(Received for publication, May 11, 1995)
From the
The collagen framework of hyaline cartilage is based on
copolymers of types II, IX, and XI collagens. Previous studies have
established specific covalent interactions between types II and IX
collagens. The present study examined cross-linking sites in type XI
collagen to define better the full heteropolymeric assembly.
Pepsin-solubilized type XI collagen was purified from fetal bovine
cartilage. The cross-linking amino acids in the preparation were
primarily divalent, borohydride-reducible structures; pyridinoline
residues were essentially absent. Individual 1(XI),
2(XI),
and
3(XI) chains were resolved by high performance liquid
chromatography. Telopeptides still attached by cross-links to helical
sites were released by periodate oxidation and identified by
microsequencing. Analysis of cross-linked peptides isolated from
trypsin digests of each
-chain identified the attachment helical
sites for the telopeptides. A high degree of interchain specificity was
evident in the cross-linking between type XI collagen molecules. The
dominant cross-links were between N-telopeptides and the COOH terminus
of the triple-helix, consistent with a head-to-tail interaction of
molecules staggered by 4D (D = 67 nm) periods. In addition,
1(II) C-telopeptide was linked to the amino-terminal site of the
1(XI) triple helix. In summary, the results show that type XI
collagen molecules are primarily cross-linked to each other in
cartilage, implying that a homopolymer is initially formed. Links to
type II collagen are also indicated, consistent with an eventual
cofibrillar assembly. Analysis of cartilage extracts showed that all
three chains,
1(XI),
2(XI), and
3(XI), had at least in
part retained their N-propeptides in cartilage matrix and that the
3(XI) chain was the IIB splicing variant product of the COL2A1
gene. Of particular note was the finding that the N-telopeptide
cross-linking site in both
1(XI) and
2(XI) is located
amino-terminal to the putative N-propeptidase cleavage site. This
structural feature provides a potential mechanism for the proteolytic
depolymerization of type XI collagen by proteases that can cleave
between the cross-link and the triple helix (e.g. stromelysin).
Hyaline cartilage contains three tissue-specific collagens, types II, IX, and XI, that form cross-linked copolymers in the tissue's fibrillar framework(1, 2, 3) . Collagen types VI, XII, and XIV are also present in small amounts but appear not to form covalently cross-linked polymers since they are extractable in denaturing solvents(4, 5) .
Type XI
collagen can be purified from 4 °C pepsin-extracts of hyaline
cartilage by precipitation from 1.2 M NaCl at pH
2-3(6) . This protein accounts for about 10% of the total
collagen of fetal cartilage but decreases to about 3% in adult
articular cartilage(7) . Native type XI collagen yields three
genetically distinct chains, the primary native molecule being the
heterotrimer, 1(XI)
2(XI)
3(XI)(8) . As articular
cartilage matures, however, the collagen
1(V) chain is detected in
the isolated pool of type XI collagen. The existence of heterotypic
molecules containing
1(V) chains is, therefore,
indicated(2, 4) . The
1(XI),
2(XI), and
1(V) chains are related in sequence (9, 10, 11, 12, 13, 14) ,
whereas
3(XI) is a product of the COL2A1 gene, differing only in
post-translational quality from
1(II)(6, 8) .
Type XI collagen is a fibril-forming molecule in the same general class as types I, II, III, and V, all of which form 67-nm banded fibrils (15) and are cross-linked by the lysyl oxidase-mediated mechanism(2) . A similar pattern of cross-linking residues has been observed in collagen types II, IX, and XI(1) .
Type II
collagen in mature cartilage is cross-linked primarily by hydroxylysyl
pyridinoline(16, 17) . These trivalent cross-links
occur at two intermolecular sites, one linking two C-telopeptides ()to residue 87 of the triple helix and the other linking
two N-telopeptides to residue 930 of the triple
helix(18, 19, 20) . Molecules of type IX
collagen have an interrupted triple helical structure. They are seen
decorating the surface of thin fibrils of type II collagen in
cartilage(21, 22) , and the formation of covalent
cross-links between type II and type IX collagen has been
established(20, 23, 24) . The relative
locations of the intermolecular cross-linking domains also demonstrated
that type IX molecules are cross-linked to each other and predicted
that the mode of interaction between types II and IX molecules is
anti-parallel(25) .
The present study focused on determining the location and structure of cross-linked domains of type XI collagen extracted from cartilage matrix to understand the polymeric properties of the protein and its potential interactions with type II collagen in cartilage.
Individual 1-,
2-, and
3-chains of
pepsin-extracted type XI collagen were resolved by reverse-phase HPLC (Fig.1). All three chains revealed borotritide-labeled residues (Fig. 1). No pyridinoline cross-links were detected in the type
XI collagen purified from fetal articular cartilage (<0.01 residues
of hydroxylysylpyridinoline/collagen molecule; data not shown).
Figure 1:
Reverse-phase
HPLC separation of the -chains of pepsin-extracted fetal bovine
type XI collagen. The [
H]NaBH
-treated
type XI collagen molecules were denatured and chromatographed on a C8
column to resolve the three type XI collagen chains (see
``Materials and Methods'' for detail). Aliquots of collected
fractions (100 µl) were assayed for tritium
activity.
Fig.2compares reverse-phase HPLC profiles of tryptic
peptides from 1(II) and
3(XI) chains of fetal cartilage. The
UV absorbance profiles are essentially the same, indicating the same
primary structure (Fig.2a), confirmed by
amino-terminal sequencing of several peaks (data not shown; see also Fig.7and Fig. 9). However,
3(XI) was quite
different from
1(II) in its cross-linking profile, lacking
pyridinolines (Fig.2b) and featuring
borohydride-reducible cross-links (Fig.2c). The
H activity of
3(XI) was four times that of
1(II).
Amino-terminal Edman degradation revealed two peptide sequences from
each of the cross-linked peptides. The molecular sites of the divalent
cross-links and pyridinolines in type II collagen were the
same(20) , with N-telopeptides (DEXAGGAQ) linked to
residue 930 and C-telopeptides (GQREXGPDP) linked to residue
87. In contrast, only one helical site of cross-linking was found to be
occupied in
3(XI), linking the sequence GLXGHR, where X is the known cross-linking hydroxylysine at residue 930, to
the N-telopeptide domain of
1(XI). The two tritium-labeled tryptic
peptides resolved from
3(XI) (Fig.2c) both gave
sequences indicating an origin at this site but differing in pepsin
cleavage site.
Figure 2:
Reverse-phase HPLC fractionation of
tryptic peptides prepared from 3(XI) and
1(II) chains. a, UV absorbance; b, fluorescence, specific for
detecting pyridinoline residues; c, tritium activity. The
sample (1 mg) was eluted from a C8 column (Brownlee Aquapore RP-300; 25
cm
4.6 mm) with a linear gradient (0-30%) of solvent B in
A over 60 min at a flow rate of 1 ml/min. Solvent A was 0.1%
trifluoroacetic acid (v/v) in water, and solvent B was 0.085%
trifluoroacetic acid (v/v) in acetonitrile-n-propanol (3:1,
v/v). Shown in c are the amino-terminal sequences of the
isolated
H-labeled cross-linked peptides. Note that the
3-chain of type XI collagen differs from type II collagen in its
cross-linking properties, containing only borohydride-reducible
cross-links and no fluorescent
pyridinolines.
Figure 7:
Amino-terminal sequences determined for
the intact 1(II) and
3(XI) chains from bovine cartilage.
Samples were treated with pyroglutamate aminopeptidase (30) to
remove the blocking pyroglutamate residues from both
1(II) and
3(XI) chains before SDS-PAGE and polyvinylidene difluoride
transblotting. Arrows locate the sequences in the
molecule.
Figure 9:
Reverse-phase HPLC of collagen XI
N-propeptides. Fractions marked by the bar in Fig.8were concentrated and eluted on reverse-phase HPLC by a
0-30% linear gradient of B in A over 60 min (see Fig.2legend). The elution positions and amino-terminal
sequences of the helical domains from each type XI chain N-propeptide
are shown. Material eluting in fractions 22-26 proved to be an
over-cleavage product of the 1(II) main helix, beginning at
residue 938.
Figure 8:
Gel filtration chromatography to isolate
collagen XI N-propeptides. The 4 M NaCl-precipitate (5 mg)
from a pepsin digest of fetal cartilage was eluted from a Bio-Gel P-60
column (Bio-Rad, less than 400 mesh, 1 50 cm) previously
equilibrated in 0.05 M Tris, 0.15 M NaCl, pH 7.5, at
25 °C. Fractions indicated by the bar were pooled for
further purification.
Fig.3shows the reverse-phase HPLC elution
profile of tryptic peptides from the 1(XI) and
2(XI) chains.
Peptides containing
H activity were sequenced either
directly or after further purification by anion exchange and
reverse-phase HPLC (chromatographic steps not shown). All
H-labeled peptides gave two running sequences on Edman
degradation (in about 1:1 molar ratio). Peptide A1 matched an
3(XI) (or
1(II)) C-telopeptide linked to the cross-linking
domain near the amino terminus of
1(XI). Peptides A2 and A3 both
gave an
2(XI) N-telopeptide sequence and a fragment containing the
putative cross-linking site near the COOH terminus of
1(XI). The
helical sequence of A3 was 39 residues longer than that of A2 at the
amino terminus by comparison with the published cDNA(9) .
Incomplete hydroxylation of lysine residue 921 resulting in partial
cleavage by trypsin can explain this.
Figure 3:
Reverse-phase HPLC fractionation of
tryptic peptides from 1(XI) and
2(XI) chains. The elution
conditions were as described in Fig.2. The tritiated peptides,
A1-A3 and B1-B3, were purified by ion-exchange (DEAE) HPLC then
reverse-phase (C8) HPLC and identified by protein microsequencing. The
paired sequences found for each peak are indicated. Chain assignments
were aided by comparison with the human cDNAs for
1(II),
1(XI), and
2(XI)(9, 10, 11, 14, 32, 44) . P*, 4-hydroxyproline; K*, hydroxylysine; X,
cross-linking hydroxylysine residue.
All three cross-linked
peptides under peaks B1, B2, and B3 were derived from a site of
covalent interaction between hydroxylysine residue 924 of the
2(XI) triple helix and an
3(XI) (or
1(II))
N-telopeptide, the latter presumably providing the hydroxylysine
aldehyde.
The individual (XI) chains isolated by reverse-phase
HPLC (Fig.1) were treated with sodium periodate to release
cross-linked telopeptides from the helical sites. The
1(XI) chain
released two
H-labeled peptides (Fig.4), one a
fragment of the
1(II) (or
3(XI)) C-telopeptide, the other of
the
2(XI) N-telopeptide (see Fig.3). The
2(XI) chain
gave two
H-labeled peptides; both were fragments of the
3(XI) (or
1(II)) N-telopeptide, one bearing an additional
amino-terminal phenylalanine. The
3(XI) chain yielded two
tritiated peptides, both from the
1(XI) N-telopeptide.
Figure 4:
Reverse-phase HPLC fractionation of
tritiated telopeptides released by periodate from isolated 1(XI),
2(XI), and
3(XI) chains. The conditions are as in Fig.2, eluting with 0.1% (v/v) trifluoroacetic acid for 5 min
and then with a linear gradient (0-35%) of solvent B in A over 35
min. The results of amino-terminal sequencing are indicated, where X was the tritium-labeled
-hydroxynorvaline residue
derived from the cross-link.
Fig.5summarizes the results identifying molecular sites of
cross-linking in collagen type XI. The molecules must be cross-linked
in cartilage matrix primarily in a head-to-tail fashion. The identified
cross-links are mostly derived from type XI to type XI interactions. Of
the two prospective cross-linking sites in the triple-helix of the
molecule, only one appears to be occupied (the one near the COOH
terminus). Each chain (1(XI),
2(XI), and
3(XI)) shows
preferential links to a specific telopeptide. Thus,
1(XI) is
linked to an
2(XI) N-telopeptide,
2(XI) to an
3(XI) (or
1(II)) N-telopeptide, and
3(XI) to an
1(XI)
N-telopeptide. Only one chain,
1(XI), was occupied at the amino
end of the helix (residue 84), where the
1(II) (or
3(XI))
C-telopeptide was attached. Because the latter sequence is identical in
1(II) and
3(XI), the molecular origin, whether type II or XI,
is uncertain.
Figure 5:
A summary of the identified cross-linking
sites in the triple-helical domain of bovine type XI collagen. Residue
numbers shown in this figure and elsewhere in the manuscript are
counted from the beginning of the main triple helix, based on the
published cDNA data for human 1(XI),
2(XI), and
1(II)
chains(9, 10, 11, 14, 32) .
The telopeptide residue numbers are counted back from the start of the
main triple helix for N-telopeptides and forward for
C-telopeptides.
The location of cross-linking hydroxylysine residues
in the N-telopeptide domains of 1(XI) and
2(XI) was an
unexpected finding. From cDNA data(11, 14) , there are
no lysines in the putative telopeptide domains as defined by the
predicted N-propeptidase cleavage site. It turns out, however, that the
non-helical cross-linking sites in the
1- and
2-chains are
located outside the putative N-propeptidase cleavage locus (Fig.6). Therefore, the N-propeptide extensions on the type XI
collagen molecule must not be cleaved at these sites in the
cross-linked extracellular protein. The following results confirmed
this.
Figure 6: Identified sites of cross-linking in the N-telopeptides of bovine type XI collagen. Solidletters indicate residues determined by sequence analysis; outlinedletters were not determined but represent the human cDNA predictions for these sites(14, 32) . The verticaldashedline marks the junction between N-telopeptides and the main triple helix. Solidarrows show the cross-linking hydroxylysine residues, and the openarrow shows the N-propeptidase cleavage site in type II collagen.
Amino-terminal sequencing of the 3(XI) chain from native
molecules of type XI collagen extracted by 1 M NaCl showed
that it retained its full N-propeptide extension. The
1(II) chain
from native type II collagen molecules in the same extract did not (Fig.7). The sequence data also showed that the
3(XI)
chain was the IIB splicing product of the COL2A1 gene, which lacks the
exon 2 coding domain(31) .
Triple helical domains from the
N-propeptides of type XI collagen were isolated from the 4 M NaCl precipitated fraction of a pepsin digest of fetal bovine
cartilage. Proteoglycans and uncross-linked collagen molecules had been
removed from the tissue by extraction in 4 M guanidine HCl
before digestion. Fig.8shows the resolution of the
N-propeptide fragments by molecular sieve chromatography. The earliest
peak included pepsin and the COL1 domains of type IX collagen. The
second peak contained mainly collagenous fragments of type XI
N-propeptides. Individual peptides in the fractions shown by the bar were isolated by reverse-phase HPLC. Peptides were
identified by amino-terminal sequencing (Fig.9). The 3(XI)
sequence was matched to that reported for human COL2A1
cDNA(32) . The yields of
1(XI),
2(XI), and
3(XI)
fragments were about 1:1:1. The results are consistent with type XI
collagen molecules in their polymeric, cross-linked state in cartilage
matrix, retaining their N-propeptides at least to the extent of their
triple helical domains.
In cartilage, collagen fibrils are strengthened by covalent intermolecular bonds. Types II, IX, and XI collagens are all present as covalently cross-linked polymers in cartilage, but their precise distribution and molecular inter-relationships are unclear. Both types II and IX collagens contain the trivalent mature pyridinoline cross-linking residues, even in the fetal tissue, but most prominently in adult cartilage (Fig.2)(20, 24, 25) . However, in type XI collagen the cross-links remain as the initial divalent ketoamines even in the mature tissue(2, 33) . Before molecular sieve purification, some type IX collagen (COL2 domain) was evident in the preparation on an SDS-PAGE immunoblot using anti-type IX collagen antiserum(34) . This contamination may in part contribute to the low content of pyridinoline residues observed in less rigorously purified type XI collagen preparations(2) . Thus, type XI collagen prepared from fetal epiphyseal cartilage contains only divalent borohydride-reducible ketoamines. No pyridinoline residues are detected, whereas collagens II and IX even from fetal cartilage contain significant amounts of pyridinoline (24, 25) (Fig.2).
Type XI collagen from
fetal cartilage consist mostly of heterotrimeric molecules of
composition (1(XI)
2(XI)
3(XI))(8) . The present
structural analyses on the cross-linking sites show that highly
specific, non-random molecular interactions had occurred in the tissue.
The microsequencing results on purified tryptic peptides that contained
the divalent cross-links and on telopeptides released by periodate
indicated that type XI collagen molecules are primarily cross-linked to
each other head-to-tail in cartilage matrix (Fig.10). The
cross-links are chain-specific between N-telopeptides and COOH-terminal
helical sites in adjacent molecules (i.e.
1 N-telopeptide
to
2 COOH-terminal helix,
2 N-telopeptide to
3
COOH-terminal helix, and
3 N-telopeptide to
1 COOH-terminal
helix; Fig.5). No firm conclusion is possible on the source of
the C-telopeptide sequence found attached to residue 84 in
1(XI), i.e. whether from type II collagen, the
3-chain of type
XI collagen, or both. Similar analyses of type V collagen of bone
showed the occurrence of heterotypic intermolecular cross-linking
between types I and V collagens, mediated by a C-telopeptide of
1(I) linked to a COOH-terminal helical site in
1(V)(7, 35) . The structure and cross-linking
properties of types V and XI collagens appear to be similar, except
that type XI associates with type II and type V associates with type I.
It has been shown by immunoelectron microscopy that type XI collagen is
codistributed with type II collagen in cartilage
matrix(3, 36) . From the type V collagen evidence, we
suspect, therefore, that the C-telopeptide sequence recovered from the
1(XI) chain originated primarily in molecules of type II collagen.
Figure 10: Molecular conclusions from the results. Most of the cross-linking bonds formed by type XI collagen molecules are homopolymeric. Molecules cross-link head to tail, staggered by 4D (D = 67 nm) periods. The three modes of chain interaction are shown. Unlike collagen types I and II, N-propeptides are retained in the cross-linked polymer. These propeptides may function in the matrix to regulate fibril growth and intermolecular cross-linking.
From cDNA data, both 1(XI) and
2(XI) include an unusually
large globular amino terminus (NC3), which together with the
collagenous domain (COL2) forms the N-propeptide. Each N-propeptide
accounts for about 25% of the total chain
mass(9, 10, 11, 14) . The present
results confirm that, in part at least, the N-propeptides of all three
chains,
1(XI),
2(XI) and
3(XI), are retained on the
molecules cross-linked in the extracellular matrix of cartilage (Fig.9)(8, 37) . This is consistent with
observations on type V collagen (38) . The propeptides
presumably confer specific properties on the matrix, for example by
regulating the intermolecular cross-linking properties of the molecule
or restricting the lateral growth of fibrils.
Prior work has indicated that type XI collagen may preferentially exist in the form of thin fibrils, which can be heteropolymeric structures embodying types II and IX collagens(3, 36, 39, 40, 41) . It has been shown that type IX collagen molecules decorate and are covalently linked to the surface of such fibrils(20, 21, 22, 23, 24, 25) , whereas type XI collagen molecules appear to be in the interior(3, 36) . The present results are consistent with the latter concept but add the knowledge that type XI collagen molecules must be cross-linked primarily to each other. It is not clear whether they form a core at the center of fibrils (3, 36) or are distributed under the surface of type II fibrils as proposed for types V and I copolymeric collagen fibrils in cornea(42) .
Previous studies have shown essentially the
same CNBr-peptide profile for 3(XI) as
1(II), except for
slower
3(XI) peptide mobilities on SDS-PAGE. This difference is
explained by post-translational
over-modification(2, 6, 8) . Another noted
difference was in the mobility of peptide CB9,7(27) . The
present results confirm identity in primary structure of
1(II) and
3(IX) but show cross-linking differences. The most likely
explanation, therefore, for the mobility shift on SDS-PAGE of CB9,7 is
the attachment of a degradation product of the N-propeptide of
1(XI) instead of
1(II) to helical residue 930 (Fig.2).
Of particular interest also was the finding that
the N-telopeptide cross-linking lysines in 1(XI) and
2(XI)
lie amino-terminal rather than COOH-terminal to the putative
N-propeptidase cleavage sites. Because stromelysin (MMP3) can cleave
native type XI collagen molecules at sites between the N-telopeptide
cross-links and the main triple-helix(26) , the findings imply
a chondrocyte mechanism for remodeling of the collagenous matrix.
Chondrocytes are capable of producing stromelysin(43) , and
stromelysin has already been shown to be capable of degrading type IX
collagen and telopeptide domains of type II collagen(26) .
Thus, selective proteolysis of type XI and type IX collagens could
bring about changes in fibril architecture without removing the bulk
fabric.