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: rjf@u.washington.edu 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]. 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Biopolymers 54, 448–463. 47. Bos, K.J., Holmes, D.F., Kadler, K.E., McLeod, D., Morris, N.P. & Bishop, P.N. (2001) Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage. J. Mol. Biol. 306, 1011–1022. 3250 R. J. Fernandes et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . 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. 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 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