Protein Structure and Folding:

Type III Collagen, a Fibril Network

Modifier in Articular Cartilage
Jiann-Jiu Wu, Mary Ann Weis, Lammy S.
Kim and David R. Eyre
J. Biol. Chem. 2010, 285:18537-18544.
doi: 10.1074/jbc.M110.112904 originally published online April 19, 2010

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 1853718544, June 11, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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 articular 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 unprocessed N-propeptides are present in the extracellular matrix of
adult human and bovine articular cartilages as covalently crosslinked 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 findings 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 (15). 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 procollagen 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 molecules in vertebrates. Type I collagen is widely expressed and

* This work was supported by National Institutes of Health Grants AR 37318

1

and AR 36794.
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

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 (79). Type III collagen 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 compliant 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 (2224).
The collagen framework of hyaline cartilage is a highly crosslinked unique heteropolymer. In essence, the bulk type II collagen 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
(2528). All three collagen types, II, IX, and XI, are heavily
cross-linked in the same fibril through the lysyl oxidase-mediated 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 extracellular 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 CollagensHuman knee joints were obtained
from Northwest Tissue Services (Seattle, WA) from donors
aged 18 75 with no obvious signs of osteoarthritis. Full thickness 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 fluoride, and 5 mM 1,10-phenanthroline) at 4 C for 48 h to remove
proteoglycans and other matrix proteins. The guanidine-insoluble tissue residue was then washed thoroughly with water and
freeze-dried. Cross-linked collagens were solubilized by digestJOURNAL OF BIOLOGICAL CHEMISTRY

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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 collagens 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 chromatography on an agarose A5m column (170 1.5 cm, 200 400
mesh, Bio-Rad), eluted with 2 M guanidine HCl, 0.05 M TrisHCl, pH 7.5.
CNBr Cleavage and Peptide ChromatographyThe washed
residues after 4 M guanidine HCl extraction were minced and
digested with CNBr in 70% formic acid under N2 at room temperature for 24 h on a shaker. The digests were diluted 15-fold
with water and freeze-dried (34). Collagen CNBr-derived peptides were resolved by molecular sieve chromatography on an
agarose A1.5m column (170 1.5 cm, 200 400 mesh, BioRad), eluted with 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5.
Bacterial Collagenase Digestion and Peptide ChromatographyAn aliquot of the washed guanidine-insoluble articular 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 monitored 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 reversephase 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 DigestionThe molecular sieve-purified type III collagen 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).
IMACOne 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

The abbreviations used are: HPLC, high performance liquid chromatography; IMAC, immobilized metal ion affinity chromatography; DTT, dithiothreitol; 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 column 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 temperature for 1 h. The beads were washed with 10 ml of IMAC
sample buffer to remove unbound peptides. The bound peptides 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) ExtractionCartilage from a nonfibrillated 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. 1,10phenanthroline (10 mM) was added to each extract to stop
the reaction.
Gel ElectrophoresisPepsin-solubilized collagens were resolved 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 dithiothreitol (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 BlottingTwo mouse monoclonal antibodies 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 YDVKSGVAVGG, where K is a cross-linked lysine. Two mouse monoclonal 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 SDSPAGE were transblotted to polyvinylidene difluoride membrane
(Bio-Rad). Western blot analyses performed with each monoclonal antibody were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, Avondale, PA) and 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium as substrate for alkaline phosphatase.
N-terminal Protein Sequence AnalysisPurified 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 SpectrometryIndividual protein bands after Coomassie Blue staining on SDS-PAGE were digested in-gel by trypsin
(32, 39). The resulting peptides were subjected to microbore C8
column liquid chromatography (0.3 mm 15 cm; Vydac) interVOLUME 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. Crosslinked peptides were analyzed manually by calculating the possible MS/MS ions and matching these to the actual MS/MS ions
(32).

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 SDSPAGE, were established beyond doubt by mass spectrometry
and N-terminal protein sequence analysis. The type III collagen
content of adult human articular cartilage varied between individuals in the range 0.5% to about 10% of total collagen, based
on the recovered dry weights and relative intensity of Coomassie Brilliant Blue-stained bands on SDS-PAGE.
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
running between them at 160 kDa not visible by Coomassie
staining (Fig. 2). This band also reacted with mAb 1C10 and
JUNE 11, 2010 VOLUME 285 NUMBER 24

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-specific antibody (2C3), type II collagen-specific antibody (1C10), or type III collagen N-propeptide-specific antibody (4G9). The type III N-propeptide/telopeptide 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.

FIGURE 3. Molecular sieve chromatography of CNBr-digested human

articular cartilage collagen. The CNBr digest of cartilage residue after 4 M
guanidine HCl extraction was chromatographed on an agarose A1.5m (BioRad) molecular sieve column (170 1.5 cm), eluted with a 0.05 M Tris-HCl
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
4G9 immunoreactivity. The result shows that the N-propeptide domain of the
type III collagen was retained in the cartilage matrix.

FIGURE 4. Interrupted SDS-PAGE/Western blot analysis of type III collagen from mature human articular cartilage. SDS-PAGE was run as in Fig. 1.
mAb 10F2 was used to probe for the presence of a fragment of pepsincleaved type II collagen C-telopeptide linked to an 1(III) chain.

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Type III Collagen in Cartilage

mAb 4G9, which shows the presence of the 1(II) chain and
an 1(III) N-propeptide domain, respectively.
From these properties, this component appears to be an
1(II) chain cross-linked to an 1(III) N-telopeptide that still

has a disulfide-bonded 1(III) N-propeptide trimer attached.

Presumably this reflects a pepsin partial-cleavage product
extracted from the cartilage matrix. The findings also imply
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 fractions from a CNBr digest of the 4 M guanidine HCl-insoluble
residue of adult cartilage (Fig. 3). The CNBr peptide components 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 85130 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 cartilage 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 disuleluant 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 molecular 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 peptides 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-crosslinked linear peptides (Fig. 6a),
but, in addition, divalent and trivalent cross-linked collagen III peptides were also isolated. A prominent 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 residue (GFP*GTP*GLP*GVK87GHR).
Telopeptides from both N and C
termini of type III collagen were also
recovered linked covalently to the
helical cross-linking sites in type III
FIGURE 6. Reverse-phase HPLC fractionation of tryptic peptides prepared from 4-year-old bovine articular cartilage bound by a copper-affinity column. Tryptic peptides that had eluted from the copper column collagen (Fig. 6b). In addition, pepwith 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
were identified. One came from
linking hydroxylysine residue; galglc, glucosylgalactosyl.

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Type III Collagen in Cartilage

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 crosslinked peptide. P*, 4-hydroxyproline; X, cross-linking hydroxylysine residue.

linkage of an N-telopeptide of type III collagen (DVXSGVAGGGIAGYP*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 crosslinked 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

FIGURE 8. Purification of a heterotypic type II/type III cross-linked peptide 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 collagen 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 polymer associated with and cross-linked to type II collagen fibril
surfaces were carried out. Initial results indicated that of various metalloproteinases tested, stromelysin-1 was the most efficient 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 cartilage 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
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Type III Collagen in Cartilage

mAb 4G9. From the mass spectrometry 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 linkages, contained an additional 188Da mass on the cross-linking residue. 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
pyridinoline residues can link three
cleavage sites and molecular features of collagen type III are illustrated.
different collagen molecules. Indeed,
our
original
proposed
mechanism of formation for pyras intact 1(II) chains (1C10 blot), but most of the type III
collagen (detectable as cross-linked large fragments on 4G9 and idinoline cross-links was an aldol addition between two neigh2C3 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 stoichiobonded 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 resorpwhich was retained on the large fragments (Fig. 9a). Fig. 9c tion linking two 2(I) N-telopeptides to a helical site (45) supillustrates the various cleavage sites and molecular features of port this.
The CNBr-derived peptides in which N-propeptide domains
cross-linked pN-type III collagen.
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 canlagen 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-linkbetween 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.
The most likely explanation for the polymeric form of type III
line cross-links predominate (34). The results also indicate that
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 filamentides from type III collagen are linked to helical cross-linking tous polymer might add cohesion to a swollen, and perhaps
weakened, existing collagen II fibril network. It is notable that
sites in type II collagen.
The Western blot analyses of type III collagen extracted from the earliest observed change in articular cartilage in experimenadult 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 choncollagen (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).
Type III collagen is distinct from types I and II collagens in
same site previously implicated for bovine articular cartilage
(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-

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Type III Collagen in Cartilage

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 cartilage (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 34 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 cartilage cross-linked to the surface of type II collagen fibrils. The
amount presumably varies between individual joints, anatomical 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 content with tissue maturation and is replaced by 1(V) (56). It is
known that type III collagen is prominent in fibrous repair tissue 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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