Báo cáo khoa học: Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line ppt

Assembly of collagen types II, IX and XI into nascent hetero-fibrils
by a rat chondrocyte cell line
Russell J. Fernandes
1
, Thomas M. Schmid
2
and David R. Eyre
1,3
1
Department of Orthopedics and Sports Medicine, University of Washington, Seattle, WA, USA;
2
Department of Biochemistry,
Rush Medical College, Rush University, Chicago, IL, USA;
3
Department of Biochemistry, University of Washington,
Seattle, WA, USA
The cell line, RCS-LTC (derived from the Swarm rat
chondrosarcoma), deposits a copious extracellular matrix in
which the collagen component is primarily a polymer of
partially processed type II N-procollagen molecules.
Transmission electron microscopy of the matrix shows no
obvious fibrils, only a mass of thin unbanded filaments. We
have used this cell system to show that the type II N-pro-
collagen polymer nevertheless is stabilized by pyridinoline
cross-links at molecular sites (mediated by N- and C-telo-
peptide domains) found in collagen II fibrils processed nor-
mally. Retention of the N-propeptide therefore does not
appear to interfere with the interactions needed to form
cross-links and mature them into trivalent pyridinoline
residues. In addition, using antibodies that recognize specific
cross-linking domains, it was shown that types IX and XI

collagens, also abundantly deposited into the matrix by this
cell line, become covalently cross-linked to the type II
N-procollagen. The results indicate that the assembly and
intertype cross-linking of the cartilage type II collagen
heteropolymer is an integral, early process in fibril assembly
and can occur efficiently prior to the removal of the collagen
II N-propeptides.
Keywords: chondrocyte; type II procollagen; pyridinoline
cross-links; collagen fibril; extracellular matrix.
The collagen framework of the extracellular matrix of
developing hyaline cartilage is assembled primarily from
three cartilage-specific collagens: type II; type IX; and type
XI [1]. These three collagens copolymerize into heterotypic
fibrils and become cross-linked intermolecularly [2,3]. The
predominant mature cross-link is the trivalent hydroxyl-
ysyl pyridinoline (HP) residue, which links at two sites
(from N-telopeptide to helix and from C-telopeptide to
helix) between head-to-tail overlapping type II collagen
molecules packed in fibrils [4]. Pyridinolines and divalent
cross-links covalently bond type IX collagen molecules to
N- and C- telopeptides on the surface of type II collagen
fibrils [1,5]. Divalent cross-links (keto-amines) also link
type XI collagen molecules to each other and to
C-telopeptides of type II collagen within the heteropoly-
mer [3]. All the cross-links are formed by the lysyl
oxidase-catalyzed mechanism. This copolymeric fibrillar
network is an essential template for the assembly of the
matrix and normal function of hyaline cartilages. Muta-
tions in any one of the genes encoding the three primary
collagen subunits can cause chondrodysplasia syndromes

and/or premature osteoarthritis [6–10].
Type II collagen, the major structural protein of cartilage,
is secreted as a procollagen molecule which is processed by
removal of its C- and N-propeptides before or during fibril
assembly in the extracellular matrix [11–13]. Although
propeptide removal is required for the normal growth of
fibril diameter [14], fibrillogenesis experiments in vitro,using
purified collagens from pig eye vitreous humor, have shown
that partially processed N-procollagen can coassemble with
fully processed type II collagen into thin fibrils [15]. Type
IIA N-procollagen, an alternatively spliced product from
the type II collagen gene, COL2A1 [16], together with type
IIB N-procollagen and fully processed type II collagen
molecules, have all been detected in bovine vitreous humor
[17]. Type IIA N-procollagen has been immunolocalized to
the surface of collagen fibrils in vitreous humor [18]. To
what degree the type II N-procollagen molecules can form
fibrils and become cross-linked, however, is unclear.
We have pursued this question using a rat chondrosar-
coma cell line, RCS-LTC. These cells and the cells from the
parental tumor lay down a copious, highly hydrated matrix
of collagen [19], aggrecan [20,21] and noncollagenous
proteins, including cartilage oligomeric matrix protein [22]
and matrilin-3 [23]. Type II collagen is the major collagen
product of the cell line, but a high proportion of collagens
IX and XI are also synthesized and deposited in the
extracellular matrix [19]. In previous reports we showed that
the form of type II collagen in the matrix was the IIB
splicing variant, all molecules of which had retained their
N-propeptides [24,25]. The RCS-LTC cell line therefore fails

to express an active collagen II N-propeptidase. In the
Correspondence to R. J. Fernandes, Orthopaedic Research Laborat-
ories, Department of Orthopædics and Sports Medicine, Box 356500,
University of Washington, Seattle, WA 98195, USA.
Fax: +1 206 685 4700; Tel.: +1 206 543 4700;
E-mail:
Abbreviations: bAPN, beta-aminoproprionitrile; HP, hydroxylysyl
pyridinoline; LP, lysyl pyridinoline; pN a1(II), type II
N-procollagen a-chains.
(Received 11 April 2003, revised 4 June 2003, accepted 9 June 2003)
Eur. J. Biochem. 270, 3243–3250 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03711.x
present study, the quality of the collagenous matrix
deposited in long-term cultures of these cells was examined.
Specifically, we determined whether mature cross-links were
formed in the polymeric type II N-procollagen and if types
IX and XI collagens were also incorporated, in order to
understand better the temporal sequence and mechanism of
assembly.
Materials and methods
Cell culture
The RCS-LTC cell line was maintained in monolayer
culture in high-glucose DMEM (Dulbecco’s modified
Eagle’s medium) (Hyclone) containing 10% iron-supple-
mented bovine calf serum (Hyclone), 10 lgÆmL
)1
L
-ascor-
bate, at 37 °Cand5%CO
2
for1–4weeks[24].Some

cultures were additionally supplemented with beta-amino-
proprionitrile (bAPN) (Sigma) to inhibit lysyl oxidase.
Metabolic radiolabeling
After 4 weeks in culture, the medium was replaced with
serum-free DMEM containing 25 lCiÆmL
)1
[
3
H]proline,
10 lgÆmL
)1
ascorbate, 100 lgÆmL
)1
bAPN, and incubation
was continued for a further 24 h. The medium was then
removed and the cell layer extracted with 0.15
M
potassium
phosphate (130 m
M
K
2
HPO
4
and 19 m
M
KH
2
PO
4

,pH 7.6)
containing 1 m
M
phenylmethanesulfonyl fluoride, 1 m
M
N-ethylmaleimide, 5 m
M
EDTA [19,26], for 24 h at 4 °C.
The medium and the cell layer extract were dialyzed against
0.4
M
NaCl, 50 m
M
Tris (pH 7.5), 5 m
M
EDTA, 2 m
M
phenylmethanesulfonyl fluoride, and stored at )20 °C until
analyzed.
Collagen extraction and purification
The cell layer was extracted with 0.15
M
potassium phos-
phate containing protease inhibitors (as described above)
for 20 h at 4 °C, to solubilize newly synthesized, noncross-
linked collagen. After centrifugation (30 min, 4 °C,
30 000 g), the pellet was suspended in buffer comprising
50 m
M
sodium acetate (pH 6.0), 0.15

M
NaCl and 2 m
M
EDTA, and digested with 0.5 mgÆmL
)1
porcine testicular
hyaluronidase (Sigma). The cross-linked collagen polymer
in the residue was solubilized by digestion with pepsin
(100 lgÆmL
)1
in 3% acetic acid). Following partial purifi-
cation of type II collagen by 1.2
M
NaCl precipitation from
3% acetic acid, the a1(II) collagen chains were purified to
homogeneity by C8 reverse-phase HPLC, monitoring
absorbance (at 220 nm) and fluorescence (excitation
297 nm, emission 396 nm). Type II collagen from rat
cartilage was run as a control.
Peptide analysis
Purified a1(II) collagen chains from the cell layer and
control tissue were digested with cyanogen bromide in 70%
formic acid. The resulting peptides were fractionated by
molecular sieve HPLC, monitoring absorbance (at 220 nm)
and fluorescence (excitation 330 nm, emission 396 nm)
[4,8].
Cross-link analysis
Purified a1(II) collagen chains, and cyanogen bromide-
derived peptides were hydrolyzed in 6
M

HCl at 108 °Cfor
24 h. HP and lysyl pyridinoline (LP) cross-links were
quantified by C-18 reverse-phase HPLC (excitation 297 nm,
emission 396 nm) [27,28].
Gel electrophoresis and Western blotting
Collagen chains and chain fragments were resolved by
PAGE [29] and staining with Coomassie Blue, or by PAGE
and transfer to a poly(vinylidene difluoride) membrane and
probing with monoclonal antibody (mAb) 10F2 (1 : 1000
dilution). The mAb 10F2 is one of several mAbs raised
against protease-generated neoepitopes in the collagen
a1(II) C-telopeptide. It recognizes a cleavage-site (neoepi-
tope) in a sequence within the C-telopeptide cross-linking
domain of type II collagen [30]. This antibody can detect the
C-telopeptides of type II collagen (even as short fragments)
when cross-linked to collagen triple-helical domains. A
polyclonal antibody to type IX collagen [31] was used to
probe for a3(IX) chains. Biotin-labeled goat anti-mouse
IgG (Jackson) was used as the secondary antibody and
streptavidin-alkaline phosphatase (Sigma) was used for
detection.
[
3
H]Proline-labeled proteins were visualized by fluoro-
graphy after gel electrophoresis using Amplify fluoro-
graphic reagent (Amersham Pharmacia Biotech) and
Biomax MS X-ray film (Kodak).
Electron microscopy
The RCS-LTC cell line and chondrocytes from the Swarm
rat chondrosarcoma parental tumor, which synthesize type

II collagen [32], were cultured in micromass or in high-
density monolayers on Thermonox tissue cover slips (Nalge
Laboratories) for 12–14 days. The cultures were fixed for
1 h at room temperature in 2% glutaraldehyde, 2%
paraformaldehyde in 0.1
M
sodium cacodylate buffer,
pH 7.4. Post-fixation was carried out using 2% osmium
tetraoxide in 0.2
M
cacodylate buffer, pH 7.4. The cultures
were then stained en bloc with 1.25% aqueous uranyl
acetate, dehydrated and embedded in plastic. Ultrathin
sections (60–70 nm) were cut perpendicular to the plane of
culture, placed on 300-mesh copper grids and stained with
Reynold’s lead citrate (4.4% lead nitrate, 5.9% sodium
citrate in distilled water, pH 12.0), and 2.5% uranyl acetate
in 50% ethanol. All sections were examined and photo-
graphed on a JEOL 100 CX transmission electron micro-
scope.
Results
Transmission electron microscopy of the cell layer iden-
tified cells that retained the organelles and morphology
typical of chondrocytes (Fig. 1). In Fig. 1A, the RCS-
LTC cells show oval-shaped mitochondria, a prominent
nucleus, abundant rough endoplasmic reticulum and
Golgi vacuoles, indicating active synthesis and secretion
of proteins. The extracellular matrix, however, featured
extensive electron-lucent areas and a distinctive lack of
3244 R. J. Fernandes et al. (Eur. J. Biochem. 270) Ó FEBS 2003

electron-dense material and collagen fibrils. Linear arrays
of extremely thin filaments (<10 nm) were observed in
the extracellular matrix (Fig. 1A). This contrasted with
the more abundant network of thin fibrils (17–20 nm),
typical of developing cartilage, surrounding the chondro-
cytes derived from the cultured parent Swarm rat
chondrosarcoma tumor cells (Fig. 1B).
The results of radiolabeling with [
3
H]proline showed that
the cultured RCS-LTC cells continued actively to synthesize
and incorporate type II N-procollagen, and types IX and XI
collagens into the matrix, even after 1 month in culture
(Fig. 2). No fully processed type II collagen chains were
detected in the medium (Fig. 2, lane 1) or cell/matrix layer
(Fig. 2, lane 2). Digestion of the cell layer collagen with
AA
B
B
250 nm
250 nm
Fig. 1. Transmission electron-microscopy of cultured chondrosarcoma cells and surrounding matrix. (A) RCS-LTC chondrocyte. Note the presence of
thin filamentous fibrils pericellularly (arrowhead). Bar ¼ 1 lm. (B) Parent Swarm rat chondrosarcoma chondrocyte. Abundant, thicker collagen
fibrils (arrowhead) are seen. Bar ¼ 1 lm. The insets show a higher magnification (digital) of the area enclosed within each box (bar ¼ 250 nm).
Ó FEBS 2003 Cartilage collagen heteropolymer assembly (Eur. J. Biochem. 270) 3245
pepsin removed the N-propeptides from the a1(II) chains
(Fig. 2, lane 3).
No fully processed type II collagen a-chains were detected
at any time in the RCS-LTC cell cultures (Fig. 3, lanes 1 and
2). In the presence of bAPN for 1 month, the amount of

type II N-procollagen extractable in 0.15
M
potassium
phosphate was increased (Fig. 3, lane 2). The pepsin extract
of the residual collagen contained less type II collagen
(Fig. 3, lane 4) than that of the non-bAPN treated cultures
(Fig. 3, lane 3). These results are consistent with the
formation of lysyl oxidase-mediated cross-links in the
polymeric type II N-procollagen.
Having established that only unprocessed type II
N-procollagen was deposited in the RCS-LTC cultures, the
cross-linking properties of the collagen were analyzed. Type
II collagen was extracted with pepsin and component a1(II)
chains were purified by RP-HPLC (Fig. 4). A peak of
fluorescence, characteristic of trivalent pyridinolines, coin-
cidedwiththea1(II) chains from control rat cartilage and
from the RCS-LTC matrix. The elution position of the
a-chains was confirmed by SDS/PAGE (Fig. 4, inset).
Fractions containing the a1(II) chains from control tissue
and RCS-LTC cultures were hydrolyzed and analyzed by
C-18 reverse-phase HPLC for HP and LP, the two forms of
pyridinoline.
As seen in Fig. 5, both HP and LP were present in the
type II collagen deposited in the matrix by 1 week in culture.
With increasing time in culture, the total pyridinoline
content increased from 0.06 molÆmole
)1
of collagen
(1 week) to 0.13 molÆmole
)1

of collagen (4 weeks). In
comparison, the concentration in rat cartilage type II
collagen was 0.24 mol pyridinoline per mol of collagen.
Figure 6A compares the molecular sieve HPLC pat-
terns of cyanogen bromide-derived peptides from type II
collagen of the RCS-LTC cell layer (4 weeks in culture)
and control rat cartilage monitored for pyridinoline
fluorescence. Two peaks of fluorescent peptide of similar
yield were evident for both. They correspond to peptides
from the two cross-linking sites in the type II collagen
molecule that have been described previously [4,8]. The
results indicate that type II N-procollagen molecules are
polymerized and cross-linked as in fully processed type II
collagen. Direct assay for pyridinolines in the hydrolyzed
fractions confirmed the presence of HP and LP residues
(Fig. 6B).
To determine whether types IX and XI collagens,
which are also synthesized by these cells [19], can
copolymerize with the type II N-procollagen polymer,
the collagen network laid down by the cells after 2 weeks
in culture was depolymerized using pepsin. Pepsin cleaves
in the telopeptide domains of type II collagen and in the
noncollagenous domains of the minor collagens, type XI
and IX, but leaves their triple helical domains intact. The
short stubs of cleaved telopeptides remain cross-linked to
Fig. 2. Type II N-procollagen synthesized by RCS-LTC cells after
1month in culture. SDS/PAGE/fluorography of [
3
H]proline-labeled
protein. No fully processed a1(II) chains were detected in either the

medium (lane 1) or cell layer (lane 2). Lane 3: pepsin treatment of the
cell layer collagen converted the type II N-procollagen molecules to
fully processed a1(II) chains. All samples were run under nonreducing
conditions. Lanes 1 and 2, 10 nCiÆlane
)1
;lane3,5nCiÆlane
)1
.
Fig. 3. Effect of beta-aminoproprionitrile (bAPN) on the extractability
of type II collagen from the cell layer. No fully processed type II col-
lagen molecules are detected with or without bAPN in the RCS-LTC
cultures (lanes 1 and 2). The yield of soluble type II N-procollagen was
less from cultures in the absence of bAPN (lane 1). Addition of bAPN
(lane 2) increased the pool of type II N-procollagen extractable in
0.15
M
KH
2
PO
4
, pH 7.6, presumably by inhibiting lysyl oxidase-
mediated cross-linking. Pepsin-extracted type II collagen from
untreated (lane 3) and bAPN-treated (lane 4) cultures. Intact disulfide-
bonded type IX collagen chains were identified by MS after in-gel
trypsin digestion. Equal volumes were loaded for each extract and run
under nonreducing conditions.
3246 R. J. Fernandes et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the triple-helical sites to which they were attached in the
matrix, and the mAb 10F2 detects the pepsin-generated
neoepitope in the a1(II) C-telopeptide wherever it is

cross-linked to intact collagen chains or to chain
fragments. The various chains and chain fragments of
types II, IX and XI collagen were resolved by SDS/
PAGE. (Fig. 7A, lanes 4 and 5). Western blot analysis
(Fig. 7A, lanes 1 and 2) of the proteins resolved in lanes
4 and 5 using mAb 10F2 showed a strong reaction with
the a1(II) chain, as expected for the derivative of a cross-
linked type II collagen polymer [C-telopeptide of type II
collagen cross-linked to residue 87 of the a1(II) collagen
chain]. The antibody also reacted with the a1(XI) chains,
indicating that some of these chains had been cross-
linked to the C-telopeptide of the a1(II) chain and/or the
a3(XI) chain of type XI collagen, as they are the product
ofthesamegene,Col2A1. A third immunoreactive band
is evident only after reduction with dithiothreitol (lanes 1
and 2). This band, from its properties on SDS/PAGE
(lanes 4 and 5) and reaction with a polyclonal antibody
to type IX collagen [31] (lane 3), is a3(IX), in which the
COL2 domain is cross-linked to a cleaved C-telopeptide
from type II collagen.
Discussion
The rat chondrosarcoma cell line, RCS-LTC, expresses type
II collagen abundantly, but fails to process it beyond the
stage of N-procollagen molecules [24]. This presents a novel
system for studying whether chondrocytes can assemble
newly synthesized type II N-procollagen molecules into a
cross-linked fibrillar network, and whether types IX and XI
collagen molecules can be incorporated and cross-linked
into the nascent fibril. Yang et al. [15] have shown, by fibril-
forming assays in vitro, that vitreous type II N-procollagen

Fig. 4. Purification of pepsin-solubilized type II collagen a-chains from
the cell layer of RCS-LTC cultures. Pepsin-solubilized type II collagen
from rat cartilage (upper panel) was resolved by reverse-phase HPLC
as a control for comparison with the RCS-LTC digest (lower panel).
Fractions marked by a bar contained a1(II) chains, as shown by SDS/
PAGE (inset), and were pooled for cross-link analysis.
Fig. 5. Detection of pyridinoline cross-links in isolated a1(II) collagen
chains. Reverse-phase HPLC analysis of the a1(II) chains from 1-week
and 1-month RCS-LTC cultures (lower panels) contain both hyd-
roxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) cross-links.
Control type II collagen prepared from rat cartilage contains only HP
cross-links (upper panel).
Ó FEBS 2003 Cartilage collagen heteropolymer assembly (Eur. J. Biochem. 270) 3247
can form thin fibrillar coassemblies when mixed with
mature type II collagen. However, their study used purified
extracted collagens, so questions on the relevance to
physiological assembly remain.
The results show that type II N-procollagen molecules
are deposited in an extracellular matrix by the RCS-LTC
cells and become cross-linked by the lysyl oxidase
mechanism. Despite the retained N-propeptides, cross-
linking also progressed to the stage of mature pyridin-
oline residues by 1 week in culture. The best explanation
for this is that the procollagen molecules were assembled
into microfibrils with the precise molecular stagger and
proximities required for complex cross-links to form,
even at this early stage of fibril formation (Fig. 5). It has
been reported that the mature vitreous contains a mix of
types IIA and IIB N-procollagens [15,17,18] in its gel-like
matrix [18,33].

HP is the predominant cross-link in type II collagen of
normal rat cartilage. The significant proportion of LP
present at the two cross-linking sites in the RCS-LTC type
II collagen molecule indicates an under-hydroxylation of
lysine residues at these two triple-helical cross-linking
residues. The rapid cell doubling time of 21 h, and the high
rate of synthesis of collagen [19], may be factors contribu-
ting to this under-hydroxylation. An under-hydroxylation
of cross-links, compared with the equivalent tissue collagen,
has been observed for type I collagen synthesized by
primary chick osteoblasts in culture [34]. In contrast, over-
hydroxylation has been observed in type I collagen
synthesized by the SAOS-2 cell line in culture, and linked
to an over-expression of lysyl hydroxylase 1 [35]. It is
unknown whether the presence of LP in place of HP confers
any distinctive properties on the collagen fibril. Despite the
presence of mature pyridinoline cross-links, usually associ-
ated with stiff, resilient connective tissues, the matrix was a
highly hydrated gel in texture [19], similar in gross appear-
ance to vitreous humor of the eye. As aggrecan is also
deposited in the matrix in large amounts by these cells [20],
the fine filamentous collagenous network was presumably
distended by the osmotic swelling pressure of entrapped
aggrecan.
The N-propeptide of type IIB collagen is essentially a
short triple-helical domain that folds back onto the
N-terminus of the main triple-helix, and so exposes the
N-propeptidase cleavage site [36,37]. Its presence could
sterically interfere with the cross-linking interactions of the
adjacent N-telopeptide domain. The RCS-LTC cell line

provides a useful model for studying whether this occurs,
as the cells deposit only type II N-procollagen (no fully
processed molecules) and the N-propeptide appears to be
folded correctly as it is cleaved by conditioned medium from
normal chondrocytes [24] and by ADAMTS-2 (the known
fibrillar collagen N-propeptidase) and ADAMTS-3 (the
putative collagen N-propeptidase of cartilage) [25]. The
present results indicate that mature trivalent pyridinoline
residues are formed in equal amounts at both ends of the
molecule, where they link two C-telopeptides to residue 87
and two N-telopeptides to residue 930. Control rat cartilage
showed a similar result (Fig. 6). This implies that the
N-propeptide of type IIB N-procollagen does not sterically
interfere with aldehyde formation by lysyl oxidase or the
linkage of the N-telopeptide to helical residue 930 and
Fig. 6. Analysis of collagen type II cyanogen bromide-derived peptides.
(A) Chromatogram of cyanogen bromide-derived peptides from
digestion of type II collagen purified from rat cartilage (upper panel)
and the RCS-LTC matrix (lower panel), as resolved by molecular sieve
HPLC. Pyridinoline fluorescence is detected in peptides that are
derived from the two cross-linking sites in type II collagen CB12 X
(C-telo)
2
and CB9,7 X (N-telo)
2
. (B) Reverse-phase HPLC analysis
confirming that hydroxylysyl pyridinoline (HP) and lysyl pyridinoline
(LP) are responsible for the fluorescence in the cross-linked cyanogen
bromide-derived peptides from RCS-LTC type II collagen.
Fig. 7. Western blot analysis showing intertype cross-linking between

collagens II, IX and XI. (A) Pepsin-solubilized RCS-LTC matrix col-
lagens [in the presence and absence of dithiothreitol (lanes 1 and 2,
respectively)] were probed using mAb 10F2, which specifically recog-
nizes the C-telopeptide domain of type II collagen. Lanes 4 and 5 show
Coomassie Blue-stained samples equivalent to those in lanes 1 and 2.
Lane 3 shows a Western blot of a sample similar to that in lane 1 but
probed with an antibody to type IX collagen. (B) Molecular inter-
pretation of the results of Western blot analysis. Antibody 10F2
reactedwiththea1(II), a1(XI) and a3(IX) chains, showing that
C-telopeptide domains of type II collagen had become cross-linked to
type II, XI and IX collagen molecules in the matrix. These heterotypic
cross-linking reactions have all been demonstrated in the collagen
heteropolymers that form the matrix of developing cartilage in vivo
[2,3].
3248 R. J. Fernandes et al. (Eur. J. Biochem. 270) Ó FEBS 2003
subsequent interaction with a second N-telopeptide to form
pyridinoline. Similarly, the cross-linking of C-telopeptides
to helical residue 87 also proceeded to pyridinoline. As
electron micrographs of the matrix showed no obvious
collagen fibrils, only fine filaments (Fig. 1), we can speculate
that the retained N-propeptides had prevented lateral
growth of the nascent type II N-procollagen assembly
beyond a limiting size. Immunolocalization of N-propep-
tides to thin fibrils of skin type I N-procollagen, but not to
thick fibrils [38], supports this speculation. It is probable
that type II N-procollagen forms fine fibrils in developing
cartilage, but this is more obvious with the RCS-LTC cells
because none of the type II N-procollagen is processed.
This system demonstrated also the incorporation of
collagens IX and XI into the collagen II heteropolymer that

characterizes developing cartilage [39], as an early, integral
process. The results of Western blotting with antibody 10F2
establish that type II collagen molecules are covalently
linked to both types IX and XI collagens in the forming
matrix (Fig. 7). This antibody specifically recognizes a
protease-generated cleavage site in the cleaved C-telopeptide
domain of type II collagen when it is cross-linked to triple-
helical sequences [40]. Hence, on electrophoresis of a pepsin
digest of cell layer collagen, the a1(II) chain is heavily
stained, but the a3(IX) COL2 domain and the a1(XI) chain
are also recognized strongly by the antibody. These results
are consistent with the known cross-linking properties and
chain-specific interactions of the collagen II C-telopeptide
domain with collagens IX and XI in cartilage (Fig. 7B) [2,3].
Cross-linking of the minor collagens, IX and XI, to the type
II N-procollagen polymer at 2 weeks and a nascent stage of
fibril growth, supports the early integration of types IX and
XI collagens during collagen II fibrillogenesis.
From the present data we can speculate that the type II
N-procollagen heteropolymer is assembled by RCS-LTC
cells, during or soon after secretion, in the form of a
microfibril. The concept of a microfibril was introduced by
Smith [41]. In the Smith microfibril, a unit of five type I
collagen molecules, staggered by 67 nm (234 amino acids),
repeats to form a microfibril of 4-nm diameter [42–46]. For
type II collagen it was further speculated that such
microfibrils associate laterally with the minor cartilage
collagen molecules (types IX and XI) [47]. On theoretical
grounds it was concluded that type II N-procollagen can
probably form such a microfiber, but that further lateral

growth will be sterically hindered by the N-propeptides [45].
Although microfibrils of 4-nm diameter have not been
visualized convincingly, the concept is consistent with
electron microscopic findings on the RCS-LTC extracellular
matrix, showing thin filaments in an otherwise amorphous
background (Fig. 1).
In summary, the findings support the integration and
intermolecular cross-linking of type II N-procollagen with
types IX and XI collagen molecules at an early stage in the
process of collagen network formation by chondrocytes.
Acknowledgements
The authors thank Kae Ellingsen for help in preparing the manuscript
and Tom Eykemans for his expertise with the graphics. This work was
supported by grants AR37318, AR36794 and AR39239 from the
National Institutes of Health.
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