©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Analysis of Cross-linking Domains in Cartilage Type XI Collagen
INSIGHTS ON POLYMERIC ASSEMBLY (*)

(Received for publication, May 11, 1995)

Jiann-Jiu Wu (§) David R. Eyre

From theDepartment of Orthopaedics, Orthopædic Research Laboratories, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha1(XI), alpha2(XI), and alpha3(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 alpha-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, alpha1(II) C-telopeptide was linked to the amino-terminal site of the alpha1(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, alpha1(XI), alpha2(XI), and alpha3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the alpha3(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 alpha1(XI) and alpha2(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).


INTRODUCTION

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, alpha1(XI)alpha2(XI)alpha3(XI)(8) . As articular cartilage matures, however, the collagen alpha1(V) chain is detected in the isolated pool of type XI collagen. The existence of heterotypic molecules containing alpha1(V) chains is, therefore, indicated(2, 4) . The alpha1(XI), alpha2(XI), and alpha1(V) chains are related in sequence (9, 10, 11, 12, 13, 14) , whereas alpha3(XI) is a product of the COL2A1 gene, differing only in post-translational quality from alpha1(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 (^1)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.


MATERIALS AND METHODS

Preparation of Type XI Collagen

Articular cartilage was sliced by scalpel from the epiphyses of 5-6-month-old fetal calves. The tissue was extracted in 1 M NaCl, 0.05 M Tris-HCl, pH 7.4, as a source of newly made, uncross-linked collagen molecules. Native types IX and XI collagens were purified from this extract by sequential precipitation with (NH(4))(2)SO(4) and then NaCl(26) . The tissue residue was then extracted in 4 M guanidine HCl, 0.05 M Tris, pH 7.4, at 4 °C for 24 h to remove proteoglycans, washed thoroughly with water, and freeze-dried. Cross-linked collagens were solubilized by digestion with pepsin at 4 °C and fractionated by precipitation at 0.7, 1.2, and 2.0 M NaCl(1) . Type XI collagen, which precipitated at 1.2 M NaCl, was further purified by redissolving in 3% acetic acid and precipitating again at 1.2 M NaCl after removing residual type II collagen at 0.9 M NaCl(27) . The purified type XI collagen was treated with NaB^3H(4) (10 Ci/mol) in 0.1 M sodium phosphate, pH 7.4, to reduce and label ketoamine cross-links(28) . After 1 h at 25 °C, the reaction mixture was acidified, dialyzed against 0.1 M (v/v) acetic acid, and freeze dried.

N-propeptides

After precipitating collagen types II, XI, and IX at 0.7 M, 1.2 M and 2.0 M NaCl, the supernatant was adjusted to 4.0 M NaCl, conditions which precipitated the N-propeptides of type XI collagen.

Column Chromatography

Collagen alpha-chains were isolated by elution from an agarose A5m molecular sieve column (170 1.5 cm, 200-400 mesh, Bio-Rad) in 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5. Type IX collagen degradation products that coeluted with the type XI alpha-chains were removed by rechromatography on the same column after reduction and carboxymethylation of cysteine residues. Individual alpha-chains of type XI collagen were resolved by reverse-phase HPLC (C8 column, Brownlee Aquapore RP-300, 4.6 mm 25 cm) using a linear gradient (23-32%) of acetonitrile:n-propanol (3:1, v/v) in aqueous 0.1% (v/v) trifluoroacetic acid at 1 ml/min over 40 min, monitoring 220 nm absorbance. Aliquots (100 µl) of collected fractions were assayed for ^3H activity by scintillation spectrometry. Peptides containing pyridinoline cross-linking residues were detected by monitoring for fluorescence (excitation, 297 nm; emission, >380 nm) using a Kratos model 970 HPLC fluorescence detector(19) . Tryptic peptides containing ^3H-labeled cross-links were purified by ion-exchange and reverse-phase HPLC. The N-propeptide collagenous extensions of type XI collagen were purified by sieving on a polyacrylamide P60 column (1.0 50 cm, <400 mesh, Bio-Rad), eluted with 0.15 M NaCl, 0.05 M Tris-HCl, pH 7.5, followed by reverse-phase HPLC.

Periodate Cleavage

Purified alpha-chains containing ^3H-labeled cross-links were reacted with sodium metaperiodate (NaIO(4)) to cleave the borohydride-reduced ketoamine cross-links(28) . After cleavage, the tritium remains with the residue, hydroxynorvaline, which is derived from the hydroxylysine aldehyde donor of the cross-link in the telopeptide sequence. The resulting tritiated telopeptides were resolved by reverse-phase HPLC and structurally identified by protein microsequencing.

Trypsin Digestion

Purified type II and XI alpha-chains were dissolved in 0.2 M NH(4)HCO(3) at 2 mg/ml, heat denatured to 70 °C for 10 min, and digested with trypsin (1:50 w/w, Boehringer Mannheim sequencing grade) for 4 h at 37 °C. Digests were acidified with acetic acid and freeze dried.

Gel Electrophoresis and Electroblotting

Collagen fractions were routinely examined by SDS-PAGE(29) . For amino-terminal microsequencing, protein bands were transblotted to polyvinylidene difluoride membrane (Westran, Schleicher & Schuell) using a MilliBlot-SDE electroblotting apparatus(25) .

Amino-terminal Sequence Analysis

Peptides were subjected to amino-terminal sequencing on a Porton 2090E gas-phase sequencer equipped with on-line HPLC analysis of phenylthiohydantoin-derivatives. To avoid losses in yield, small peptides were covalently attached to Sequelon-AA membrane by carbodiimide activation as described by the manufacturer (Millipore). For amino-terminal sequencing of intact alpha1(II) and alpha3(XI) chains, blocking amino-terminal pyroglutamate residues were removed enzymatically before SDS-PAGE(30) .


RESULTS

Individual alpha1-, alpha2-, and alpha3-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 alpha-chains of pepsin-extracted fetal bovine type XI collagen. The [^3H]NaBH(4)-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 alpha1(II) and alpha3(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, alpha3(XI) was quite different from alpha1(II) in its cross-linking profile, lacking pyridinolines (Fig.2b) and featuring borohydride-reducible cross-links (Fig.2c). The ^3H activity of alpha3(XI) was four times that of alpha1(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 alpha3(XI), linking the sequence GLXGHR, where X is the known cross-linking hydroxylysine at residue 930, to the N-telopeptide domain of alpha1(XI). The two tritium-labeled tryptic peptides resolved from alpha3(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 alpha3(XI) and alpha1(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 ^3H-labeled cross-linked peptides. Note that the alpha3-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 alpha1(II) and alpha3(XI) chains from bovine cartilage. Samples were treated with pyroglutamate aminopeptidase (30) to remove the blocking pyroglutamate residues from both alpha1(II) and alpha3(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 alpha1(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 alpha1(XI) and alpha2(XI) chains. Peptides containing ^3H activity were sequenced either directly or after further purification by anion exchange and reverse-phase HPLC (chromatographic steps not shown). All ^3H-labeled peptides gave two running sequences on Edman degradation (in about 1:1 molar ratio). Peptide A1 matched an alpha3(XI) (or alpha1(II)) C-telopeptide linked to the cross-linking domain near the amino terminus of alpha1(XI). Peptides A2 and A3 both gave an alpha2(XI) N-telopeptide sequence and a fragment containing the putative cross-linking site near the COOH terminus of alpha1(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 alpha1(XI) and alpha2(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 alpha1(II), alpha1(XI), and alpha2(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 alpha2(XI) triple helix and an alpha3(XI) (or alpha1(II)) N-telopeptide, the latter presumably providing the hydroxylysine aldehyde.

The individual alpha(XI) chains isolated by reverse-phase HPLC (Fig.1) were treated with sodium periodate to release cross-linked telopeptides from the helical sites. The alpha1(XI) chain released two ^3H-labeled peptides (Fig.4), one a fragment of the alpha1(II) (or alpha3(XI)) C-telopeptide, the other of the alpha2(XI) N-telopeptide (see Fig.3). The alpha2(XI) chain gave two ^3H-labeled peptides; both were fragments of the alpha3(XI) (or alpha1(II)) N-telopeptide, one bearing an additional amino-terminal phenylalanine. The alpha3(XI) chain yielded two tritiated peptides, both from the alpha1(XI) N-telopeptide.


Figure 4: Reverse-phase HPLC fractionation of tritiated telopeptides released by periodate from isolated alpha1(XI), alpha2(XI), and alpha3(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 (alpha1(XI), alpha2(XI), and alpha3(XI)) shows preferential links to a specific telopeptide. Thus, alpha1(XI) is linked to an alpha2(XI) N-telopeptide, alpha2(XI) to an alpha3(XI) (or alpha1(II)) N-telopeptide, and alpha3(XI) to an alpha1(XI) N-telopeptide. Only one chain, alpha1(XI), was occupied at the amino end of the helix (residue 84), where the alpha1(II) (or alpha3(XI)) C-telopeptide was attached. Because the latter sequence is identical in alpha1(II) and alpha3(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 alpha1(XI), alpha2(XI), and alpha1(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 alpha1(XI) and alpha2(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 alpha1- and alpha2-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 alpha3(XI) chain from native molecules of type XI collagen extracted by 1 M NaCl showed that it retained its full N-propeptide extension. The alpha1(II) chain from native type II collagen molecules in the same extract did not (Fig.7). The sequence data also showed that the alpha3(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 alpha3(XI) sequence was matched to that reported for human COL2A1 cDNA(32) . The yields of alpha1(XI), alpha2(XI), and alpha3(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.


Discussion

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 (alpha1(XI)alpha2(XI)alpha3(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. alpha1 N-telopeptide to alpha2 COOH-terminal helix, alpha2 N-telopeptide to alpha3 COOH-terminal helix, and alpha3 N-telopeptide to alpha1 COOH-terminal helix; Fig.5). No firm conclusion is possible on the source of the C-telopeptide sequence found attached to residue 84 in alpha1(XI), i.e. whether from type II collagen, the alpha3-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 alpha1(I) linked to a COOH-terminal helical site in alpha1(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 alpha1(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 alpha1(XI) and alpha2(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, alpha1(XI), alpha2(XI) and alpha3(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 alpha3(XI) as alpha1(II), except for slower alpha3(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 alpha1(II) and alpha3(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 alpha1(XI) instead of alpha1(II) to helical residue 930 (Fig.2).

Of particular interest also was the finding that the N-telopeptide cross-linking lysines in alpha1(XI) and alpha2(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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AR36794. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Orthopædics, Box 356500, University of Washington, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax: 206-685-4700.

^1
The abbreviations used are: C- and N-telopeptides, short sequences that form the carboxyl and amino ends of types I, II, III, V, and XI collagen chains; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; D-period, the 67-nm repeating periodicity of types I, II, III, V, and XI collagen fibrils.


ACKNOWLEDGEMENTS

We thank Ryan Wilkerson for preparing the figures and Kae Pierce for preparing the manuscript.


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