity of
the precursor pools were the same and incorporations
were constant during incubation, the similarity in accumulation of collagen in these tissues must be due to
differences in the turnover of this macromolecule.
Because the neonate cornea continuously remodels
its extracellular matrix to accommodate growth, the
collagen turnover is very high.31-32 The scar tissue, on
the other hand, synthesizes collagen at a slower rate
and remodels the scar gradually,13'3334 indicating its
collagen turnover is also gradual. Although our hypothesis is consistent with the results of this and previous studies, future studies are needed to test collagen turnover in these tissues more directly.
Type VI collagen has been extracted from numerous tissues and subjected to several analyses.1635"38
Among the prominent features of this collagen are its
abundance of interchain disulfide cross-links, which
may be responsible for its resistance to bacterial collagenase36-39; its unique polymeric structure within
the extracellular matrix, identifying it with microfibrillar structures 171837 ; its unusual carbohydrate
composition of mannose and fucose residues39; and
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Table 4. Semiquantitative analysis of collagen
synthesized in organ culture of neonate
cornea and corneal scar*
Collagen polypeptide ratio
Tissues and
fractions
Corneal scar
Pepsin-soluble^:
Pepsin-insoluble
Neonate
Pepsin-soluble
Pepsin-insoluble
al(I)
al(V)
W
a2(l)
a2(I)
a2(I)
2.52
2.70
0.76
1.03
—
1.43
2.14
1.98
0.25
1.18
—
1.35
* Based on the relative band densities in SDS-PAGE fluorographs of collagen polypeptides from 3-day neonate corneas and 2-week-old corneal scar
tissue. Type I collagen polypeptides are calculated from a 1(1) and a2(l) only.
t The sum of SCI, SC2 and SC3 polypeptides of type VI collagen.
% Pepsin-soluble, NaCI-precipitable collagen.
its large terminal nonhelical portions of the molecules in the completed structure, constituting over
50% of the molecular weight.40 Although structural
details of type VI collagen are still not clear, chemical,
ultrastructural and analytical ultracentrifugation
studies have suggested that the monomer of type VI
collagen, 110-145 Kd, is a 105-nm-long triple helix
terminated by globular domains on each end. Monomers associate to form dimers with a lateral 30 nm
stagger and align themselves in an antiparallel fashion. The outer segments of two parallel dimers are
covalently linked to form tetramers. Finally, tetramers are assembled end-to-end with overlap between
outer segments to form a microfibrillar structure.373841
Although indirect immunofluorescence has demonstrated the presence of type VI collagen in many
tissues and cell cultures,l9-36-41-42 cartilage, basement
membranes, elastin and cross-striated collagen fibrils
do not appear to contain this collagen.16 The present
results adds to the list of tissues containing type VI
collagen and suggest that this collagen is deposited at
a slower rate in corneal scars than in normal neonate
tissue.
Current concepts of corneal transparency require
that small distances between the collagen fibrils be
maintained.43 If indeed type VI occupies the spaces
between collagen fibrils,171844 is it possible that the
opacity of the scar is partially maintained by the
space-filling type VI collagen? Clearly, further studies
are required to determine the ultrastructural distribution of type VI collagen within scar tissue.
Key words: cornea, collagen, wound healing, development,
type VI collagen
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