THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp.
18537–18544, June 11, 2010
Type III Collagen, a Fibril Network Modifier in Articular Cartilage*
Received for publication, February 9, 2010, and in revised form, April 12, 2010 Published, JBC Papers in Press, April 19, 2010, DOI 10.1074/jbc.M110.112904
Jiann-Jiu Wu1, Mary Ann Weis, Lammy S. Kim, and David R. Eyre
From the Department of Orthopedics and Sports Medicine, University of Washington, Seattle, Washington 98195
The collagen framework of hyaline cartilages, including artic-
ular cartilage, consists largely of type II collagen that matures
from a cross-linked heteropolymeric fibril template of types II,
IX, and XI collagens. In the articular cartilages of adult joints,
type III collagen makes an appearance in varying amounts
superimposed on the original collagen fibril network. In a study
to understand better the structural role of type III collagen in
cartilage, we find that type III collagen molecules with unproc-
essed N-propeptides are present in the extracellular matrix of
adult human and bovine articular cartilages as covalently cross-
linked polymers extensively cross-linked to type II collagen.
Cross-link analyses revealed that telopeptides from both N and
C termini of type III collagen were linked in the tissue to helical
cross-linking sites in type II collagen. Reciprocally, telopeptides
from type II collagen were recovered cross-linked to helical sites
in type III collagen. Cross-linked peptides were also identified in
which a trifunctional pyridinoline linked both an ␣1(II) and an
␣1(III) telopeptide to the ␣1(III) helix. This can only have arisen
from a cross-link between three different collagen molecules,
types II and III in register staggered by 4D from another type III
molecule. Type III collagen is known to be prominent at sites of
healing and repair in skin and other tissues. The present find-
ings emphasize the role of type III collagen, which is synthesized
in mature articular cartilage, as a covalent modifier that may add
cohesion to a weakened, existing collagen type II fibril network
as part of a chondrocyte healing response to matrix damage.
Fibrillar collagens are the most abundant vertebrate proteins.
They provide the extracellular framework and mechanical
strength of most animal tissues. There are seven collagens in
the fibrillar collagen family, types I, II, III, V, XI, XXIV, and
XXVII, encoded by 11 distinct genes (for review see Ref. 1).
Based on phylogenic analysis, fibrillar collagen genes can be
subdivided into three distinct groups or clades (1–5). A-clade
comprises ␣1(I), ␣1(II), ␣1(III), ␣2(I), and ␣2(V); B-clade is
␣1(V), ␣3(V), ␣1(XI), and ␣2(XI); and C-clade is ␣1(XXIV) and
␣1(XXVII). All fibrillar collagens are synthesized as procolla-
gen molecules consisting of a long uninterrupted triple-helical
domain (each ␣ chain contains about 1000 amino acid residues)
with globular extensions at both N and C termini and a minor
triple-helical domain in the removable N-propeptide (1, 6).
Collagen types I, II, and III are the main fibril-forming mol-
ecules in vertebrates. Type I collagen is widely expressed and
* This work was supported by National Institutes of Health Grants AR 37318
and AR 36794.
1
To whom correspondence should be addressed: 1959 NE Pacific St., HSB
BB1052, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax: 206-685-4700;
E-mail: wujj@u.washington.edu.
JUNE 11, 2010 • VOLUME 285 • NUMBER 24
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
prominent in skin, tendon, bone and ligaments, and many other
tissues but not in hyaline cartilages. The type I molecule is a
heterotrimer of two ␣1(I) chains and one ␣2(I) chain (6). Type
II collagen is restricted to cartilages, vitreous and intervertebral
disc, and is a homotrimer of ␣1(II) chains (7–9). Type III colla-
gen is also a homotrimer of ␣1(III) and appears to function as a
copolymer with type I collagen in many tissues, including skin,
tendon, ligament, vascular walls, periodontal ligament, and
synovial membranes and is most prominent in highly compli-
ant connective tissues (10 –17). As with types I and II collagens,
the strength of polymeric type III collagen depends on covalent
cross-links formed by the lysyl oxidase mechanism (18 –20). In
addition to cross-links between the type III collagen molecules
themselves, intertype cross-links also form to type I collagen,
for example, in aorta which is rich in both collagens I and III
(21). A small but significant amount of type III collagen
becomes deposited in articular cartilage of mature joints, where
it can be detected by immunofluorescence concentrated in the
matrix surrounding chondrocytes throughout the depth of the
tissue and particularly prominent in human osteoarthritic
joints (22–24).
The collagen framework of hyaline cartilage is a highly cross-
linked unique heteropolymer. In essence, the bulk type II colla-
gen is polymerized on a template of type XI collagen, and type
IX collagen covalently decorates the surface type II molecules of
the nascent fibrillar networks most prominently in young tissue
(25–28). All three collagen types, II, IX, and XI, are heavily
cross-linked in the same fibril through the lysyl oxidase-medi-
ated mechanism (29 –32). In a study to understand better the
structural role of type III collagen in cartilage, we have revealed
that pN-type III collagen molecules are present in the extracel-
lular matrix of adult human and bovine articular cartilages as
covalently cross-linked polymers extensively cross-linked to
the surface of type II collagen fibrils, suggesting a role in matrix
reinforcement and a healing response to tissue damage.
EXPERIMENTAL PROCEDURES
Preparation of Collagens—Human knee joints were obtained
from Northwest Tissue Services (Seattle, WA) from donors
aged 18 –75 with no obvious signs of osteoarthritis. Full thick-
ness articular cartilage was sliced from the femoral and tibia
chondyles and from an equivalent site in a 4-year-old cow
(bovine) knee. Minced tissue was extracted in 4 M guanidine
HCl, 0.05 M Tris-HCl, pH 7.4, containing protease inhibitors (2
mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl flu-
oride, and 5 mM 1,10-phenanthroline) at 4 °C for 48 h to remove
proteoglycans and other matrix proteins. The guanidine-insol-
uble tissue residue was then washed thoroughly with water and
freeze-dried. Cross-linked collagens were solubilized by digest-
JOURNAL OF BIOLOGICAL CHEMISTRY 18537
Type III Collagen in Cartilage
ing the washed residue with pepsin (1:10 w/w by dry weight) in
3% acetic acid (v/v) for 24 h at 4 °C. Different collagen fractions
were then precipitated from the acid solution at 0.7 M, 1.2 M and
2.0 M NaCl to separate types II/III, type XI, and type IX collag-
ens respectively (33, 34).
The 0.7 M NaCl precipitate from a pepsin digest of articular
cartilage, which contains type II and any type III collagen in the
solubilized pool, was dissolved in 4 M guanidine HCI, 0.05 M
Tris-HCl, pH 7.5, and resolved by molecular sieve chromatog-
raphy on an agarose A5m column (170 ⫻ 1.5 cm, 200 – 400
mesh, Bio-Rad), eluted with 2 M guanidine HCl, 0.05 M Tris-
HCl, pH 7.5.
CNBr Cleavage and Peptide Chromatography—The washed
residues after 4 M guanidine HCl extraction were minced and
digested with CNBr in 70% formic acid under N2 at room tem-
perature for 24 h on a shaker. The digests were diluted 15-fold
with water and freeze-dried (34). Collagen CNBr-derived pep-
tides were resolved by molecular sieve chromatography on an
agarose A1.5m column (170 ⫻ 1.5 cm, 200 – 400 mesh, Bio-
Rad), eluted with 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5.
Bacterial Collagenase Digestion and Peptide Chromato-
graphy—An aliquot of the washed guanidine-insoluble articu-
lar cartilage residue was suspended in 0.05 M Tris-HCl, 0.1 M
CaCl2 buffer, pH 7.5, containing 0.001% thimerosol at 10
mg/ml, heat denatured at 70 °C for 10 min, and digested with
bacterial collagenase (Sigma, type IA) at an enzyme:substrate
ratio of 1:100 (w/w) at 37 °C for 24 h. Resulting digests were
resolved on a BioGel P-10 molecular sieve column (100 ⫻ 1.5
cm) equilibrated with 10% acetic acid (v/v) at a flow rate of 11.4
ml/h collecting 5.7 ml/fraction. Collected fractions were mon-
itored for pyridinoline-specific fluorescence (excitation, 297
nm; emission, 395 nm) (34).
Peptides containing pyridinoline cross-links were further
purified by sequential DEAE ion-exchange and C8 reverse-
phase HPLC2 (29). Ion-exchange HPLC was performed on a
DEAE 5-PW column (75 ⫻ 7.5 mm; Bio-Rad), eluting with a
linear gradient of 0 – 0.2 M NaCl in 40 ml of 0.02 M Tris-HCl, pH
7.5, containing 10% (v/v) acetonitrile at 1 ml/min over 40 min.
Pooled DEAE fractions were dried and chromatographed by
reverse-phase HPLC on a C8 column (Brownlee Aquapore
RP-300, 4.6 mm ⫻ 25 cm) with a linear gradient of acetonitrile:
n-propyl alcohol (3:1, v/v) in aqueous 0.1% (v/v) trifluoroacetic
acid from 0 to 40% at 1 ml/min over 60 min.
Trypsin Digestion—The molecular sieve-purified type III col-
lagen preparation from pepsin-solubilized articular cartilage
was dissolved in 0.05 M Tris-HCl, 0.15 M NaCl, pH 8.0, at 5
mg/ml, heat-denatured at 60 °C for 10 min, and digested with
trypsin (Boehringer sequencing grade) at an enzyme:substrate
ratio of 1:200 (w/w) at 37 °C for 20 h. Tryptic peptides were
fractionated by immobilized metal ion affinity chromatography
(IMAC).
IMAC—One ml of HiTrap chelating HP Sepharose beads
(GE Healthcare) were packed into a column (1 ⫻2 cm). The
beads were charged with 3 ml of 0.5 M CuCl2. After washing
2 sie Blue staining on SDS-PAGE were digested in-gel by trypsin
The abbreviations used are: HPLC, high performance liquid chromatogra-
phy; IMAC, immobilized metal ion affinity chromatography; DTT, dithio-
threitol; mAb, monoclonal antibody; MS/MS, tandem mass spectrometry.
18538 JOURNAL OF BIOLOGICAL CHEMISTRY
with 10 ml of Milli Q H2O to remove unbound CuCl2, the col-
umn was equilibrated with 0.05 M Tris-HCl, pH 8.0, containing
0.15 M NaCl (IMAC sample buffer). Trypsin-digested collagen
was incubated with the copper-chelated beads at room temper-
ature for 1 h. The beads were washed with 10 ml of IMAC
sample buffer to remove unbound peptides. The bound pep-
tides were eluted from the column sequentially with 4 ml of 0.1
M sodium acetate, pH 6.35, then 4 ml of 0.1 M sodium acetate,
pH 4.6, and finally with 4 ml of 0.1 M sodium acetate, pH 4.6,
containing 0.2 M imidazole. Eluted peptides were resolved on
C8 reverse-phase HPLC and identified by N-terminal protein
sequence analysis and mass spectrometry.
Stromelysin-1 (MMP3) Extraction—Cartilage from a non-
fibrillated surface of a knee joint removed at replacement
surgery (59-year-old female) was used to study the extraction
of collagen type III by stromelysin-1 (MMP3) compared with
pepsin extraction. The washed guanidine-insoluble residue
was extracted with MMP3 (prostromelysin-1 (35) activated
by trypsin as described (36)) at an enzyme:substrate ratio
of 1:90 (w/w) in 0.05 M Tris-HCl, 0.2 M NaCl, 10 mM CaC12,
1 mM ZnC12, pH 7.5, at 37 °C. Two serial 24-h extractions
were carried out, removing the supernatant after 24 h, and
adding fresh enzyme for the second 24-h extraction. 1,10-
phenanthroline (10 mM) was added to each extract to stop
the reaction.
Gel Electrophoresis—Pepsin-solubilized collagens were re-
solved on 6% SDS-PAGE according to the method of Laemmli
(37) using a Bio-Rad mini protean 3 system. Delayed reduction
of disulfide bonds was performed by adding 10 ␮l of 0.5 M dithio-
threitol (DTT) in 10% glycerol (v/v) to each sample well after
15 min of electrophoresis at 150 V. Stromelysin extracts were
run on 10% SDS-PAGE.
Antisera and Western Blotting—Two mouse monoclonal anti-
bodies to human type III collagen were used. mAb 4G9 is specific
to a conformational epitope in the globular N-propeptide
domain (24). mAb 2C3 is specific to a proteolytic neoepitope at
the C terminus of the ␣1(III) N-telopeptide sequence YDVKS-
GVAVGG, where K is a cross-linked lysine. Two mouse mono-
clonal antibodies to type II collagen were also used. mAb 10F2
recognizes a proteolytic neoepitope at the C terminus of a
cleaved type II C-telopeptide sequence generated by pepsin
(38), and mAb 1C10 is specific to a denatured epitope in the
triple-helical domain of ␣1(II) near the C-terminal end (31).
For Western blotting, collagen fractions resolved by SDS-
PAGE were transblotted to polyvinylidene difluoride membrane
(Bio-Rad). Western blot analyses performed with each mono-
clonal antibody were developed using alkaline phosphatase-con-
jugated goat anti-rabbit IgG (Jackson ImmunoResearch, Avon-
dale, PA) and 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium as substrate for alkaline phosphatase.
N-terminal Protein Sequence Analysis—Purified cross-linked
peptides were identified by N-terminal sequence analysis on a
Porton 2090E gas phase sequencer with on-line HPLC analysis
of phenylthiohydantoin derivatives (29).
Mass Spectrometry—Individual protein bands after Coomas-
(32, 39). The resulting peptides were subjected to microbore C8
column liquid chromatography (0.3 mm ⫻ 15 cm; Vydac) inter-
VOLUME 285 • NUMBER 24 • JUNE 11, 2010
 Type III Collagen in Cartilage

FIGURE 1. Interrupted SDS-PAGE to detect ␣1(III) chains in pepsin

extracts of human articular cartilage from three tissue donor knees. Lane
1, 18-year-old; lane 2, 60-year-old; lane 3, 73-year-old. For the reduced sample
lanes (⫹DTT), DTT was added 15 min after starting the electrophoresis to
resolve the ␣1(III) chain from ␣1(II) chain. The band immediately below ␣1(II)
in lane 1 is a pepsin overcleavage product of ␣1(II).
faced directly to a ThermoFinnnigan LCQ Deca XP tandem
mass spectrometer equipped with an electrospray ionization
source. For protein identification, peptide fragments were
compared with the NCBI nonredundant protein database using
SEQUEST, an automated database search algorithm designed
for use with tandem mass spectrometry (MS/MS) data. Cross-
linked peptides were analyzed manually by calculating the pos-
sible MS/MS ions and matching these to the actual MS/MS ions
(32).
FIGURE 2. SDS-PAGE/Western blot analysis to screen for type III collagen
fragments covalently attached to type II collagen. Pepsin-solubilized type
II collagens from mature human (H) and bovine (B) articular cartilages were
resolved on SDS-PAGE without reducing disulfide bonds. Gels were either
stained with Coomassie Brilliant Blue or electroblotted to polyvinylidene
difluoride membrane and probed with a type III collagen N-telopeptide-spe-
cific antibody (2C3), type II collagen-specific antibody (1C10), or type III colla-
gen N-propeptide-specific antibody (4G9). The type III N-propeptide/te-
lopeptide is detected cross-linked to both human and bovine ␣1(II) chains.
The arrow shows the position of the 160 kDa band that reacts with all three
antibodies.

RESULTS
Using interrupted SDS-PAGE with delayed reduction of
disulfide bonds to resolve ␣1(III) from ␣1(II) chains, we were
able to identify type III collagen in pepsin-solubilized material
from all adult human articular cartilage samples examined
(results from three representative donor joints are shown in Fig.
1). The chain identities, indicated by their migration on SDS-
PAGE, were established beyond doubt by mass spectrometry FIGURE 3. Molecular sieve chromatography of CNBr-digested human
and N-terminal protein sequence analysis. The type III collagen articular cartilage collagen. The CNBr digest of cartilage residue after 4 M
guanidine HCl extraction was chromatographed on an agarose A1.5m (Bio-
content of adult human articular cartilage varied between indi- Rad) molecular sieve column (170 ⫻ 1.5 cm), eluted with a 0.05 M Tris-HCl
viduals in the range 0.5% to about 10% of total collagen, based buffer, pH 7.5, containing 2 M guanidine HCl, at a flow rate of 6 ml/h, collecting
3.0-ml fractions. Aliquots of collected fractions (4 ␮l) were assayed for mAb
on the recovered dry weights and relative intensity of Coomas- 4G9 immunoreactivity. The result shows that the N-propeptide domain of the
sie Brilliant Blue-stained bands on SDS-PAGE. type III collagen was retained in the cartilage matrix.
Fig. 2 shows a Western blot analysis using mAb 2C3 to
probe the collagen chains extracted from adult human and
bovine articular cartilage for a covalently attached type III
collagen N-telopeptide. The 2C3 antibody is specific to
human collagen III and does not cross-react with bovine
collagen. Because collagen samples were not treated with
DTT to reduce disulfide bonds prior to electrophoresis, the
intramolecularly disulfide-bonded type III collagen chains
ran near the top of the separating gel. From human cartilage
three bands can be seen stained with 2C3, all of which were
also stained by 1C10, the type II collagen-specific antibody.
All three therefore were based on an ␣1(II) triple-helix but
had an ␣1(III) N-telopeptide cross-linked to them. The gel
shows that in addition to the signal from the main ␣ and ␤
chains of type II collagen seen by Coomassie Brilliant Blue
staining in the pepsin digest, 2C3 also binds to a minor band FIGURE 4. Interrupted SDS-PAGE/Western blot analysis of type III colla-
gen from mature human articular cartilage. SDS-PAGE was run as in Fig. 1.
running between them at 160 kDa not visible by Coomassie mAb 10F2 was used to probe for the presence of a fragment of pepsin-
staining (Fig. 2). This band also reacted with mAb 1C10 and cleaved type II collagen C-telopeptide linked to an ␣1(III) chain.

JUNE 11, 2010 • VOLUME 285 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 18539
Type III Collagen in Cartilage
mAb 4G9, which shows the presence of the ␣1(II) chain and has a disulfide-bonded ␣1(III) N-propeptide trimer attached.
an ␣1(III) N-propeptide domain, respectively. Presumably this reflects a pepsin partial-cleavage product
From these properties, this component appears to be an extracted from the cartilage matrix. The findings also imply
␣1(II) chain cross-linked to an ␣1(III) N-telopeptide that still that relatively large amounts of the N-propeptide domain of
type III collagen are present in the extracellular matrix of adult
cartilage. The presence of collagen type III N-propeptides in
articular cartilage was confirmed by mAb 4G9 enzyme-linked
immunosorbent assay across molecular sieved column frac-
tions from a CNBr digest of the 4 M guanidine HCl-insoluble
residue of adult cartilage (Fig. 3). The CNBr peptide compo-
nents containing the N-propeptide domain eluted early in the
chromatogram. Based on their elution positions on molecular
sieve chromatogram and migration position on SDS-PAGE, the
4G9-reactive peptides in elution volume 85–130 ml have
molecular masses of 100 kDa and above. The results indicate
that the type III collagen N-propeptide domain is retained by
most molecules of type III deposited and polymerized in carti-
lage matrix.
Fig. 4 shows that mAb 10F2 reacted with the ␣1(III) chain
FIGURE 5. Molecular sieve chromatography of pepsin-extracted bovine resolved on interrupted electrophoresis, indicating that ␣1(II)
types II and III collagens. Collagen recovered in the 0.7 M NaCl fraction (36 C-telopeptides were attached to some of the ␣1(III) chains.
mg) from pepsin-solubilized 4-year-old cow articular cartilage collagen was
dissolved in 1.8 ml of 4 M guanidine HCI, 0.05 M Tris-HCI, pH 7.5, and chromato- Such heterotypic cross-linking between type III collagen and
graphed on an agarose A5m (Bio-Rad) molecular sieve column (170 ⫻ 1.5 cm) type II collagen was also identified in extracts of adult bovine
to resolve type III collagen from the bulk of type II collagen ␣ and ␤ chains. The articular cartilage as follows. Because ␣1(III) chains are disul-
eluant was 2 M guanidine HCI, 0.05 M Tris-HCI, pH 7.5, at a flow rate of 6.9 ml/h,
collecting 2.3-ml fractions. The bar indicates fractions enriched in type III col- fide-bonded intramolecularly, they can be resolved from the
lagen that were pooled and digested with trypsin and purified by IMAC. bulk type II collagen ␣ and ␤ chains in a pepsin digest by molec-
ular sieve column chromatography
(Fig. 5). Collagen recovered from
the indicated pooled fractions
enriched in type III collagen was
digested with trypsin. Cross-linked
peptides were further purified by
IMAC and reverse-phase HPLC.
Using Cu2⫹ IMAC, several pep-
tides containing histidine residues
were selectively bound from the
trypsin digest of the enriched type
III collagen pool (Fig. 5). Four
of these peptides were non-cross-
linked linear peptides (Fig. 6a),
but, in addition, divalent and tri-
valent cross-linked collagen III pep-
tides were also isolated. A promi-
nent divalent cross-linked peptide
was derived from the C-telopeptide
of type II collagen (EKGPDPLQ)
linked to the type II helical sequence
that contained the residue 87
hydroxylysine cross-linking resi-
due (GFP*GTP*GLP*GVK87GHR).
Telopeptides from both N and C
termini of type III collagen were also
recovered linked covalently to the
FIGURE 6. Reverse-phase HPLC fractionation of tryptic peptides prepared from 4-year-old bovine artic-
helical cross-linking sites in type III
ular cartilage bound by a copper-affinity column. Tryptic peptides that had eluted from the copper column collagen (Fig. 6b). In addition, pep-
with 0.1 M sodium acetate buffer, pH 4.6 (a) and 0.1 M sodium acetate buffer, pH 4.6, containing 0.2 M imidazole tides from heterotypic cross-links
(b) were resolved on a C8 reverse-phase column (for details, see “Experimental Procedures”). Purified peptides
were identified by N-terminal sequence analysis and by mass spectrometry. P*, 4-hydroxyproline; X, cross- between types II and III collagens
linking hydroxylysine residue; galglc, glucosylgalactosyl. were identified. One came from

18540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 24 • JUNE 11, 2010

FIGURE 7. Heterotypic cross-linking between types II and III collagens in
bovine articular cartilage. a, MS/MS identified a pyridinoline cross-linked
peptide in fraction 45 (Fig. 6a) that resulted from heterotypic cross-linking
between types II and III collagens. b, structure and origin of the purified cross-
linked peptide. P*, 4-hydroxyproline; X, cross-linking hydroxylysine residue.
linkage of an N-telopeptide of type III collagen (DVXSGV-
AGGGIAGYP*GPAGPP*—) to the helical 930 site in type II
collagen (GLXGHR) (Fig. 6b). The structure of this heterotypic
cross-linked peptide was confirmed beyond doubt by MS/MS
(24). Another heterotypic cross-linked peptide was purified
from fraction 45 (Fig. 6a) and identified as a trivalent cross-
linked peptide linking the ␣1(II) C-telopeptide (EXGPDPLQ)
to an ␣1(III) C-telopeptide (IAGVGAEXAGGFAGY) and the
helical cross-linking site Lys87 in type III collagen (Fig. 7). In
addition to links to the helix of type III collagen, C-telopeptides
of collagens II and III were also found that were covalently
linked to the helix of type II collagen through a pyridinoline
residue. Thus, an ␣1(II) C-telopeptide (LGPREXGPDPL)
sequence and an ␣1(III) C-telopeptide sequence (IAGIGGEXA)
were found linked through pyridinoline to the type II collagen
helical cross-linking site at residue 87 in a peptide isolated from
JUNE 11, 2010 • VOLUME 285 • NUMBER 24
Type III Collagen in Cartilage
FIGURE 8. Purification of a heterotypic type II/type III cross-linked pep-
tide from human articular cartilage collagen. a, DEAE HPLC fractionation
of fluorescent cross-linked peptides from bacterial collagenase digested
human articular cartilage. Fractions indicated by the bar were pooled and
resolved by reverse-phase HPLC. b, reverse-phase HPLC isolation of a colla-
gen type II/type III heterotypic cross-linked peptide linking the sequences
shown. c, structure of the purified cross-linked peptide. X, cross-linking
hydroxylysine residue.
a bacterial collagenase digest of adult human articular cartilage
(Fig. 8).
Experiments designed to test whether the cartilage matrix
type III collagen was readily available for extraction as a poly-
mer associated with and cross-linked to type II collagen fibril
surfaces were carried out. Initial results indicated that of vari-
ous metalloproteinases tested, stromelysin-1 was the most effi-
cient under native conditions in extracting the type III collagen
pool without extracting significant amounts of type II collagen.
Fig. 9 compares the results of two serial 24-h extractions by
recombinant MMP3 at 37 °C (Fig. 9a) of minced articular car-
tilage from an osteoarthritic joint with pepsin extraction (Fig.
9b). The results of Western blotting using three different
monoclonal antibodies on replicate lanes show that MMP3
extracted very little type II collagen, which was detectable only
JOURNAL OF BIOLOGICAL CHEMISTRY 18541
Type III Collagen in Cartilage
mAb 4G9. From the mass spec-
trometry results, all of the divalent
cross-linked peptides identified in
type III collagen of bovine cartilage,
either from III to III or III to II link-
ages, contained an additional 188-
Da mass on the cross-linking resi-
due. Such an adduct has been shown
to be a maturation product of a pool
of ketoimine cross-links in type II
collagen of bovine cartilages that do
not mature to pyridinolines. It
results from ketoimine oxidation
and arginine addition (40).
The results also reveal trivalent
cross-linked peptides between mixed
FIGURE 9. Selective extraction of collagen type III from human articular cartilage by MMP3. a, minced
cartilage after 4 M guanidine HCl extraction was serially extracted twice for 24 h with recombinant MMP3. telopeptides of both types II and
Replicate aliquots of each extract were run on 10% SDS-PAGE ⫾ DTT, stained directly with Coomassie Blue, or III collagens to helical residue 87
immunoblotted using three different mAbs, 4G9, 2C3, or 1C10, which recognize the ␣1(III) N-propeptide, ␣1(III)
N-telopeptide proteolytic neoepitope, and ␣1(II) triple-helical domain, respectively. Bands in the lower half of in either collagen II or collagen III
the Coomassie-stained lanes are from the enzyme preparation. Lane 1, first 24-h extract; lane 2, second 24-h (Figs. 6 and 7). This finding is
extract. b, another sample of the same cartilage guanidine-HCl residue was digested with pepsin, the digest important because it confirms that
fractionated into 0.8 M, 1.2 M, and 2.2 M NaCl precipitates, and each run on 6% SDS-PAGE ⫾ DTT. c, proteolytic
cleavage sites and molecular features of collagen type III are illustrated. pyridinoline residues can link three
different collagen molecules. In-
as intact ␣1(II) chains (1C10 blot), but most of the type III deed, our original proposed mechanism of formation for pyr-
collagen (detectable as cross-linked large fragments on 4G9 and idinoline cross-links was an aldol addition between two neigh-
2C3 blots). Pepsin removed all of the type III collagen too, but boring ketoimine cross-links within the microenvironment of
cleaved mostly between the ␣1(III) N-propeptide (disulfide- the molecular packing arrangement of a fibril (41). The stoichio-
bonded trimer) and the main triple helix, whereas MMP3 did metry from C14-lysine labeling of cartilage in vivo and in vitro
not cleave and release the free ␣1(III) N-propeptide trimer, (42– 44) and the finding of a urinary peptide from bone resorp-
which was retained on the large fragments (Fig. 9a). Fig. 9c tion linking two ␣2(I) N-telopeptides to a helical site (45) sup-
illustrates the various cleavage sites and molecular features of port this.
cross-linked pN-type III collagen. The CNBr-derived peptides in which N-propeptide domains
of type III collagen were detected have estimated molecular
DISCUSSION masses ⬎100 kDa (Fig. 3). This is larger than the monomeric
The results confirm that significant amounts of type III col- processed N-propeptide trimer (⬃60 kDa) and therefore can-
lagen are present in adult human articular cartilage cross- not represent simply processed collagen III N-propeptides after
linked covalently to other type III collagen molecules, suggest- synthesis, but the retention of N-propeptides on cross-linked
ing their presence in the matrix as homotypic polymers of type pN-type III collagen molecules in cartilage matrix. Retained
III collagen presumably in the form of fine filaments of head- N-propeptides will prevent type III collagen from forming thick
to-tail cross-linked molecules. Most of the cross-links formed collagen fibrils (46 – 48), but will not impair divalent cross-link-
between the type III collagen molecules are of the divalent vari- ing internally in the polymer or trivalent bonds at the interface
ety, in contrast to type II collagen in which trivalent pyridino- with type II collagen fibrils.
line cross-links predominate (34). The results also indicate that The most likely explanation for the polymeric form of type III
in addition to type III-to-type III cross-links, the polymeric type collagen in cartilage matrix is a thin filamentous polymer of
III collagen is also heavily cross-linked to type II collagen. pN-type III molecules cross-linked head to tail at 4D-staggered
Telopeptides from type II collagen are linked to the helical sites but heavily cross-linked laterally to the surfaces of type II
cross-linking sites in type III collagen and, vice versa, telopep- collagen fibrils wherever they interact. In effect, such a filamen-
tides from type III collagen are linked to helical cross-linking tous polymer might add cohesion to a swollen, and perhaps
sites in type II collagen. weakened, existing collagen II fibril network. It is notable that
The Western blot analyses of type III collagen extracted from the earliest observed change in articular cartilage in experimen-
adult human and bovine articular cartilages also revealed that a tal animal models of osteoarthritis is a swelling of the collagen
fraction of the molecules had been covalently linked to type II fibril network (49) and that collagen III is expressed by chon-
collagen (Fig. 2). A major site of linkage was between the C-he- drocytes of human osteoarthritic cartilage (50), in which its
lix (Lys930) of ␣1(II) and the ␣1(III) N-telopeptide. This is the content has been reported to be enriched (51).
same site previously implicated for bovine articular cartilage Type III collagen is distinct from types I and II collagens in
(24). The results in Fig. 2 show that this cross-linkage in fact lacking 3-hydroxyproline in the triple helix, which we suspect
occurs between a longer form of the ␣1(III) N-telopeptide in may be related to an inability to form thick, homotypic fibrils
which the N-propeptide extension is retained and detected by (52). In skin and other tissues, immunogold electron micros-

18542 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 24 • JUNE 11, 2010

FIGURE 10. Illustrated concept of collagen type III polymeric filaments
interwoven with and cross-linked to a collagen type II fibrillar network.
The collagen III polymer is susceptible to depolymerization and selective
extraction by MMP3 cleavage in the triple helix and telopeptide domains as
indicated by the scissors symbols.
copy showed that collagen III with retained N-propeptides is
present on the surface of type I collagen fibrils (13). Similarly,
collagen type III with retained N-propeptides has been detected
on the surface of type II collagen fibrils in human articular car-
tilage (23). The results in Fig. 9 show that type III collagen can
be readily extracted by stromelysin (MMP3) digestion of
human articular cartilage under native conditions, suggesting
that it is accessible as a cross-linked polymer external to type II
collagen fibrils. This concept is shown in Fig. 10.
The size of the fragments extracted by MMP3 with retained
N-propeptide domains is consistent with depolymerization by
cleavage in the main triple helix, most likely at the 3⁄4 length
collagenase-cleavage domain, which is especially susceptible in
type III collagen to proteases other than collagenase including
MMP3 (53, 54). Taken together, the results show that type III
collagen molecules accumulate in mature human articular car-
tilage cross-linked to the surface of type II collagen fibrils. The
amount presumably varies between individual joints, anatomi-
cal location, and tissue microanatomy, perhaps dependent on
the history of injuries and the wear and tear experienced by a
normal joint. If so, the content will tend to increase with age. It
has also been noted that as articular cartilage matures and ages,
the collagen fibrils become thicker, and the content of types IX
and XI collagens decreases relative to type II collagen (55). The
␣2(XI) chain of type XI collagen progressively decreases in con-
tent with tissue maturation and is replaced by ␣1(V) (56). It is
known that type III collagen is prominent in fibrous repair tis-
sue in skin and other tissues (57). Therefore, it seems likely that
type III collagen is synthesized as a modifier of existing fibril
networks in response to tissue and matrix damage.
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