Supplemental Material can be found at:

http://www.jbc.org/content/suppl/2011/02/18/M110.169813.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 15, pp. 1301113022, April 15, 2011
2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Effects of Ultraviolet-A and Riboflavin on the Interaction of

Collagen and Proteoglycans during Corneal Cross-linking*
S

Received for publication, July 28, 2010, and in revised form, January 31, 2011 Published, JBC Papers in Press, February 18, 2011, DOI 10.1074/jbc.M110.169813

Yuntao Zhang1, Abigail H. Conrad, and Gary W. Conrad

From the Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901

The cornea is the transparent, dome-shaped tissue covering

the front of the eye. It is a powerful refracting surface, providing
6575% of the eyes focusing power, and is made up of three
distinct layers: epithelium, stroma, and endothelium. The
stroma comprises about 90% of cornea thickness in humans (1).

* This work was supported, in whole or in part, by National Institutes of Health

Grant R01EY000952 (to G. W. C.). This work was also supported by the
Research Career Development Core (Brychta) in the Division of Biology at
Kansas State University GOBO000657 (to G. W. C.), by National Science
Foundation Major Research Instrumentation Program Grant 0521587 (to
Kansas State University), and by the Terry C. Johnson Center for Basal Cancer Research at Kansas State University.

S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1.
1
To whom correspondence should be addressed: Division of Biology, Ackert
Hall, Kansas State University, Manhattan, KS 66506-4901. Tel.: 785-5326553; Fax: 785-532-6653; E-mail: ytz@ksu.edu.

APRIL 15, 2011 VOLUME 286 NUMBER 15

Although it is very highly innervated, it does not contain any

blood vessels (2 4). Collagen gives the cornea its strength, elasticity, and form (5). The unique molecular shape, paracrystalline arrangement, and very regular fine diameter of the evenly
spaced collagen fibrils are essential in producing a transparent
cornea (6, 7).
In the corneal stromal extracellular matrix, glycosaminoglycan (GAG)2 polysaccharides are the most abundant negatively
charged macromolecules and are classified on the basis of their
repeating disaccharide structures into four main groups: keratan sulfate (KS), chondroitin sulfate/dermatan sulfate (CS/DS),
heparan sulfate and heparin, and hyaluronan. Glycosaminoglycans, normally covalently attached to proteoglycan (PG) core
proteins, play important roles in corneal transparency, nerve
growth cone guidance, and cell adhesion, largely dependent on
their patterns of sulfation or lack of it (8, 9). The corneal stroma
is composed primarily of collagen fibrils, each of which consists
of a core of type V collagen coated with type I collagen (10, 11),
coated in turn by two classes of PGs (12): keratan sulfate proteoglycan and chondroitin sulfate/dermatan sulfate proteoglycan. Through N-linked oligosaccharides, KS chains
are attached to three core proteins, lumican, keratocan, and
mimecan, to form keratan sulfate proteoglycans (1315). These
three core proteins belong to a class of proteins known as small
leucine-rich repeat proteins (16 18). Through O-linked oligosaccharide, CS/DS chains are attached to core protein decorin
(19, 20). In the case of decorin, a single CS/DS linkage site is
present near the amino terminus of the core protein (21, 22),
whereas lumican and keratocan possess four or five potential
KS attachment sites in the central leucine-rich repeat region of
each core protein molecule (13, 23, 24), and mimecan has two
potential KS attachment sites (25, 26). Current molecular models of the corneal stroma suggest that these proteoglycan core
proteins wrap themselves laterally around the collagen fibrils in
a manner that folds their hydrophobic domains inside, against
the collagen fibrils (27). In contrast, the highly sulfated GAG
chains (together with their associated water molecules of
hydration) are thought to stick out laterally away from the sides
of the collagen fibrils, forming an exterior hydrophilic shell.
The thickness of that shell matches the thickness of the shell
surrounding adjoining fibrils, producing a very precise centerto-center spacing between the collagen fibrils characteristic of
2

The abbreviations used are: GAG, glycosaminoglycan; RF, riboflavin; UVA,

ultraviolet-A; PG, proteoglycan; KER, keratocan; LUM, lumican; MIM, mimecan; DEC, decorin; Ab, antibody; Coll, collagen; KS, keratan sulfate; CS,
chondroitin sulfate; GHCl, guanidine HCl; DS, dermatan sulfate; NMWL,
normal molecular weight limit; RFUVA, riboflavin and ultraviolet-A; rcf, relative centrifuge force.

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Corneal cross-linking using riboflavin and ultraviolet-A

(RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican,
mimecan, and decorin, core proteins of major proteoglycans
(PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified,
non-glycosylated PG core proteins in solution in vitro has been
compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen
cross-linking. Irradiation with UVA and riboflavin cross-links
collagen and chains into larger polymers. In addition,
RFUVA cross-links PG core proteins, forming higher molecular
weight polymers. When collagen type I is mixed with individual
purified, non-glycosylated PG core proteins in solution in vitro
and subjected to RFUVA, both keratocan and lumican strongly
inhibit collagen cross-linking. However, mimecan and decorin
do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal
glycosaminoglycans, keratan sulfate and chondroitin sulfate, in
isolation from their core proteins, are not cross-linked by
RFUVA and do not form cross-links with collagen. Significantly,
when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form
cross-links with collagen. Thus, RFUVA causes cross-linking of
collagen molecules among themselves and PG core proteins
among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as
a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan
and decorin.

Collagen and Proteoglycans in Corneal Cross-linking

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EXPERIMENTAL PROCEDURES
MaterialsBovine eyeballs were freshly collected from Alta
Vista Locker (Alta Vista, KS). Collagen type I from bovine skin
was purchased from INAMED (Fremont, CA). Collagen type III
and type IV from human placenta and RF 5-monophosphate
sodium salt were purchased from Sigma (St. Louis, MO). KS
from bovine cornea was purchased from Seikagaku America
(East Falmouth, MA) and was purified further by chromatography and chondroitinase ABC treatment (sodium salt), as
described in detail below. The average molecular mass of KS
was 15 kDa. CS from bovine trachea was purchased from
Sigma. CS was purified by chemical treatment, as described in
detail below. Recombinant human keratocan and human lumican proteins were purchased from Abnova Corp. (Taipei, Taiwan). Recombinant mouse mimecan and recombinant human
decorin proteins were purchased from R&D Systems (Minneapolis, MN). Anti-human keratocan polyclonal antibody was
purchased from Abnova Corp. Anti-human lumican antibody,
anti-mouse mimecan antibody, and anti-human decorin antibody were purchased from R&D Systems (Minneapolis, MN).
Rabbit polyclonal antibody to collagen type I (ab34710) was
purchased from Abcam Inc. (Cambridge, MA). NuPAGE
Novex BisTris 4 12% precast gels (8 cm 8 cm 1.5 mm),
NuPAGE Novex Tris acetate 3 8% precast gels (8 cm 8
cm 1.5 mm), and chromogenic Western blot kits were purchased from Invitrogen (Carlsbad, CA).
Preparation of Free KS Polysaccharide ChainsBased on the
manufacturers protocol for isolation of KS, there is residual
core peptide at the reducing terminus. N-Glycanase was
employed to release N-linked KS chains from the short peptides. Following the procedure suggested by Prozyme, the mixture of KS and N-Glycanase was incubated overnight at 37 C.
Enzymatic digestions were terminated by heating for 10 min in
boiling water and then cooled to room temperature. Subsequently, 1 volume of 100% ice-cold trichloroacetic acid (TCA)
was added to 4 volumes of the digestion sample, mixed, and
kept at 4 C overnight. The digestion sample was centrifuged at
14,000 rpm and at 4 C for 30 min to remove precipitated N-glycanase. The supernatant, containing KS, was transferred to
Ultra free-MC centrifugal filter units (5,000 NMWL; Millipore)
and washed extensively with water to remove salt, and then the
retentate was lyophilized.
Preparation of Free CS Polysaccharide ChainsTo obtain CS
chains free of residual core protein peptides for cross-linking
analysis, an alkaline -elimination reaction was carried out in
the presence of NaBH4. The CS sample was adjusted to 1.0 M
NaBH4, 0.05 M NaOH, incubated at 45 C for 24 h (52), neutralized with 1 M acetic acid, washed extensively with water using
Ultra free-MC centrifugal filter units (5,000 NMWL; Millipore)
to remove salt, and then the retentate, containing CS, was
lyophilized.
Cross-linking Model SystemPurified collagen type I was
used to assess the mechanisms of cross-linking induced by the
photosensitizer RF and UVA under conditions that resemble
those used for clinical treatment of progressive keratoconus.
The mixture of purified collagen type I (1 g/l in PBS) and RF
(0.1% in PBS), in solution, was irradiated with UVA of 370 nm
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the corneal stroma and necessary for its transparency (28).

Through this interaction with collagen (mostly with type I),
PGs play important biological roles in collagen fibrillogenesis
and matrix assembly (29). They also may serve as binders of
other proteins, some of which may be neurorepellants and neuroattractants (30).
Keratoconus is a vision disorder of unknown molecular etiology but with some genetic basis that occurs when the normally round cornea begins to bulge outward. This abnormal
shape, arising as the central region becomes thinner, prevents
the light entering the eye from being focused correctly on the
retina and causes distortion of vision (31). Keratoconus may
progress for 10 20 years and then slow in its progression. Each
eye may be affected differently. Keratoconus affects 1 in 2000
people (32). Treatment options include conservative approaches, aimed at maintaining visual acuity, such as rigid contact lenses placed to straighten corneal aberrations (33). Alternatively, more invasive methods are applied using intrastromal
corneal implants (34) or performing anterior lamellar keratoplasty or penetrating keratoplasty in extreme cases (35, 36).
However, all of these techniques only correct the refractive
errors of keratoconus and do not treat the cause underlying the
corneal ectasia and therefore do not stop the progression of
keratoconus.
Recently, a new technique of corneal cross-linking was
devised that directly improves the biomechanical rigidity of the
corneal stroma (37). This approach consists of irradiation with
ultraviolet-A (UVA) in the presence of the photosensitizer,
riboflavin (RF), as a chromophore, to stop progression of the
keratoconus syndrome. The basic principle of corneal crosslinking is thought to be that in the presence of UVA at 370 nm,
riboflavin is excited into its triplet state, generating singlet oxygen, which can react further with various molecules, inducing
chemical covalent bonds bridging amino groups of collagen
fibrils (38). In brief, the central 7 mm of the corneal epithelium
is removed to allow better diffusion of riboflavin into the
stroma. Before irradiation with UVA, a 0.1% riboflavin solution
(10 mg of riboflavin 5-phosphate in 10 ml of dextran 20% solution) is applied onto the debrided central cornea every 5 min for
30 min. Then, as the application of riboflavin continues, the
irradiation is performed from a 1-cm distance for 30 min with
UVA at 370 nm and irradiance of 3 milliwatt/cm2 (5.4 J/cm2)
(39). Clinical studies have shown that the progression of keratoconus is effectively stopped (40), and the biomechanical
strength of a keratoconus cornea is significantly increased by
70 300% (41, 42). In the anterior stroma, average collagen fibril
diameter in the treated cornea is significantly increased by
12.2%, and in the posterior stroma, it is increased by 4.6% (43).
Currently, laboratory studies are mainly focused on documenting biomechanical effects (44), thermomechanical effects (45),
morphological changes (46), effects on keratocytes (47), localization of cross-linking (48), and effects on collagenase resistance (49, 50). However, the chemical mechanisms of collagen
and PG interactions catalyzed by riboflavin UVA treatment
during corneal cross-linking have not been elucidated except in
recent work demonstrating that singlet oxygen and carbonyl
groups are required (51). Some additional mechanisms are
reported here.

Collagen and Proteoglycans in Corneal Cross-linking

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Protein transfer buffer was prepared following instructions of

BioTrans: pH 8.4, 48 mM Tris base, 39 mM glycine hydrochloride, and 1.3 mM SDS in 20% methanol. Voltage was set at 150 V.
Transfer time was 45 min. Subsequently, chromogenic immunodetection was performed following the kit protocol for small
membranes (Invitrogen). Collagen type I and core proteins
were identified using anti-collagen type I antibody (ab34710),
anti-keratocan antibody (Abnova), anti-lumican antibody
(AF2846), anti-mimecan antibody (AF2949), and anti-decorin
antibody (AF143), respectively. The specificity of each of these
four PG core protein antibodies was confirmed by Western
blotting of electrophoretic gels of all four core proteins (supplemental Fig. S1).
Analysis of the Interaction of GAGs and Collagen Type I with
Mass SpectrometryWhen gel electrophoresis was finished,
the areas containing cross-linked collagen (the area from 150
kDa up to the base of the gel sample well) and KS/CS (the area
from 100 kDa down to the bottom of the gel) were each cut off
and eluted from unstained SDS gels with electrophoretic elution (model 422 Electro-Eluter, Bio-Rad). Eluted molecules
were centrifuged through an Ultra free-MC centrifugal filter
unit (5,000 NMWL; Millipore) to remove buffer salts. For analysis of KS-sulfated disaccharides, the retentates were recovered
in 100 l of 0.1 M ammonium acetate buffer (pH 6.0) containing
0.1 milliunits/l keratanase II and digested for 24 h at 37 C. For
analysis of CS-sulfated disaccharides, the retentates instead
were recovered in 100 l of 50 mM ammonium acetate buffer
(pH 8.0) containing 1 milliunit/l chondroitinase ABC and
digested for 24 h at 37 C. The enzymes were then inactivated at
100 C for 5 min. Digest solutions (10 l) of each set were
diluted by adding 70 l of MeOH, 5 l of 5 mM ammonium
acetate buffer (pH 7.5), 5 l of 2 mM (NH4)2SO4, and 10 l of
water. The mixtures were centrifuged at 3,800 rcf for 30 min at
4 C (Ultra free-MC centrifugal filter unit, 5,000 NMWL; Millipore) with subsequent analysis of the retentates by electrospray ionization-MS/MS (55, 56). Mass spectra were obtained
using an electrospray ionization source on a quadrupole ion
trap instrument (Bruker Daltonics Esquire 3000). Parameters
used for analysis of all internal standards and corneal stromal
tissue digests were as follows: spray voltage, 3.5 kV; dry gas
(nitrogen), flow, 5.0 liters/min; drying temperature, 180 C. The
mass range scanned was m/z 50 1000. Data acquisition software used was Bruker Daltonics Data Analysis version 3.0.
Evaluation of Collagen Cross-linking in Whole CorneasAntibodies to collagen type I and to each of the PG core proteins
were used to assess the cross-linking of collagen and PG core
proteins. Cleavage of collagen chains and non-collagen proteins at methionine residues with cyanogen bromide (CNBr)
was performed in 70% formic acid under argon covered at room
temperature as described previously (57). Samples were treated
with N-glycanase or O-glycanase according to the manufacturers protocols (ProZyme), to remove intact chains of KS or
CS/DS, respectively. Briefly, 2 l of N-glycanase or O-glycanase
was added to the reaction mixture (50 l) and incubated for
24 h at 37 C. Other samples were digested with keratanase II
(100 milliunits/ml in 0.1 M ammonium acetate buffer, pH 6.0)
or with chondroitinase ABC (1000 milliunits/ml in 50 mM
ammonium acetate buffer, pH 8.0) for 24 h at 37 C, respectively
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for 30 min at a distance of 5 cm from the light source (UV-X

Radiation System for Corneal Cross-linking CXL; IROC Medical, Zurich, Switzerland). The volume of reaction solution was
20 l in 0.5-ml plastic centrifugal tubes. The sample solutions
were irradiated with the UVA shining down directly onto the
surface of the solutions, mixed two times by vortexing at
10-min intervals while it was being irradiated.
Cross-linking Treatment on Intact Whole Corneas ex Vivo
The cross-linking procedure was performed as described previously (51). In brief, the bovine corneal epithelium was
mechanically removed using a blunt knife, and riboflavin 0.1%
(w/v) solution (RF PBS) was applied beginning 30 min before
irradiation and continuing every 5 min during irradiation. The
corneas were irradiated with UVA of 370 nm for 30 min at a
distance of 5 cm from the light source.
Guanidine HCl Extraction of Corneal TissueThe de-epithelialized bovine corneas (0.6 g wet weight), treated or
untreated with RFUVA, were frozen by liquid nitrogen, pulverized, homogenized further in 4 M guanidine HCl (GHCl) containing protease inhibitors (53), and then incubated for 24 h at
0 4 C with gentle agitation. The tissue residue was removed
by centrifugation at 10,000 g for 30 min, and the supernatant
was retained as the extract. The tissue residue pellet was re-extracted for a second 24 h with fresh 4 M GHCl solution. The two
extracts were combined together and then applied to an Amicon Ultra centrifugal filter (regenerated cellulose, 3,000 Mr cutoff; Millipore), centrifuged to desalt, and concentrated to onefifth of the original volume. The retentates that did not pass
through the filter were used for collagen cross-linking evaluation without any further processing.
Pepsin Extraction of Corneal TissueFor pepsin extraction,
the frozen, pulverized, and homogenized corneal tissue (0.6 g
wet weight) was extracted for 48 h in 20 volumes of 0.5 M acetic
acid with 10 mg/ml pepsin (800 2,500 units/mg, EC 3.4.23.1;
Sigma) with gentle stirring movement at 0 4 C (54). The tissue residue was removed by centrifugation at 10,000 g for 30
min. The supernatant was collected and neutralized by the
addition of NaOH and then desalted and concentrated as
described above. These retentates were used for collagen crosslinking evaluation.
SDS-PAGEFor analysis of collagen and PG cross-linking, 5
l of NuPAGE LDS sample buffer (Invitrogen) and 2 l of
NUPAGE reducing agent (Invitrogen) were added into each
sample solution, heated at 70 C for 10 min, and then loaded
onto NuPAGE Novex BisTris 4 12% gels (8 cm 8 cm 1.5
mm precast gel; Invitrogen) and subjected to electrophoresis
(100 mA/gel for 60 min) under reducing conditions. After electrophoresis, the gels were stained with 0.1% (w/v) Coomassie
Brilliant Blue R-250.
Western Blot Analysis of Collagen Interaction with Proteoglycan Core ProteinsFor detection of possible cross-linking
between collagen and proteoglycan core proteins, samples were
loaded onto NuPAGE Novex Tris acetate 3 8% polyacrylamide gels (8 cm 8 cm 1.5 mm precast gel; Invitrogen) and
subjected to electrophoresis (40 mA/gel for 60 min) under
reducing conditions. Following electrophoresis, proteins were
transferred to nitrocellulose (Fisher) by electroblotting (BioTrans (Ann Arbor, MI) semidry electrophoretic transfer unit).

Collagen and Proteoglycans in Corneal Cross-linking

(56) to depolymerize chains of KS or CS/DS while leaving their

linkage region sugars still attached to the core protein. Enzymatic digestions were terminated by heating for 10 min in boiling water and then lyophilized. The same proportions of each
digested sample were subjected to electrophoresis on 4 12%
SDS gels and then to Western blot analysis, as described above.

RESULTS
Establishment of Cross-linking Model SystemThree purified collagens, type I, type III, and type IV, were chosen to assay
the reactivity of collagen cross-linking under the clinical conditions of treatment of keratoconus patients with UVA and riboflavin. The reaction was monitored by SDS-PAGE. The results
showed that collagen types I, III, and IV each display a banding
pattern distinct from the other two. For collagen type I, RFUVA
causes (a) 1, 2, and chains to almost disappear; (b) chains
to increase slightly in intensity; and (c) protein staining at the
base of the well to increase (Fig. 1, lane 3). For collagen types III
and IV, similar results were observed, as shown for the RFUVA
treatment in Fig. 1, lane 5 (Type III) and lane 7 (Type IV). These
results suggest that collagen cross-linking definitely happens
when irradiating with UVA in the presence of riboflavin. The
anterior corneal stroma is the location of the major effects of
riboflavin UVA treatment, and the stromal fibrils are primarily composed of collagen type I. This collagen type therefore
was chosen for the following experiments. Based on the initial

13014 JOURNAL OF BIOLOGICAL CHEMISTRY

experiment shown in Fig. 1, we used the following standard

reaction conditions in the rest of this study (unless stated otherwise): collagen type I (1 g/l) in 0.1 M PBS (pH 7.2), 0.1%
riboflavin in 0.1 M PBS (pH 7.2), and UVA at 370 nm for 30 min
at a distance of 5 cm from the light source. This reaction model
system was in a 0.5-ml plastic centrifuge tube and mixed two
times by vortexing at 10-min intervals while it was being irradiated from directly above the sample solution.
Core Protein Cross-linkingUnder experimental conditions
described above for Fig. 1, core proteins keratocan, lumican,
mimecan, and decorin (all at a concentration of 0.2 g/l) each
migrate as a monomer band. However, after exposure to
RFUVA, each such monomer band virtually disappears (Fig. 2).
For keratocan, lumican, and mimecan, RFUVA does not cause a
distinct higher molecular mass band to form but rather causes
formation of polymers of a very wide range of molecular mass
values from 100/150 kDa up to the bottom of the gel sample
well (Fig. 2, lanes 3, 5, and 7). In contrast, in the case of decorin,
RFUVA causes an entirely new band to form of approximately
twice the size of the monomer band (Fig. 2, lane 9).
Interaction of Collagen and Core ProteinsTo detect possible collagen interactions with core proteins during RFUVA,
collagen type I in solution, in the presence or absence of PG
core proteins, was irradiated by UVA in the presence of RF. The
relative intensity profiles of individual bands of SDS gels (Figs. 3
and 4) are summarized in Table 1 as a percentage of the total
protein in each scanned sample. When collagen type I in solution (Fig. 3, lane 2) was exposed to RFUVA (Fig. 3, lane 3), the
relative intensity of collagen chains and chains significantly
decreased, and the band at the very top of the gel increased.
However, when the same reaction was performed in the presence of keratocan (Fig. 3, lane 5) or lumican (Fig. 3, lane 7), the
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FIGURE 1. SDS-PAGE patterns of cross-linking collagens. Collagen types I

(lane 2), III (lane 4), and IV (lane 6) each displayed a banding pattern distinct
from the other two. RFUVA causes collagen type I (lane 3) and chains to
almost disappear, chains to increase slightly in intensity, and protein staining at the base of the gel well to increase. A similar pattern of band disappearance is seen also with type III collagen (lane 5) and type IV collagen (lane 7),
with increased staining at the top of the gel (base of the sample well). Lane 1,
molecular mass standards. Gels were stained with Coomassie Blue.

FIGURE 2. SDS-PAGE patterns of cross-linking core proteins. RFUVA

caused the monomer band of each PG to virtually disappear. For keratocan,
lumican, and mimecan, RFUVA PG did not cause a higher Mr band to form,
instead forming new higher Mr polymers of a wide range of sizes from 100/
150 kDa to the base of the gel well (lanes 3, 5, and 7). For decorin, RFUVA
caused an entirely new band to form of approximately twice the size of the
monomer band (lane 9). Gels were stained with Coomassie Blue.

Collagen and Proteoglycans in Corneal Cross-linking

FIGURE 4. Interaction of core protein decorin with type I collagen. In the

presence of RFUVA (lane 7), decorin did not interfere with collagen crosslinking, as evidenced by the virtual disappearance of and chains, the
increased density of chains, and the same increased density of staining in
the upper region of the gel as in the absence of decorin (lane 3). Gels were
stained with Coomassie Blue.

relative intensity of collagen chains and chains did not

decrease as significantly as in their absence, suggesting that
keratocan and lumican strongly inhibit the effect of RFUVA. In
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FIGURE 3. Interaction of core proteins keratocan, lumican, and mimecan

with type I collagen. Presence of keratocan or lumican prevented RFUVA from
causing diminished staining of chains and chains of collagen (lanes 5 and 7). In
contrast, mimecan did not interfere with collagen cross-linking, thus allowing
chains and chains to be cross-linked to form high Mr polymers near chains in
size and toward the top range of the gel, and staining intensity increased sharply
at the base of the sample well (lane 9). Gels were stained with Coomassie Blue.

other words, keratocan and lumican both appear to inhibit collagen cross-linking with other collagen molecules. However, in
contrast to keratocan and lumican, in the presence of mimecan,
the intensity of collagen and chains significantly decreased
after RFUVA, as if mimecan were not present (Fig. 3, compare
lane 3 versus lane 9), indicating that mimecan does not interfere
with collagen cross-linking (Fig. 3, compare lane 3 with lane 9),
resulting in 40% decrease in the relative intensities of chains
and chains, whereas the relative intensity of chains (Fig. 3,
lane 3 versus lane 9) was increased 20%. Similarly to mimecan, decorin does not interfere with collagen cross-linking in
the presence of RFUVA (Fig. 4, lane 3 versus lane 7), suggesting
that neither mimecan nor decorin interferes with RFUVA collagen cross-linking.
Western Blot Analysis of Collagen Interaction with Core
ProteinsIn the SDS-PAGE experiments, the core protein-tocollagen ratio was 1:5. These same ratios were used in all Western blot experiments. In case of keratocan (Fig. 5A), keratocan
partly inhibits RFUVA cross-linking of collagen I (lane 2 versus
lane 4), yet keratocan does not appear to become bound to
collagen I by RFUVA (lane 5 versus lane 7 versus lane 8). Interestingly, if RFUVA is applied to lumican in the presence of
collagen I (Fig. 5B, lanes 4 and 8), lumican displays a pattern of
interaction with collagen similar to that of keratocan in that
RFUVA cross-linking of collagen I is partly inhibited (Fig. 5B,
lane 2 versus lane 4). Thus, keratocan and lumican interact with
collagen I and respond to RFUVA in ways that correspond
closely with one another. In sharp contrast with that pattern,
mimecan does not prevent RFUVA from causing collagen I to
undergo cross-linking (Fig. 5C, lane 2 versus lane 4). Conversely, however, the presence of collagen does affect the crosslinking response of mimecan to RFUVA (Fig. 5C, lane 7 versus
lane 8). Thus, in the absence of collagen (Fig. 5C, lane 7), mimecan in the presence of RFUVA forms higher molecular weight
regions, with an especially strong one at the bottom of the sample well. However, in the presence of collagen I (Fig. 5C, lane 8),
mimecan is found in two higher molecular weight regions,
especially in the upper region of the gel, including in bands in
the same position as chains of collagen (arrowhead). In comparison with mimecan, the responses of decorin to RFUVA are
even more dramatic in the new banding patterns created when
collagen is also present, especially in the appearance not only of
the apparent dimer form seen in Fig. 4 (lane 4 versus lanes 5 and
7) but also of three even higher molecular weight forms (Fig.
5D, lane 7). The presence of collagen and RFUVA causes
decorin to migrate even less as its single monomer band, leaves
the dimer band approximately the same, but instead, causes
two of its four higher molecular weight polymers to display
greatly diminished staining intensity, and causes a new group of
bands to form near the top of the gel co-migrating with the
chains of collagen (arrowhead) (Fig. 5D, lane 8). Thus, although
mimecan and decorin do not inhibit RFUVA-induced crosslinking of collagen I with itself as do keratocan and lumican,
both mimecan and decorin do respond significantly to RFUVA
and to the simultaneous presence of collagen I in ways that
correspond closely with one another and contrast greatly with
the pattern shown by keratocan and lumican. Thus, the pattern
of interaction of keratocan and lumican with collagen appears

Collagen and Proteoglycans in Corneal Cross-linking

TABLE 1
Scanning density analysis of SDS-PAGE bands using Image Quantity Software TL (band intensity, percentage of total detected protein stain in
each sample, n 3)
The density of each band is compared with the integrated total of all the bands (and diffuse staining) in that individual sample. For any individual sample, the sum of staining
from the 1, 2, , and bands will be less than 100%; the remainder represents the diffuse staining in other Mr regions of that sample.
Collagen I
Collagen I RFUVA
Collagen I KER RFUVA
Collagen I LUM RFUVA
Collagen I MIM RFUVA
Collagen I DEC RFUVA

10.75 0.39
5.89 0.41
7.71 0.16
7.58 0.19
3.22 0.22
2.25 0.28

24.37 0.43
10.99 0.39
17.63 0.29
19.13 0.41
7.28 0.20
5.11 0.22

27.36 0.60
15.34 0.61
32.56 0.76
28.21 0.23
11.76 0.58
15.11 0.35

23.06 0.16
44.47 1.07
42.34 0.79
34.83 0.78
54.07 0.61
59.86 1.69

FIGURE 5. Western blot of proteoglycan core protein interaction with type I

collagen induced by RFUVA. Western blot is shown. A, the presence of keratocan prevented RFUVA from causing collagen type I to undergo cross-linking (lane
4 versus lane 2). The intensities of and chains (lane 4) were almost the same as
the corresponding bands in lane 1. B, lumican inhibited collagen cross-linking
(lane 4 versus lane 2). C, mimecan did not inhibit RFUVA-induced collagen crosslinking (lane 2 versus lane 4), whereas mimecan formed a new band near chains
and in the top range of the gel (lane 8 versus lane 7). D, decorin did not inhibit
RFUVA-induced collagen cross-linking (lane 2 versus lane 4), whereas decorin
formed a new band near chains (lane 8 versus lane 7). Decorin dimers and
oligomers induced by RFUVA were observed (lane 7).

to be very different from the pattern of interaction of mimecan

and decorin with collagen.
Fig. 6 shows that keratocan and lumican not only interfere
with RFUVA cross-linking of collagen but also interfere with
RFUVA cross-linking of both mimecan and decorin. In the
absence of keratocan and lumican, but even in the simultaneous
presence of both mimecan and decorin, collagen undergoes
cross-linking ( and chains almost disappear) (Fig. 6A, lane 1
versus lanes 2 and 3). Decorin undergoes cross-linking in the
presence (Fig. 6, lane 5) or absence (Fig. 6, lane 4) of collagen I,
but in its presence, decorin also occurs in a band in the same
position as collagen I chains (arrowhead), suggesting that
some decorin is bound to the chain, three-chain polymer
form of collagen I. Similar to decorin, mimecan also occurs in a
band in the same position as collagen I chains (arrowheads),

13016 JOURNAL OF BIOLOGICAL CHEMISTRY

suggesting that some mimecan is bound to the polymer form

of collagen I (Fig. 6A, lane 7). In the presence of keratocan, as
well as mimecan and decorin, collagen I cross-linking by
RFUVA is partly inhibited (Fig. 6B, lane 3 versus lane 2). Moreover, for both decorin and mimecan, RFUVA-induced crosslinking in the presence or absence of collagen I is inhibited by
the presence of keratocan (Fig. 6B, lane 4 versus lane 5 and lane
6 versus lane 7 in comparison with those same lanes in Fig. 6A).
Thus, the presence of mimecan or decorin in the same position
as collagen I chains is not seen in Fig. 6B, lanes 5 and 7 (arrowheads), in comparison with those same lanes in Fig. 6A (arrowheads), suggesting that keratocan can prevent both decorin and
mimecan from cross-linking with themselves and also can prevent their interaction with collagen. Importantly, lumican displays the same inhibitory ability as keratocan (Fig. 6C, lane 4
versus lane 5 and lane 6 versus lane 7, arrowheads).
Interaction of Collagen with KS and CSTo determine if KS
or CS might be involved in RFUVA-induced collagen crosslinking, mass spectrometry was used to analyze the SDS gel
samples. As can be seen from Fig. 7, AD, a strong signal intensity detected at m/z 462.0 corresponds to KS-monosulfated
disaccharide released by keratanase II from gel slices (area from
100 kDa down to the bottom of the gel), whereas no signal at
m/z 462.0 (Fig. 7, EH) was observed with the corresponding
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FIGURE 6. Effect of keratocan or lumican on RFUVA cross-linking of collagen in the simultaneous presence of both mimecan and decorin. Western
blot is shown. A, in the simultaneous presence of both mimecan and decorin,
collagen cross-linking in response to RFUVA is not inhibited, and both mimecan and decorin each undergo their respective patterns of cross-linking. B, in
contrast, in the presence of keratocan, as well as mimecan and decorin, collagen I cross-linking by RFUVA was partly inhibited (lane 3 versus lane 2). In
addition, RFUVA-induced cross-linking of both decorin and mimecan in the
presence or absence of collagen I are inhibited (lane 4 versus lane 5 and lane 6
versus lane 7). C, similarly, in the presence of lumican, collagen I cross-linking
by RFUVA was partly inhibited (lane 3 versus lane 2). In addition, RFUVA-induced cross-linking of both decorin and mimecan in the presence or absence
of collagen I is inhibited (lane 4 versus lane 5 and lane 6 versus lane 7).

Collagen and Proteoglycans in Corneal Cross-linking

gel slice area from 150 kDa up to the base of the gel sample well,
suggesting an absence of higher molecular polymer forms
involving KS. As can be seen from Fig. 8, AD, in the case of
CS, a strong signal intensity was detected at m/z 458.0 for CSmonosulfated disaccharide (4S/6S) from gel slices (area from
100 kDa down to the bottom of the gel), whereas no signal at
m/z 458.0 (Fig. 8, EH) was observed with the corresponding
gel slice area from 150 kDa up to the base of the gel sample well,
suggesting an absence of higher molecular polymer forms
involving CS. These results indicate that neither KS nor CS is
involved in RFUVA-induced corneal cross-linking.
Collagen and Core Proteins Cross-linking in Whole Cornea ex
VivoAntibodies to collagen type I and to each of the PG core
proteins were used to assay for possible cross-linking between
collagen and PG core proteins in native cornea with RFUVA.
Fig. 9A shows the electrophoretic migration pattern of collagen
type I from whole cornea treated by RFUVA and then extracted
with either GHCl or with pepsin; the latter, by cleaving only
APRIL 15, 2011 VOLUME 286 NUMBER 15

their telopeptide domains, leaves the triple-chain helical

domains of collagen molecules intact. Compared with control
samples (Fig. 9A, lanes 1 and 3), the staining intensities of 1
chains, 2 chains, and chains in the presence of RFUVA were
decreased, whereas the staining intensities of chains were
significantly increased (Fig. 9A, lanes 2 and 4), thus indicating
that collagen molecules were undergoing cross-linking even in
the presence of the normal mixture of all four natively GAGglycosylated PGs ex vivo.
Fig. 9C indicates that, in the absence of RFUVA treatment,
incubation of samples of purified collagen with CNBr causes
collagen type I to be cleaved at methionine residues, generating
the collagen type I-specific pattern of lower molecular size peptides expected (Fig. 9C, lane 3). However, CNBr incubation of
collagen type I that was first treated with RFUVA demonstrates
that all domains of the collagen type I molecule are involved in
cross-linking into higher Mr molecules, as evidenced by the
absence of low Mr collagen peptides and, instead, the appearJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 7. Analysis to detect possible interaction of KS and collagen type I, using mass spectrometry. Peaks at m/z 462.0 (AD) correspond to the
characteristic KS-monosulfated disaccharide released by keratanase II from gel slices (area from 100 kDa down to the bottom of the SDS gel). There are no peaks
at m/z 462.0 (EH) observed with gel slices in the area from 150 kDa up to the base of the gel sample well.

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FIGURE 8. Analysis to detect possible interaction of CS and collagen type I, using mass spectrometry. Peaks at m/z 458.0 (AD) correspond to the
characteristic CS monosulfated disaccharide (4S/6S) released by chondroitinase ABC from gel slices (area from 100 kDa down to the bottom of the SDS gel).
There are no peaks at m/z 458.0 (EH) observed with gel slices in the area from 150 kDa to the base of the gel sample well.

ance of a broad range of higher Mr molecules (Fig. 9C, lane 4).

Fig. 9B indicates that regardless of whether whole corneal samples were extracted from corneas using the GHCl protocol
standard for extracting PGs (53) or the pepsin protocol standard
for extracting the triple-helical domains of collagens (54), when
such samples are derived from corneas treated with RFUVA,
chemical cleavage with CNBr reveals a range of collagen peptides
that migrate in a high MW range (Fig. 9B, lanes 2 and 4), just as do
the CNBr peptides from collagen type I (Fig. 9C, lane 4).
Fig. 10, AD, displays the profiles of each individual PG core
protein extracted with GHCl from whole corneas with or without exposure to RFUVA and with or without treatments to
remove GAG chains from their core proteins. Exposure of
whole corneas to RFUVA causes some proportions of all proteoglycan core proteins to migrate in the same band positions
as collagen type I chains (Fig. 9), shown here as arrowheads,

13018 JOURNAL OF BIOLOGICAL CHEMISTRY

regardless of whether glycosaminoglycan chains of KS were

intact (untreated lanes) or were removed with N-glycanase or
with keratanase II, consistent with PGs in solutions containing
type I collagen also co-migrating with collagen chains (Figs. 5
and 6). Moreover, in corneas exposed to RFUVA, even after
cleavage of core proteins to small peptides with CNBr, a range
of reactivity was detected in the high Mr region (arrowheads),
whereas none of the core proteins were detected in that high Mr
region in samples from control corneas treated with CNBr.
Thus, these results suggest that RFUVA causes some crosslinking to occur between PG core proteins and collagen I in
whole cornea, ex vivo.

DISCUSSION
Corneal cross-linking by RFUVA represents a new method
for treatment of progressive keratoconus in Europe (40) and
VOLUME 286 NUMBER 15 APRIL 15, 2011

Collagen and Proteoglycans in Corneal Cross-linking

currently is under clinical study in the United States. Here, we

have used both an in vitro and an ex vivo model reaction system
to investigate the effects of RFUVA specifically on the interaction of collagen and PGs, an interaction very likely to occur
during corneal cross-linking. Our data demonstrate that irradiation with UVA in the presence of riboflavin cross-links collagen and chains into high molecular weight polymers. In
addition, RFUVA can induce purified, non-glycosylated PG
core proteins themselves to cross-link to form higher Mr polymers. When both collagen and PG core proteins are present
together in solution, both of the corneal PG core proteins, keratocan and lumican, inhibit collagen cross-linking. However, in
sharp contrast, two other corneal PG core proteins, mimican
and decorin, not only do not interfere with collagen cross-linking, they in fact form cross-links with collagen to form new
bands in high molecular weight regions of electrophoretic gels.
When studied in isolation from their normal PG core proteins,
the corneal GAGs, KS and CS, are not induced by RFUVA to
cross-link among themselves or to form cross-links with collagen. Thus, based on interactions between these purified proteins in solution in vitro, RFUVA mainly appears to involve
cross-linking of collagen molecules with themselves, PG core
proteins with themselves, and collagen molecules with the two
specific PG core proteins, mimecan and decorin. These results
suggest that in the absence of RFUVA, collagen interacts noncovalently with keratocan and lumican very differently from the
way interacts with mimecan and decorin. Data from ex vivo
exposure of whole corneas to RFUVA lead to these same conclusions. This reveals two sharply distinct styles of molecular
interactions between collagen and PGs.
Type I collagen, the most abundant collagen in cornea (11), is
composed of three polypeptide chains and two types of single
chains, 1 and 2, both of which are accessible to polymerization through intra- and intermolecular bonds (58). Dimers of
chains are called -components (composed of 1 2 or 1 1
chains). Trimers of three chains are called -components. In
this study, RFUVA mainly causes 1, 2, 11, and 12 chains to
APRIL 15, 2011 VOLUME 286 NUMBER 15

FIGURE 10. Patterns of cross-linking collagens and PG core proteins in

whole cornea ex vivo. Western blot is shown. A, interaction of keratocan and
collagen type I from corneas (with or without RFUVA treatment) treated by
CNBr (lanes 4 and 5), N-glycanase (NGase) (lanes 6 and 7), and keratanase II
(KSase) (lanes 8 and 9). B, interaction of lumican and collagen type I from
corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5),
N-glycanase (lanes 6 and 7), and keratanase II (lanes 8 and 9). C, interaction of
mimecan and collagen type I from corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5), N-glycanase (lanes 6 and 7), and keratanase II (lanes 8 and 9). D, interaction of decorin and collagen type I from
corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5),
O-glycanase (OGase) (lanes 6 and 7), and chondroitinase ABC (ABCase) (lanes 8
and 9). All four PG core proteins in the cornea show some molecules that
co-migrate in the same region as collagen chains (arrowheads).

cross-link into macromolecules at least as large as -components, and larger (Fig. 1, lane 3) and thus to disappear from their
normal and chain positions on electrophoretic gels. Intermicrofibrillar cross-links significantly affect the mechanical
properties of the corneal collagen tissue (59). The strength of
the collagen fibers depends on the formation of covalent crosslinks between the telopeptide terminal domains and adjacent
helical domains of collagen molecules (60). Riboflavin-sensitized photodynamic modification of collagen causes formation
of cross-linked molecules, in which an energy transfer between
the excited riboflavin and molecular oxygen produces singlet
oxygen that then interacts with the collagen fibrils in an oxidation reaction to form physical, covalent cross-links (61). Results
presented here provide new and direct evidence that covalent
cross-links between collagen molecules, especially involving 1,
2, 11, and 12 chains, are initiated by RFUVA and are consistent with earlier data indicating that singlet oxygen plays a primary role in corneal cross-linking (51).
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FIGURE 9. Effect of RFUVA on collagen type I cross-linking in whole cornea ex vivo versus collagen type I in solution. A, patterns of collagen type I
from whole cornea treated by RFUVA. Lanes 1 and 2, GHCl extraction; lanes 3
and 4, pepsin extraction. B, patterns of collagen type I from whole cornea
treated by RFUVA and then digested with CNBr. Lanes 1 and 2, GHCl extraction; lanes 3 and 4, pepsin extraction. C, patterns of electrophoretic migration
of purified collagen type I in solution, treated by RFUVA, and digested with
CNBr. All peptide domains of collagen type I participate in cross-linking to
form a range of high Mr molecules. A and B, Western blot; C, Coomassie Blue
staining.

Collagen and Proteoglycans in Corneal Cross-linking

13020 JOURNAL OF BIOLOGICAL CHEMISTRY

and purified, non-glycosylated keratocan or lumican in solution

in vitro. The mechanism by which keratocan and lumican so
drastically inhibit collagen cross-linking with itself by RFUVA
therefore suggests a mechanism of steric blockage that does not
put collagen molecules in reactive juxtaposition with either
non-glycosylated PG core protein. In contrast, although both
mimecan and decorin fail to inhibit cross-linking of collagen
with itself, they each produce a new band at 300 kDa (Fig. 5, C
(lane 8) and D (lane 8)) that appears to represent co-polymers of
collagen-PG core protein that have not been described
previously.
Binding antibodies to collagen type I and to each of the PG
core proteins (in both glycosylated and non-glycosylated
forms) allowed an independent way to assess degrees of crosslinking between collagen and PG core proteins. In the absence
of core proteins, collagen type I in solution in vitro forms higher
molecular weight polymers, especially leading both to the disappearance of most collagen chains and chains and to some
enhancement of triple-chain polymers ( polymers; 300 kDa)
(Fig. 5, lane 2 in each gel). In contrast, the simultaneous presence of purified non-glycosylated keratocan or lumican in solution in vitro virtually eliminated such collagen cross-linking
with itself (Fig. 5, A and B, lanes 4). In those same solutions,
keratocan and lumican do not undergo cross-linking in the
absence of RFUVA (lanes 5), but they do appear in a range of
higher molecular weight polymers in response to RFUVA, in
the absence (lanes 7) or presence of collagen type I (lanes 8).
Thus, no purified, non-glycosylated keratocan or lumican
appears to be bound to collagen during RFUVA in solution in
vitro, although those core proteins form cross-links with themselves. In sharp contrast, although neither mimecan nor
decorin inhibit the ability of collagen molecules to cross-link
with themselves in response to RFUVA, both of those purified,
non-glycosylated core proteins in the simultaneous presence of
collagen form higher molecular weight polymer forms with
themselves and even larger ones in the presence of collagen
(Fig. 5, C and D, lanes 8) than in its absence (lanes 7), suggesting
that they cross-link to collagen type I.
When collagen type I was exposed to RFUVA in solution in
vitro in the presence of both mimecan and decorin, together
with either keratocan or mimecan, (all as purified, non-glycosylated proteins), collagen cross-linking with itself was inhibited (as expected, because of the presence of keratocan and
lumican) (Fig. 6, A versus B or A versus C, lanes 3) but not as
much as if mimecan and decorin had been absent. In the
absence of keratocan or mimecan, both mimecan and decorin
epitopes are detected in macromolecules (equal to and 300 kDa)
in the presence of collagen (Fig. 6A, lanes 5 and 7). However, if
either keratocan (gel B) or lumican (gel C) is present in that
mixture in solution in vitro, the amount of mimecan and
decorin detected in the very high molecular size molecules is
reduced although not eliminated. Thus, in a complete cornea in
vivo with abundant collagen type I and all four PG core proteins
in glycosylated form, some collagen molecules would undergo
cross-linking with mimecan and decorin and might contribute
substantively to tissue strength, even if such collagen-PG core
protein cross-links did not represent a large proportion of the
molar reaction yield of product.
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Data presented here indicate that collagen types I, III, and IV,
in isolation from one another and from PGs, undergo crosslinking reactions that all share some characteristics (Fig. 1).
Both and chains virtually disappear, presumably because of
their covalent cross-linking into higher molecular weight polymers that either migrate as chains (300 kDa) to some extent
or as even larger aggregates, some of which barely enter the gel
at the bottom of the sample well (increased density at top of lane
3 in Fig. 1). In response to RFUVA, normal corneal PG core
proteins, keratocan, lumican, mimecan, and decorin (but nonglycosylated (i.e. lacking their normal GAG chains)), similarly
appear to undergo cross-linking reactions with themselves that
cause their virtual disappearance as monomer molecules (Fig.
2). One PG core protein, decorin, appears to form distinct
dimer molecules (80 kDa) (Fig. 2, lane 9). When collagen type
I is subjected to RFUVA in the simultaneous presence of these
PG core proteins (Fig. 3), collagen cross-linking is drastically
inhibited if either keratocan or lumican is present (Fig. 3, lanes
5 and 7). In sharp contrast, however, the presence of mimecan
does not inhibit collagen cross-linking (Fig. 3, lane 9). In those
same solutions containing collagen type I, PG core proteins
keratocan and lumican appear to undergo almost normal crosslinking with themselves (as indicated by a diminution of their
monomer bands) as a result of RFUVA (Fig. 3, lanes 5 and 7), as
does mimecan (Fig. 3, lane 9). These results suggest that keratocan and lumican can block those sites on collagen type I that
normally allow collagen to cross-link with itself. However, even
when any one of those three PG core proteins is present,
RFUVA treatment still produces large molecular aggregates
that are present at the bottom of the sample wells (Fig. 3, lanes
5, 7, and 9), as it does in their absence (Fig. 3, lane 3), perhaps
representing some of the cross-linked collagen seen in Fig. 1.
Thus, none of the core proteins appears to produce molecular
aggregates with collagen type I, at least in the region in which
molecules can enter the gel (with the possible exception of
mimecan (Fig. 3, lane 9)). Decorin (Fig. 4), like mimecan, does
not inhibit the cross-linking of collagen type I (Fig. 4, lane 7) in
response to RFUVA; likewise, DEC undergoes its normal crosslinking with itself in response to RFUVA, forming a prominent
dimer molecule in the absence (lane 5) and presence (Fig. 4,
lane 7) of collagen type I. Also similar to mimecan, decorin
appears to form a new macromolecule (300 kDa) when
exposed to RFUVA in the presence of collagen type I (Fig. 4,
lane 7). Altogether, the results in Figs. 1 4 indicate that keratocan and lumican interact with collagen type I in solution very
differently than do mimecan and decorin.
In cornea and in other tissues, any of these core proteins may
exist either in forms lacking glycosylation or with glycosylation
consisting of GAG chains. Such glycosaminoglycan polysaccharides (which do not appear to participate in RFUVA crosslinking, as shown here in Figs. 79) may sterically hold PG core
proteins at sufficient distances from collagen molecules in adjacent fibrils as to prevent formation of cross-links between them
in response to RFUVA in vivo. However, such PG core proteins,
even when heavily glycosylated with GAG chains, are modeled
as binding with their leucine-rich domains directly to the side of
collagen fibrils (62, 63), physical proximity that apparently does
not facilitate RFUVA cross-linking, at least between collagen

Collagen and Proteoglycans in Corneal Cross-linking

APRIL 15, 2011 VOLUME 286 NUMBER 15

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Evidence presented in Fig. 8 suggests that the GAG components of native PG molecules do not themselves participate in
RFUVA cross-linking. The smallest molecules were categorized as those in a size range arbitrarily below 100 kDa (Figs. 8
(AD) and 9 (AD)), whereas the largest were in a size range of
150 kDa and above (to the bottom of the sample well). These
analyses probed for evidence of cross-linking to keratan sulfate
(Fig. 8, AH), chondroitin sulfate (Fig. 9, AH), or any protein,
but no such new indicator molecules were detected in response
to exposure to RFUVA, consistent with an apparent absence of
RFUVA causing visible cross-linking of corneal GAGs with
themselves or with collagen.
Data presented in Fig. 9 indicate that collagen type I, present
in the corneal stroma and examined here ex vivo, displays
RFUVA cross-linking in the presence of the normal mixture of
all four native PGs (i.e. PG core proteins with their GAG chains
attached). Although the presence of the purified, non-glycosylated core proteins, keratocan and lumican, suppresses collagen
cross-linking when both molecules are in solution in vitro (Figs.
1, 3, and 4), when those same molecules are present in the
polymerized gel of the corneal stroma and in their native, glycosylated form, cross-linking of these PG core proteins to collagen is detected (Fig. 10). Future work will be needed to determine if this difference in the ability of both keratocan and
lumican to form RFUVA cross-links with collagen type I arises
because the presence of KS chains affects the conformation of
these two core proteins as they bind to collagen (enhancing
their RFUVA cross-linking to collagen) or because the higher
densities of molecules in the native stroma bring collagen and
these two core proteins close enough together to undergo
RFUVA cross-linking. In contrast to the behavior of both keratocan and lumican (in solution in vitro versus in native glycosylated form ex vivo), both mimecan and decorin appear able to
form RFUVA cross-links with collagen in solution in vitro (Fig.
5, C (lane 8) and D (lane 8)) and also in their native glycosylated
form ex vivo (Fig. 10, C and D, lane 9). These results strikingly
demonstrate that, as revealed here by using RFUVA as a type of
molecular diagnostic probe, both keratocan and lumican interact with collagen type I very differently than do both mimecan
and decorin. Elucidating the exact nature of those two styles of
molecular interaction with collagen type I will be the subject of
future experiments.
Protocols involving treatment of corneas with RFUVA are
being used for an ever wider set of clinical conditions (40, 64,
65). It therefore is essential to elucidate as completely as possible the precise molecular effects of that treatment, not only on
the cellular populations exposed (66) but also on the component molecules of the extracellular matrix. That has been the
purpose of the experiments performed here. Many other interactions catalyzed by RFUVA in the cornea remain to be characterized in this same manner (e.g. possible interactions
between collagens type I and type V within individual collagen
fibrils and interactions between the collagen type IV of the epithelial basal lamina and laminin and the stromal proteoglycans). Using RFUVA reactivity as a molecular diagnostic probe
may therefore prove useful in formulating new hypotheses
about the native conformations of molecules in the extracellular matrix in general.

Collagen and Proteoglycans in Corneal Cross-linking

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