Kim and David R. EyreJiann-Jiu Wu, Mary Ann Weis, Lammy S.  Modifier in Articular CartilageType III Collagen, a Fibril NetworkProtein Structure and Folding:

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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. EyreFrom the Department of Orthopedics and Sports Medicine, University of Washington, Seattle, Washington 98195

The collagen framework of hyaline cartilages, including artic-ular cartilage, consists largely of type II collagen that maturesfrom 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 amountssuperimposed on the original collagen fibril network. In a studyto understand better the structural role of type III collagen incartilage, we find that type III collagen molecules with unproc-essed N-propeptides are present in the extracellular matrix ofadult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen.Cross-link analyses revealed that telopeptides from both N andC termini of type III collagen were linked in the tissue to helicalcross-linking sites in type II collagen. Reciprocally, telopeptidesfrom type II collagenwere recovered cross-linked to helical sitesin type III collagen.Cross-linkedpeptideswere also identified inwhich a trifunctional pyridinoline linked both an �1(II) and an�1(III) telopeptide to the�1(III) helix. This can only have arisenfrom a cross-link between three different collagen molecules,types II and III in register staggered by 4D from another type IIImolecule. Type III collagen is known to be prominent at sites ofhealing and repair in skin and other tissues. The present find-ings emphasize the role of type III collagen, which is synthesizedinmature articular cartilage, as a covalentmodifier thatmay addcohesion to a weakened, existing collagen type II fibril networkas part of a chondrocyte healing response to matrix damage.

Fibrillar collagens are themost abundant vertebrate proteins.They provide the extracellular framework and mechanicalstrength of most animal tissues. There are seven collagens inthe fibrillar collagen family, types I, II, III, V, XI, XXIV, andXXVII, encoded by 11 distinct genes (for review see Ref. 1).Based on phylogenic analysis, fibrillar collagen genes can besubdivided into three distinct groups or clades (1–5). A-cladecomprises �1(I), �1(II), �1(III), �2(I), and �2(V); B-clade is�1(V), �3(V), �1(XI), and �2(XI); and C-clade is �1(XXIV) and�1(XXVII). All fibrillar collagens are synthesized as procolla-gen molecules consisting of a long uninterrupted triple-helicaldomain (each� chain contains about 1000 amino acid residues)with globular extensions at both N and C termini and a minortriple-helical domain in the removable N-propeptide (1, 6).Collagen types I, II, and III are the main fibril-forming mol-

ecules in vertebrates. Type I collagen is widely expressed and

prominent in skin, tendon, bone and ligaments, andmany othertissues but not in hyaline cartilages. The type I molecule is aheterotrimer of two �1(I) chains and one �2(I) chain (6). TypeII collagen is restricted to cartilages, vitreous and intervertebraldisc, and is a homotrimer of �1(II) chains (7–9). Type III colla-gen is also a homotrimer of �1(III) and appears to function as acopolymer with type I collagen in many tissues, including skin,tendon, ligament, vascular walls, periodontal ligament, andsynovial membranes and is most prominent in highly compli-ant connective tissues (10–17). As with types I and II collagens,the strength of polymeric type III collagen depends on covalentcross-links formed by the lysyl oxidase mechanism (18–20). Inaddition to cross-links between the type III collagen moleculesthemselves, 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 collagenbecomes deposited in articular cartilage ofmature joints, whereit can be detected by immunofluorescence concentrated in thematrix surrounding chondrocytes throughout the depth of thetissue and particularly prominent in human osteoarthriticjoints (22–24).The collagen framework of hyaline cartilage is a highly cross-

linked unique heteropolymer. In essence, the bulk type II colla-gen is polymerized on a template of type XI collagen, and typeIX collagen covalently decorates the surface type IImolecules ofthe nascent fibrillar networksmost prominently in young tissue(25–28). All three collagen types, II, IX, and XI, are heavilycross-linked in the same fibril through the lysyl oxidase-medi-ated mechanism (29–32). In a study to understand better thestructural role of type III collagen in cartilage, we have revealedthat pN-type III collagen molecules are present in the extracel-lular matrix of adult human and bovine articular cartilages ascovalently cross-linked polymers extensively cross-linked tothe surface of type II collagen fibrils, suggesting a role in matrixreinforcement and a healing response to tissue damage.

EXPERIMENTAL PROCEDURES

Preparation of Collagens—Human knee joints were obtainedfrom Northwest Tissue Services (Seattle, WA) from donorsaged 18–75 with no obvious signs of osteoarthritis. Full thick-ness articular cartilage was sliced from the femoral and tibiachondyles and from an equivalent site in a 4-year-old cow(bovine) knee. Minced tissue was extracted in 4 M guanidineHCl, 0.05 M Tris-HCl, pH 7.4, containing protease inhibitors (2mMEDTA, 5mMbenzamidine, 2mMphenylmethylsulfonyl flu-oride, and 5mM1,10-phenanthroline) at 4 °C for 48 h to removeproteoglycans and other matrix proteins. The guanidine-insol-uble tissue residue was then washed thoroughly with water andfreeze-dried. Cross-linked collagens were solubilized by digest-

* This work was supported by National Institutes of Health Grants AR 37318and AR 36794.

1 To whom correspondence should be addressed: 1959 NE Pacific St., HSBBB1052, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax: 206-685-4700;E-mail: wujj@u.washington.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 18537–18544, June 11, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JUNE 11, 2010 • VOLUME 285 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 18537

ing the washed residue with pepsin (1:10 w/w by dry weight) in3% acetic acid (v/v) for 24 h at 4 °C. Different collagen fractionswere then precipitated from the acid solution at 0.7 M, 1.2 M and2.0 M NaCl to separate types II/III, type XI, and type IX collag-ens respectively (33, 34).The 0.7 M NaCl precipitate from a pepsin digest of articular

cartilage, which contains type II and any type III collagen in thesolubilized pool, was dissolved in 4 M guanidine HCI, 0.05 M

Tris-HCl, pH 7.5, and resolved by molecular sieve chromatog-raphy on an agarose A5m column (170 � 1.5 cm, 200–400mesh, Bio-Rad), eluted with 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5.CNBr Cleavage and Peptide Chromatography—The washed

residues after 4 M guanidine HCl extraction were minced anddigested with CNBr in 70% formic acid under N2 at room tem-perature for 24 h on a shaker. The digests were diluted 15-foldwith water and freeze-dried (34). Collagen CNBr-derived pep-tides were resolved by molecular sieve chromatography on anagarose A1.5m column (170 � 1.5 cm, 200–400 mesh, Bio-Rad), eluted with 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5.Bacterial Collagenase Digestion and Peptide Chromato-

graphy—An aliquot of the washed guanidine-insoluble articu-lar cartilage residue was suspended in 0.05 M Tris-HCl, 0.1 M

CaCl2 buffer, pH 7.5, containing 0.001% thimerosol at 10mg/ml, heat denatured at 70 °C for 10 min, and digested withbacterial collagenase (Sigma, type IA) at an enzyme:substrateratio of 1:100 (w/w) at 37 °C for 24 h. Resulting digests wereresolved on a BioGel P-10 molecular sieve column (100 � 1.5cm) equilibrated with 10% acetic acid (v/v) at a flow rate of 11.4ml/h collecting 5.7 ml/fraction. Collected fractions were mon-itored for pyridinoline-specific fluorescence (excitation, 297nm; emission, 395 nm) (34).Peptides containing pyridinoline cross-links were further

purified by sequential DEAE ion-exchange and C8 reverse-phase HPLC2 (29). Ion-exchange HPLC was performed on aDEAE 5-PW column (75 � 7.5 mm; Bio-Rad), eluting with alinear gradient of 0–0.2 MNaCl in 40ml of 0.02 MTris-HCl, pH7.5, containing 10% (v/v) acetonitrile at 1 ml/min over 40 min.Pooled DEAE fractions were dried and chromatographed byreverse-phase HPLC on a C8 column (Brownlee AquaporeRP-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) trifluoroaceticacid from 0 to 40% at 1 ml/min over 60 min.TrypsinDigestion—Themolecular sieve-purified type III col-

lagen preparation from pepsin-solubilized articular cartilagewas dissolved in 0.05 M Tris-HCl, 0.15 M NaCl, pH 8.0, at 5mg/ml, heat-denatured at 60 °C for 10 min, and digested withtrypsin (Boehringer sequencing grade) at an enzyme:substrateratio of 1:200 (w/w) at 37 °C for 20 h. Tryptic peptides werefractionated by immobilizedmetal ion affinity chromatography(IMAC).IMAC—One ml of HiTrap chelating HP Sepharose beads

(GE Healthcare) were packed into a column (1 �2 cm). Thebeads were charged with 3 ml of 0.5 M CuCl2. After washing

with 10 ml of Milli Q H2O to remove unbound CuCl2, the col-umn was equilibrated with 0.05 M Tris-HCl, pH 8.0, containing0.15 M NaCl (IMAC sample buffer). Trypsin-digested collagenwas incubatedwith the copper-chelated beads at room temper-ature for 1 h. The beads were washed with 10 ml of IMACsample buffer to remove unbound peptides. The bound pep-tides were eluted from the column sequentially with 4 ml of 0.1M 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 onC8 reverse-phase HPLC and identified by N-terminal proteinsequence analysis and mass spectrometry.Stromelysin-1 (MMP3) Extraction—Cartilage from a non-

fibrillated surface of a knee joint removed at replacementsurgery (59-year-old female) was used to study the extractionof collagen type III by stromelysin-1 (MMP3) compared withpepsin extraction. The washed guanidine-insoluble residuewas extracted with MMP3 (prostromelysin-1 (35) activatedby trypsin as described (36)) at an enzyme:substrate ratioof 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 extractionswere carried out, removing the supernatant after 24 h, andadding fresh enzyme for the second 24-h extraction. 1,10-phenanthroline (10 mM) was added to each extract to stopthe reaction.Gel Electrophoresis—Pepsin-solubilized collagens were re-

solved on 6% SDS-PAGE according to the method of Laemmli(37) using a Bio-Rad mini protean 3 system. Delayed reductionof disulfide bonds was performed by adding 10 �l of 0.5 M dithio-threitol (DTT) in 10% glycerol (v/v) to each sample well after15 min of electrophoresis at 150 V. Stromelysin extracts wererun on 10% SDS-PAGE.Antisera and Western Blotting—Two mouse monoclonal anti-

bodies to human type III collagen were used. mAb 4G9 is specificto a conformational epitope in the globular N-propeptidedomain (24). mAb 2C3 is specific to a proteolytic neoepitope atthe C terminus of the �1(III) N-telopeptide sequence YDVKS-GVAVGG, where K is a cross-linked lysine. Twomouse mono-clonal antibodies to type II collagen were also used. mAb 10F2recognizes a proteolytic neoepitope at the C terminus of acleaved type II C-telopeptide sequence generated by pepsin(38), and mAb 1C10 is specific to a denatured epitope in thetriple-helical domain of �1(II) near the C-terminal end (31).For Western blotting, collagen fractions resolved by SDS-

PAGE were transblotted to polyvinylidene difluoride membrane(Bio-Rad). Western blot analyses performed with each mono-clonal antibody were developed using alkaline phosphatase-con-jugated goat anti-rabbit IgG (Jackson ImmunoResearch, Avon-dale, PA) and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as substrate for alkaline phosphatase.N-terminal Protein SequenceAnalysis—Purified cross-linked

peptides were identified by N-terminal sequence analysis on aPorton 2090E gas phase sequencer with on-line HPLC analysisof phenylthiohydantoin derivatives (29).Mass Spectrometry—Individual protein bands after Coomas-

sie Blue staining on SDS-PAGE were digested in-gel by trypsin(32, 39). The resulting peptides were subjected tomicrobore C8column liquid chromatography (0.3mm� 15 cm;Vydac) inter-

2 The abbreviations used are: HPLC, high performance liquid chromatogra-phy; IMAC, immobilized metal ion affinity chromatography; DTT, dithio-threitol; mAb, monoclonal antibody; MS/MS, tandem mass spectrometry.

Type III Collagen in Cartilage

18538 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 24 • JUNE 11, 2010

faced directly to a ThermoFinnnigan LCQ Deca XP tandemmass spectrometer equipped with an electrospray ionizationsource. For protein identification, peptide fragments werecomparedwith theNCBI nonredundant protein database usingSEQUEST, an automated database search algorithm designedfor use with tandem mass spectrometry (MS/MS) data. Cross-linked peptides were analyzed manually by calculating the pos-sibleMS/MS ions andmatching these to the actualMS/MS ions(32).

RESULTS

Using interrupted SDS-PAGE with delayed reduction ofdisulfide bonds to resolve �1(III) from �1(II) chains, we wereable to identify type III collagen in pepsin-solubilized materialfrom all adult human articular cartilage samples examined(results from three representative donor joints are shown in Fig.1). The chain identities, indicated by their migration on SDS-PAGE, were established beyond doubt by mass spectrometryandN-terminal protein sequence analysis. The type III collagencontent of adult human articular cartilage varied between indi-viduals in the range 0.5% to about 10% of total collagen, basedon the recovered dry weights and relative intensity of Coomas-sie 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 andbovine articular cartilage for a covalently attached type IIIcollagen N-telopeptide. The 2C3 antibody is specific tohuman collagen III and does not cross-react with bovinecollagen. Because collagen samples were not treated withDTT to reduce disulfide bonds prior to electrophoresis, theintramolecularly disulfide-bonded type III collagen chainsran near the top of the separating gel. From human cartilagethree bands can be seen stained with 2C3, all of which werealso stained by 1C10, the type II collagen-specific antibody.All three therefore were based on an �1(II) triple-helix buthad an �1(III) N-telopeptide cross-linked to them. The gelshows that in addition to the signal from the main � and �chains of type II collagen seen by Coomassie Brilliant Bluestaining in the pepsin digest, 2C3 also binds to a minor bandrunning between them at 160 kDa not visible by Coomassiestaining (Fig. 2). This band also reacted with mAb 1C10 and

FIGURE 1. Interrupted SDS-PAGE to detect �1(III) chains in pepsinextracts of human articular cartilage from three tissue donor knees. Lane1, 18-year-old; lane 2, 60-year-old; lane 3, 73-year-old. For the reduced samplelanes (�DTT), DTT was added 15 min after starting the electrophoresis toresolve 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).

FIGURE 2. SDS-PAGE/Western blot analysis to screen for type III collagenfragments covalently attached to type II collagen. Pepsin-solubilized typeII collagens from mature human (H) and bovine (B) articular cartilages wereresolved on SDS-PAGE without reducing disulfide bonds. Gels were eitherstained with Coomassie Brilliant Blue or electroblotted to polyvinylidenedifluoride membrane and probed with a type III collagen N-telopeptide-spe-cific antibody (2C3), type II collagen-specific antibody (1C10), or type III colla-gen N-propeptide-specific antibody (4G9). The type III N-propeptide/te-lopeptide is detected cross-linked to both human and bovine �1(II) chains.The arrow shows the position of the 160 kDa band that reacts with all threeantibodies.

FIGURE 3. Molecular sieve chromatography of CNBr-digested humanarticular cartilage collagen. The CNBr digest of cartilage residue after 4 M

guanidine HCl extraction was chromatographed on an agarose A1.5m (Bio-Rad) molecular sieve column (170 � 1.5 cm), eluted with a 0.05 M Tris-HClbuffer, pH 7.5, containing 2 M guanidine HCl, at a flow rate of 6 ml/h, collecting3.0-ml fractions. Aliquots of collected fractions (4 �l) were assayed for mAb4G9 immunoreactivity. The result shows that the N-propeptide domain of thetype III collagen was retained in the cartilage matrix.

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

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mAb 4G9, which shows the presence of the �1(II) chain andan �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 productextracted from the cartilage matrix. The findings also implythat relatively large amounts of the N-propeptide domain oftype III collagen are present in the extracellular matrix of adultcartilage. The presence of collagen type III N-propeptides inarticular cartilage was confirmed by mAb 4G9 enzyme-linkedimmunosorbent assay across molecular sieved column frac-tions from a CNBr digest of the 4 M guanidine HCl-insolubleresidue of adult cartilage (Fig. 3). The CNBr peptide compo-nents containing the N-propeptide domain eluted early in thechromatogram. Based on their elution positions on molecularsieve chromatogram andmigration position on SDS-PAGE, the4G9-reactive peptides in elution volume 85–130 ml havemolecular masses of 100 kDa and above. The results indicatethat the type III collagen N-propeptide domain is retained bymost molecules of type III deposited and polymerized in carti-lage matrix.Fig. 4 shows that mAb 10F2 reacted with the �1(III) chain

resolved on interrupted electrophoresis, indicating that �1(II)C-telopeptides were attached to some of the �1(III) chains.Such heterotypic cross-linking between type III collagen andtype II collagen was also identified in extracts of adult bovinearticular cartilage as follows. Because �1(III) chains are disul-fide-bonded intramolecularly, they can be resolved from thebulk type II collagen� and� chains in a pepsin digest bymolec-

ular sieve column chromatography(Fig. 5). Collagen recovered fromthe indicated pooled fractionsenriched in type III collagen wasdigested with trypsin. Cross-linkedpeptides were further purified byIMAC and reverse-phase HPLC.Using Cu2� IMAC, several pep-

tides containing histidine residueswere selectively bound from thetrypsin digest of the enriched typeIII collagen pool (Fig. 5). Fourof these peptides were non-cross-linked linear peptides (Fig. 6a),but, in addition, divalent and tri-valent cross-linked collagen III pep-tides were also isolated. A promi-nent divalent cross-linked peptidewas derived from the C-telopeptideof type II collagen (EKGPDPLQ)linked to the type II helical sequencethat contained the residue 87hydroxylysine cross-linking resi-due (GFP*GTP*GLP*GVK87GHR).Telopeptides from both N and Ctermini of type III collagenwere alsorecovered linked covalently to thehelical cross-linking sites in type IIIcollagen (Fig. 6b). In addition, pep-tides from heterotypic cross-linksbetween types II and III collagenswere identified. One came from

FIGURE 5. Molecular sieve chromatography of pepsin-extracted bovinetypes II and III collagens. Collagen recovered in the 0.7 M NaCl fraction (36mg) from pepsin-solubilized 4-year-old cow articular cartilage collagen wasdissolved in 1.8 ml of 4 M guanidine HCI, 0.05 M Tris-HCI, pH 7.5, and chromato-graphed on an agarose A5m (Bio-Rad) molecular sieve column (170 � 1.5 cm)to resolve type III collagen from the bulk of type II collagen � and � chains. Theeluant 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-lagen that were pooled and digested with trypsin and purified by IMAC.

FIGURE 6. Reverse-phase HPLC fractionation of tryptic peptides prepared from 4-year-old bovine artic-ular cartilage bound by a copper-affinity column. Tryptic peptides that had eluted from the copper columnwith 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(b) were resolved on a C8 reverse-phase column (for details, see “Experimental Procedures”). Purified peptideswere identified by N-terminal sequence analysis and by mass spectrometry. P*, 4-hydroxyproline; X, cross-linking hydroxylysine residue; galglc, glucosylgalactosyl.

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18540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 24 • JUNE 11, 2010

linkage of an N-telopeptide of type III collagen (DVXSGV-AGGGIAGYP*GPAGPP*—) to the helical 930 site in type IIcollagen (GLXGHR) (Fig. 6b). The structure of this heterotypiccross-linked peptide was confirmed beyond doubt by MS/MS(24). Another heterotypic cross-linked peptide was purifiedfrom fraction 45 (Fig. 6a) and identified as a trivalent cross-linked peptide linking the �1(II) C-telopeptide (EXGPDPLQ)to an �1(III) C-telopeptide (IAGVGAEXAGGFAGY) and thehelical cross-linking site Lys87 in type III collagen (Fig. 7). Inaddition to links to the helix of type III collagen, C-telopeptidesof collagens II and III were also found that were covalentlylinked to the helix of type II collagen through a pyridinolineresidue. 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 collagenhelical cross-linking site at residue 87 in a peptide isolated from

a bacterial collagenase digest of adult human articular cartilage(Fig. 8).Experiments designed to test whether the cartilage matrix

type III collagen was readily available for extraction as a poly-mer associated with and cross-linked to type II collagen fibrilsurfaces were carried out. Initial results indicated that of vari-ous metalloproteinases tested, stromelysin-1 was the most effi-cient under native conditions in extracting the type III collagenpool without extracting significant amounts of type II collagen.Fig. 9 compares the results of two serial 24-h extractions byrecombinant MMP3 at 37 °C (Fig. 9a) of minced articular car-tilage from an osteoarthritic joint with pepsin extraction (Fig.9b). The results of Western blotting using three differentmonoclonal antibodies on replicate lanes show that MMP3extracted very little type II collagen, which was detectable only

FIGURE 7. Heterotypic cross-linking between types II and III collagens inbovine articular cartilage. a, MS/MS identified a pyridinoline cross-linkedpeptide in fraction 45 (Fig. 6a) that resulted from heterotypic cross-linkingbetween types II and III collagens. b, structure and origin of the purified cross-linked peptide. P*, 4-hydroxyproline; X, cross-linking hydroxylysine residue.

FIGURE 8. Purification of a heterotypic type II/type III cross-linked pep-tide from human articular cartilage collagen. a, DEAE HPLC fractionationof fluorescent cross-linked peptides from bacterial collagenase digestedhuman articular cartilage. Fractions indicated by the bar were pooled andresolved by reverse-phase HPLC. b, reverse-phase HPLC isolation of a colla-gen type II/type III heterotypic cross-linked peptide linking the sequencesshown. c, structure of the purified cross-linked peptide. X, cross-linkinghydroxylysine residue.

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as intact �1(II) chains (1C10 blot), but most of the type IIIcollagen (detectable as cross-linked large fragments on 4G9 and2C3 blots). Pepsin removed all of the type III collagen too, butcleaved mostly between the �1(III) N-propeptide (disulfide-bonded trimer) and the main triple helix, whereas MMP3 didnot cleave and release the free �1(III) N-propeptide trimer,which was retained on the large fragments (Fig. 9a). Fig. 9cillustrates the various cleavage sites and molecular features ofcross-linked pN-type III collagen.

DISCUSSION

The results confirm that significant amounts of type III col-lagen are present in adult human articular cartilage cross-linked covalently to other type III collagen molecules, suggest-ing their presence in the matrix as homotypic polymers of typeIII collagen presumably in the form of fine filaments of head-to-tail cross-linked molecules. Most of the cross-links formedbetween the type III collagenmolecules are of the divalent vari-ety, in contrast to type II collagen in which trivalent pyridino-line cross-links predominate (34). The results also indicate thatin addition to type III-to-type III cross-links, the polymeric typeIII collagen is also heavily cross-linked to type II collagen.Telopeptides from type II collagen are linked to the helicalcross-linking sites in type III collagen and, vice versa, telopep-tides from type III collagen are linked to helical cross-linkingsites in type II collagen.TheWestern blot analyses of type III collagen extracted from

adult human and bovine articular cartilages also revealed that afraction of the molecules had been covalently linked to type IIcollagen (Fig. 2). A major site of linkage was between the C-he-lix (Lys930) of �1(II) and the �1(III) N-telopeptide. This is thesame site previously implicated for bovine articular cartilage(24). The results in Fig. 2 show that this cross-linkage in factoccurs between a longer form of the �1(III) N-telopeptide inwhich the N-propeptide extension is retained and detected by

mAb 4G9. From the mass spec-trometry results, all of the divalentcross-linked peptides identified intype III collagen of bovine cartilage,either from III to III or III to II link-ages, contained an additional 188-Da mass on the cross-linking resi-due. Such an adduct has been shownto be amaturation product of a poolof ketoimine cross-links in type IIcollagen of bovine cartilages that donot mature to pyridinolines. Itresults from ketoimine oxidationand arginine addition (40).The results also reveal trivalent

cross-linked peptides between mixedtelopeptides of both types II andIII collagens to helical residue 87in either collagen II or collagen III(Figs. 6 and 7). This finding isimportant because it confirms thatpyridinoline residues can link threedifferent collagen molecules. In-

deed, our original proposed mechanism of formation for pyr-idinoline cross-links was an aldol addition between two neigh-boring ketoimine cross-links within the microenvironment ofthe molecular packing arrangement of a fibril (41). The stoichio-metry from C14-lysine labeling of cartilage in vivo and in vitro(42–44) and the finding of a urinary peptide from bone resorp-tion linking two �2(I) N-telopeptides to a helical site (45) sup-port this.The CNBr-derived peptides in whichN-propeptide domains

of type III collagen were detected have estimated molecularmasses �100 kDa (Fig. 3). This is larger than the monomericprocessed N-propeptide trimer (�60 kDa) and therefore can-not represent simply processed collagen IIIN-propeptides aftersynthesis, but the retention of N-propeptides on cross-linkedpN-type III collagen molecules in cartilage matrix. RetainedN-propeptideswill prevent type III collagen from forming thickcollagen fibrils (46–48), but will not impair divalent cross-link-ing internally in the polymer or trivalent bonds at the interfacewith type II collagen fibrils.Themost likely explanation for the polymeric formof type III

collagen in cartilage matrix is a thin filamentous polymer ofpN-type III molecules cross-linked head to tail at 4D-staggeredsites but heavily cross-linked laterally to the surfaces of type IIcollagen fibrils wherever they interact. In effect, such a filamen-tous polymer might add cohesion to a swollen, and perhapsweakened, existing collagen II fibril network. It is notable thatthe earliest observed change in articular cartilage in experimen-tal animal models of osteoarthritis is a swelling of the collagenfibril network (49) and that collagen III is expressed by chon-drocytes of human osteoarthritic cartilage (50), in which itscontent has been reported to be enriched (51).Type III collagen is distinct from types I and II collagens in

lacking 3-hydroxyproline in the triple helix, which we suspectmay be related to an inability to form thick, homotypic fibrils(52). In skin and other tissues, immunogold electron micros-

FIGURE 9. Selective extraction of collagen type III from human articular cartilage by MMP3. a, mincedcartilage after 4 M guanidine HCl extraction was serially extracted twice for 24 h with recombinant MMP3.Replicate aliquots of each extract were run on 10% SDS-PAGE � DTT, stained directly with Coomassie Blue, orimmunoblotted 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 ofthe Coomassie-stained lanes are from the enzyme preparation. Lane 1, first 24-h extract; lane 2, second 24-hextract. b, another sample of the same cartilage guanidine-HCl residue was digested with pepsin, the digestfractionated into 0.8 M, 1.2 M, and 2.2 M NaCl precipitates, and each run on 6% SDS-PAGE � DTT. c, proteolyticcleavage sites and molecular features of collagen type III are illustrated.

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copy showed that collagen III with retained N-propeptides ispresent on the surface of type I collagen fibrils (13). Similarly,collagen type III with retainedN-propeptides has been detectedon the surface of type II collagen fibrils in human articular car-tilage (23). The results in Fig. 9 show that type III collagen canbe readily extracted by stromelysin (MMP3) digestion ofhuman articular cartilage under native conditions, suggestingthat it is accessible as a cross-linked polymer external to type IIcollagen 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 bycleavage in the main triple helix, most likely at the 3⁄4 lengthcollagenase-cleavage domain, which is especially susceptible intype III collagen to proteases other than collagenase includingMMP3 (53, 54). Taken together, the results show that type IIIcollagen molecules accumulate in mature human articular car-tilage cross-linked to the surface of type II collagen fibrils. Theamount presumably varies between individual joints, anatomi-cal location, and tissue microanatomy, perhaps dependent onthe history of injuries and the wear and tear experienced by anormal joint. If so, the content will tend to increase with age. Ithas also been noted that as articular cartilagematures and ages,the collagen fibrils become thicker, and the content of types IXand XI collagens decreases relative to type II collagen (55). The�2(XI) chain of typeXI collagen progressively decreases in con-tent with tissue maturation and is replaced by �1(V) (56). It isknown that type III collagen is prominent in fibrous repair tis-sue in skin and other tissues (57). Therefore, it seems likely thattype III collagen is synthesized as a modifier of existing fibrilnetworks in response to tissue and matrix damage.

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