Macromolecular Specificity of Collagen Fibrillogenesis

Suprastructures of the extracellular matrix, such as banded collagen fibrils, microfibrils, filaments, or networks, are composites comprising more than one type of macromolecule. The suprastructural diversity reflects tissue-specific requirements and is achieved by formation of macromolecular composites that often share their main molecular components alloyed with minor components. Both, the mechanisms of formation and the final macromolecular organizations depend on the identity of the components and their quantitative contribution. Collagen I is the predominant matrix constituent in many tissues and aggregates with other collagens and/or fibril-associated macromolecules into distinct types of banded fibrils. Here, we studied co-assembly of collagens I and XI, which co-exist in fibrils of several normal and pathologically altered tissues, including fibrous cartilage and bone, or osteoarthritic joints. Immediately upon initiation of fibrillogenesis, the proteins co-assembled into alloy-like stubby aggregates that represented efficient nucleation sites for the formation of composite fibrils. Propagation of fibrillogenesis occurred by exclusive accretion of collagen I to yield composite fibrils of highly variable diameters. Therefore, collagen I/XI fibrils strikingly differed from the homogeneous fibrillar alloy generated by collagens II and XI, although the constituent polypeptides of collagens I and II are highly homologous. Thus, the mode of aggregation of collagens into vastly diverse fibrillar composites is finely tuned by subtle differences in molecular structures through formation of macromolecular alloys. Suprastructures of the extracellular matrix, such as banded collagen fibrils, microfibrils, filaments, or networks, are composites comprising more than one type of macromolecule. The suprastructural diversity reflects tissue-specific requirements and is achieved by formation of macromolecular composites that often share their main molecular components alloyed with minor components. Both, the mechanisms of formation and the final macromolecular organizations depend on the identity of the components and their quantitative contribution. Collagen I is the predominant matrix constituent in many tissues and aggregates with other collagens and/or fibril-associated macromolecules into distinct types of banded fibrils. Here, we studied co-assembly of collagens I and XI, which co-exist in fibrils of several normal and pathologically altered tissues, including fibrous cartilage and bone, or osteoarthritic joints. Immediately upon initiation of fibrillogenesis, the proteins co-assembled into alloy-like stubby aggregates that represented efficient nucleation sites for the formation of composite fibrils. Propagation of fibrillogenesis occurred by exclusive accretion of collagen I to yield composite fibrils of highly variable diameters. Therefore, collagen I/XI fibrils strikingly differed from the homogeneous fibrillar alloy generated by collagens II and XI, although the constituent polypeptides of collagens I and II are highly homologous. Thus, the mode of aggregation of collagens into vastly diverse fibrillar composites is finely tuned by subtle differences in molecular structures through formation of macromolecular alloys. Extracellular matrix aggregates, despite their functional and morphological diversity, often contain the same type of macromolecules as their major constituent. In tendons, for example, large and strongly banded fibrils of variable width are aggregated into bundles that resist extreme tensile forces in one dimension. By contrast, thin fibrils with uniform diameter and spacing are organized into orthogonal sheets forming the translucent corneal stromal matrix that withstands high traction in two dimensions. However, the same protein, i.e. collagen I, is the quantitatively predominant component in both suprastructures (1Birk D.E. Fitch J.M. Babiarz J.P. Linsenmayer T.F. J. Cell Biol. 1988; 106: 999-1008Crossref PubMed Scopus (294) Google Scholar, 2Birk D.E. Micron. 2001; 32: 223-237Crossref PubMed Scopus (324) Google Scholar). The major cartilage collagen is type II, but this protein, too, is common to fibrils with different structures and functions (3Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (403) Google Scholar, 4Hagg R. Bruckner P. Hedbom E. J. Cell Biol. 1998; 142: 285-294Crossref PubMed Scopus (100) Google Scholar). Finally, collagens I and II occur together in fibrocartilage (5Nerlich A.G. Boos N. Wiest I. Aebi M. Virchow's Arch. Int. J. Pathol. 1998; 432: 67-76Crossref PubMed Scopus (101) Google Scholar), a tissue characterized by a high resistance to both compressive and tensile forces and that contains fibrous bundles in addition to an amorphous extrafibrillar matrix rich in proteoglycans. Collagens I and II are very similar in their molecular structure. They both contain three polypeptides with a central sequence of 1014 amino acids in which strictly every third residue is glycine. In addition, these glycine residues are frequently preceded by hydroxyproline and/or followed by proline. The (Gly-Xaa-Yaa) n sequences are flanked at both ends by short non-periodic sequences, called telopeptides. Collagen I is a heterotrimer containing two α1(I) chains and one α2(I) chain, whereas collagen II is a homotrimer of α1(II) chains. However, the three primary structures within the (Gly-Xaa-Yaa) n domains of the three polypeptides are more than 90% identical (6Baldwin C.T. Reginato A.M. Smith C. Jimenez S.A. Prockop D.J. Biochem. J. 1989; 262: 521-528Crossref PubMed Scopus (80) Google Scholar), and both collagen I and II form very similar triple helices with a length of 300 nm, turning the molecules into highly elongated rods of considerable stiffness. The molecules are incorporated into fibrils by lateral aggregation and a longitudinal stagger with a periodicity called D yielding the characteristic banding patterns of collagen fibrils observed by electron microscopy. The suprastructural diversity of collagen fibrils has been investigated by several approaches, including x-ray diffraction. High resolution fibril diffraction requires regularly ordered, crystalline-like tissue domains of sufficient expansion. These prerequisites are met in only two paradigmatic tissues, i.e. the rat tail tendon and the notochord sheath in a primitive chordate, the lamprey (7Brodsky B. Eikenberry E.F. Fleischmajer R. Olsen B.R. Kühn K. Biology, Chemistry, and Pathology of Collagen. 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The D-periodicity of skin fibrils was 64 nm (67 nm in rat tail tendon). Likewise, the center-to-center spacings between collagen molecules were larger in cartilage (1.7 nm) than in tendons (1.1 nm). However, further suprastructural details could not be resolved. The question arises how distinct matrix suprastructures can be formed and/or stabilized. The answers still are incomplete, but it is known that collagen fibrils in situ comprise not only several types of collagens (1Birk D.E. Fitch J.M. Babiarz J.P. Linsenmayer T.F. J. Cell Biol. 1988; 106: 999-1008Crossref PubMed Scopus (294) Google Scholar, 3Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (403) Google Scholar, 13Henkel W. Glanville R.W. Eur. J. Biochem. 1982; 122: 205-213Crossref PubMed Scopus (139) Google Scholar, 14Müller-Glauser W. Humbel B. Glatt M. Sträuli P. Winterhalter K.H. Bruckner P. J. 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We have demonstrated recently that incorporation of collagen IX is required for the macromolecular alloy stability in prototypic cartilage fibrils (25Blaschke U.K. Eikenberry E.F. Hulmes D.J.S. Galla H.J. Bruckner P. J. Biol. Chem. 2000; 275: 10370-10378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The other minor collagen in cartilage fibrils, i.e. collagen XI, forms efficient nuclei of fibrillogenesis and, thereby, stringently controls fibril shape by co-polymerizing with collagen II. However, polypeptides of collagen XI are not unique to cartilage but are present in a wide variety of non-cartilaginous tissues, which in part contain collagen I as the major fibrillar collagen (26Niyibizi C. Eyre D.R. FEBS Lett. 1989; 242: 314-318Crossref PubMed Scopus (92) Google Scholar, 27Nah H.D. Barembaum M. Upholt W.B. J. Biol. Chem. 1992; 267: 22581-22586Abstract Full Text PDF PubMed Google Scholar, 28Kleman J.-P. Hartmann D.J. Ramirez F. van der Rest M. Eur. J. 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Presumably for this reason, patients with Stickler syndrome harboring mutations in the α2(XI) chains do not exhibit the eye involvement characteristic for Stickler patients with mutations in the α1(XI) chains (33Vikkula M. Mariman E.C.M. Lui V.C.H. Zhidkova N.I. Tiller G.E. Goldring M.B. Van Beersum S.E.C. De Waal Malefijt M.C. Van den Hoogen F.H.J. Ropers H.-H. Mayne R. Cheah K.S.E. Olsen B.R. Warman M.L. Brunner H.G. Cell. 1995; 80: 431-438Abstract Full Text PDF PubMed Scopus (305) Google Scholar). Similarly, the type XI collagen fraction isolated from articular cartilage was reported to contain α1(V) chains in amounts increasing with age (34Eyre D. Wu J.-J. Mayne R. Burgeson R.E. Structure and Function of Collagen Types. Academic Press, New York1987: 261-281Google Scholar). In addition, these observations suggest that collagens V and XI should not be regarded as separate collagen types. Rather, minor collagens composed of polypeptides derived from the genes encoding α chains of collagens V or XI may exert discrete functions in organ- or tissue-specific fibrillogenesis. Therefore, we addressed the question whether the collagen XI-controlling growth of collagen II-containing cartilage fibrils is effective also in fibrillogenesis of collagen I. Here, we found that mixtures of collagens I and XI aggregated into specific composites, too. Strikingly, however, collagen I/XI fibrils were not only structurally distinct from those of collagens II and XI but also arose by different mechanisms. Materials—All chemicals were of reagent grade. DEAE-cellulose (DE52) was from Whatman International. Polyclonal anti-collagen I antibodies AB752P were from Chemicon International. Goat anti-rabbit IgG conjugated with horseradish peroxidase was from Sigma. Goat anti-rabbit IgG conjugated with 18-nm gold particles was from Jackson ImmunoResearch. Buffer Definitions—The buffers used were as follows: storage buffer: 100 mm Tris-HCl, pH 7.4, 400 mm NaCl; buffer A: 50 mm Tris-HCl, pH 7.4, 2 m urea, 200 mm NaCl; buffer B: 50 mm Tris-HCl, pH 7.4, 2 m urea, 20 mm NaCl; TBS 1The abbreviations used are: TBS, Tris-buffered saline; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; OI, osteogenesis imperfecta.: 20 mm Tris-HCl, pH 7.4, 150 mm NaCl; blocking buffer: 5% dried skim milk in TBS; blot buffer: TBS, containing 0.04% Tween 20 and 2.5% dried skim milk; and PBS: 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. Collagen Purification—The cartilage version of collagen XI comprising α1(XI), α2(XI), and α3(XI) chains was purified in native and fibrillogenesis-competent form from cultures of chick embryo sternal chondrocytes in agarose gels as described previously (25Blaschke U.K. Eikenberry E.F. Hulmes D.J.S. Galla H.J. Bruckner P. J. Biol. Chem. 2000; 275: 10370-10378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Collagens I and V were obtained from tarso-metatarsal tendons of 17-day-old chick embryos. Tissue from 150 embryos was accumulated, frozen at –20 °C until use, thawed, and extracted overnight with 15 volumes of 0.5 m acetic acid at 4 °C. This extraction procedure was repeated, and the extracts were combined. Collagens were precipitated by adding one-fourth the extract volume of 4.3 m NaCl. The suspensions were stirred overnight, and, after centrifugation, crude pelleted collagens were dissolved in a minimal amount of buffer A. After extensive dialysis against buffer A, the samples were passed over a DEAE-cellulose column (3.5 × 12 cm) equilibrated in buffer A. Mixtures of collagen I and V were recovered from the breakthrough fraction and were dialyzed exhaustively against buffer B. Final separation of collagens I and V was achieved by re-chromatography on DEAE-cellulose equilibrated in buffer B. Collagen I was recovered from the breakthrough fraction and was pure as judged by SDS-PAGE. Collagen V bound to the column and was eluted from the column by switching the eluent to buffer A. Purified proteins were dialyzed extensively against 0.1 m Tris-HCl, pH 7.4, containing 1 m NaCl, and were precipitated by addition of solid NaCl to a final concentration of 4.5 m. After centrifugation, pellets were redissolved in storage buffer at appropriate concentrations, dialyzed against storage buffer, and clarified by centrifugation. The purity of the collagens was judged by SDS-PAGE after staining with Coomassie Blue. Antisera to Native Collagen XI—0.5 mg of purified, native collagen XI was suspended in 1 ml of 0.01 m hydrochloric acid, and was mixed with 1 mg of keyhole limpet hemocyanin (Sigma) in 0.1 ml of H2O. After titration to pH 6.8 with 1 m NaOH, conjugation was initiated by addition of 0.05 ml of 1% glutaraldehyde. The reaction was allowed to proceed for 3 h at room temperature, and the sample was exhaustively dialyzed at 4 °C against 0.05 m Tris-HCl, pH 7.4, containing 0.15 m NaCl to remove excess glutaraldehyde and to inactivate free aldehyde groups. Antisera were raised by immunizing rabbits with 0.5 mg of conjugate suspended in Freund's complete and incomplete adjuvant following established protocols (35Timpl R. Methods Enzymol. 1982; 82: 472-498Crossref PubMed Scopus (130) Google Scholar). The reactivity and specificity of the antisera was assessed by ELISA and immunoblotting. Pepsin Digestion—To obtain molecules without non-helical terminal domains, purified collagen XI was digested with pepsin (1 mg/ml) in 0.5 m acetic acid, pH 2.5, for 24 h at 4 °C. The pH was adjusted to pH 7.4 to inactivate pepsin. The digested collagen XI was precipitated with 3 volumes of ethanol and dissolved in SDS-PAGE sample buffer. Determination of the Collagen Concentration—The collagen concentration was determined using the bicinchoninic acid assay according to the instructions of the manufacturer (Pierce, Rockford, IL). Pepsintreated collagen II in acetic acid was used as standard. The concentrations of the standard solutions were determined by circular dichroism at 221 nm (specific ellipticity [θ]221 = 8550 deg × cm2 × decimole–1 (36Bächinger H.P. Bruckner P. Timpl R. Prockop D.J. Engel J. Eur. J. Biochem. 1980; 106: 619-632Crossref PubMed Scopus (204) Google Scholar)). SDS-PAGE and Immunoblots—Protein samples for electrophoresis were prepared by precipitation with cold ethanol. The precipitates were dissolved in SDS-PAGE sample buffer and electrophoresed in 4.5–15% polyacrylamide gradient gels. The proteins were electrotransferred to nitrocellulose filters, and the membranes were treated with blocking buffer for 1 h at room temperature. This was followed by incubation at 4 °C overnight with an antiserum to collagen XI diluted 1:1000 in blot buffer, washing, and incubation for 2 h at room temperature with peroxidase-conjugated goat-anti-rabbit IgG (Sigma, Germany) in blot buffer. Immunoreactivity was revealed by chemiluminescence (ECL-kit, Amersham Biosciences, UK). In Vitro Fibrillogenesis—Solutions in storage buffer of pure collagens I and XI, or defined mixtures thereof were degassed under vacuum. 100-μl samples were transferred to microcuvettes (Multicell, light path, 1 cm, Beckman, Palo Alto, CA) and were mixed with 100 μl of distilled water at 4 °C. The cuvettes were sealed and placed immediately into a spectrophotometer (Beckman UV 640, equipped with a Multicell holder, Micro Auto 12), connected to a water bath at 37 °C. In some experiments, collagen I solutions in storage buffer were diluted with equal volumes of water and were added to collagen XI fibrils preformed in half-strength storage buffer for 180 min at 37 °C. Aggregation was monitored by turbidity development at 313 nm. Electron Microscopy and Immunoelectron Microscopy—10-μl aliquots of reconstitution mixtures were spotted onto sheets of Parafilm. Copper grids coated with Formvar/carbon were floated on the drops for 5 min to allow adsorption of aggregates and were washed with distilled water. For transmission electron microscopy, fibrils on grids were negatively stained with 2% uranyl acetate for 10 min. For immunoelectron-microscopy, the grids were treated for 30 min with 2% (w/v) dried skim milk in PBS and, subsequently, for 2 h with the same buffer containing antiserum to collagen XI or polyclonal antibodies to collagen I (dilution 1:100 and 1:400, respectively). After extensive washing with PBS, the grids were put on drops of 0.2% dried skim milk in PBS, containing colloidal gold particles (18 nm) coated with goat antibodies to rabbit immunoglobulins at dilutions recommended by the manufacturer (Jackson ImmunoResearch). Finally, the grids were washed with distilled water and negatively stained with 2% uranyl acetate for 10 min. Negative controls were done without first antibody treatment. Electron micrographs were taken at 60 kV with an EM 410 electron microscope (Philips) or at 80 kV with a CM 10 electron microscope (Philips). Purification of Collagens—Native, fibrillogenesis-competent collagen XI was purified from cultures of chick embryo sternal chondrocytes embedded into agarose and cultured in the presence of the lysyl oxidase inhibitor β-aminopropionitrile. As described previously (25Blaschke U.K. Eikenberry E.F. Hulmes D.J.S. Galla H.J. Bruckner P. J. Biol. Chem. 2000; 275: 10370-10378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), the pure protein produced electrophoretic patterns in SDS-PAGE that corresponded to several variants of α1(XI)-polypeptides, as well as the α2(XI) and α3(XI) chains (Fig. 1, lane 1). This complex polypeptide composition results from both alternative splicing (37Oxford-Thom J.R. Doege K.J. Morris N.P. J. Biol. Chem. 1995; 270: 9478-9485Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 38Zhidkova N.I. Justice S.K. Mayne R. J. Biol. Chem. 1995; 270: 9486-9493Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) as well as incomplete proteolytic processing of the amino-terminal propeptide domain (39Thom J.R. Morris N.P. J. Biol. Chem. 1991; 266: 7262-7269Abstract Full Text PDF PubMed Google Scholar, 40Rousseau J.C. Farjanel J. Boutillon M.M. Hartmann D.J. van der Rest M. Moradi-Améli M. J. Biol. Chem. 1996; 271: 23743-23748Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Collagen I was purified by ion exchange chromatography on DEAE-cellulose from a mixture of collagens extracted from leg tendons of 17-day-old chicken embryos. The purified collagen I yielded the expected banding pattern in SDS-PAGE and consisted mostly of monomeric α1(I) and α2(I) chains, in addition to small amounts of cross-linked β-components (Fig. 1, lane 5). Thus, the preparations were considered as pure and, in particular, free of collagen V. Antisera to Native Collagen XI—Because collagens V and XI are highly similar proteins, they tend to elicit cross-reactive immune responses. In addition, because the α3(XI) chain is genetically identical to the α1(II) chain, most antisera to collagen XI also recognize collagen II. Previously, we have immunized rabbits with pepsin-treated collagen XI coupled to keyhole limpet hemocyanin, and, even without affinity purification, the resulting antisera did not react with collagen II. However, a potential cross-reaction with collagen V was not investigated (3Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (403) Google Scholar). Therefore, rabbits were immunized with a hemocyanin-conjugate of native collagen XI retaining its non-triple helical domains. As demonstrated by ELISA, the crude antiserum from one of several animals specifically recognized chicken collagen XI, but not the types I, II, or V (Fig. 2). The predominant reactivity in immunoblotting was associated with the α1(XI) chains (Fig. 1, lane 2). A similar reactivity of collagen XI chains was observed after treatment with pepsin (Fig. 1, compare lanes 3 and 4). In Vitro Fibrillogenesis—All aggregation studies described here were conducted with native collagens that had not previously been subjected to limited proteolysis by pepsin. Collagens I or XI were exhaustively dialyzed against 100 mm Tris HCl, pH 7.4, containing 400 mm NaCl (storage buffer). To adjust for identical final concentrations of collagen I, mixtures of the two collagens were appropriately diluted with storage buffer. The concentrations of the stock solutions were determined by a colorimetric assay calibrated with pepsin-treated collagen II at concentrations evaluated by circular dichroism at 221 nm ([Θ]221 nm = 8550 deg × cm2 × dmol–1 (36Bächinger H.P. Bruckner P. Timpl R. Prockop D.J. Engel J. Eur. J. Biochem. 1980; 106: 619-632Crossref PubMed Scopus (204) Google Scholar)). Fibrillogenesis was initiated by dilution with an equal volume of distilled water followed by immediate warming of the reaction mixtures to 37 °C. Equilibration of these aggregation conditions was achieved within less than 3 min. The kinetics of fibrillogenesis was monitored by turbidity development at 313 nm. At given intervals, the newly formed aggregates were examined in parallel samples by transmission electron and/or immunoelectron microscopy. Pure collagen I produced sigmoidal turbidity curves at all concentrations tested, essentially as described previously (24Birk D.E. Fitch J.M. Babiarz J.P. Doane K.J. Linsenmayer T.F. J. Cell Sci. 1990; 95: 649-657Crossref PubMed Google Scholar, 41Gelman R.A. Williams B.R. Piez K.A. J. Biol. Chem. 1979; 254: 180-186Abstract Full Text PDF PubMed Google Scholar). Unlike collagen II, which precipitates from solution (25Blaschke U.K. Eikenberry E.F. Hulmes D.J.S. Galla H.J. Bruckner P. J. Biol. Chem. 2000; 275: 10370-10378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), collagen I formed homogeneous viscous suspensions allowing an unambiguous determination of final plateau levels of turbidity. Aggregate structures recognizable by electron microscopy were not formed during the initial lag phase of turbidity development (see also Fig. 6A). The length of this lag period decreased, whereas the final plateau level increased with the concentration of collagen I. After completion of fibrillogenesis, the protein was incorporated into large fibrils with a strong D-periodic banding pattern of 67 nm visualized by electron microscopy after negative staining (Fig. 3, A and B). At 90 μg/ml or at 200 μg/ml collagen I, the fibrils exhibited a broad diameter distribution ranging from 50 to 300 nm (solid bars in Fig. 4, A and B, respectively). An example of a very large fibril characteristically formed by pure collagen I is shown in Fig. 3B. Therefore, we concluded that in vitro fibrillogenesis of collagen I was not subject to a stringent lateral growth control. Instead, fibrils were formed by stochastical nucleation followed by propagation accretion of single collagen molecules, consistently with the model proposed by Silver et al. (42Silver D. Miller J. Harrison R. Prockop D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9860-9864Crossref PubMed Scopus (60) Google Scholar).Fig. 3Fibrils reconstituted from collagens I or XI, or from mixtures thereof. Transmission electron micrograph of fibrils formed in vitro after completion of turbidity development. A and B, collagen I alone (90 μg/ml); C, collagen XI alone (70 μg/ml); D, a mixture of collagen I (90 μg/ml) and collagen XI (13 μg/ml). Note: thin fibrils with a weak banding pattern resembling those shown in panel C are absent in A and B but co-existed with large fibrils with a strong D-periodic banding pattern in D. Bar, 200 nm (100 nm in inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Mass-weighted diameter distributions of fibrils reconstituted in vitro. Fibrils were reconstituted from pure collagen I, or from mixtures of collagens I and XI. The concentrations of collagen I was kept constant, and the concentrations of collagen XI were varied. Diameters of all fibrils formed by a given mixture were measured in several, arbitrarily selected micrographs. A, collagen I (90 μg/ml) and collagen XI: 0 (solid bars), 13 μg/ml (f I/XI = [collagen I]/[collagen XI] = 7, gray bars), or 53 μg/ml (f I/XI = 1.7, open bars). B, collagen I (200 μg/ml) and collagen XI: 0 (solid bars) or 38 μg/ml (f I/XI = 5.25, gray bars). Note: mean fibril diameters were reduced in the presence of collagen XI.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Confirming our earlier results (25Blaschke U.K. Eikenberry E.F. Hulmes D.J.S. Galla H.J. Bruckner P. J. Biol. Chem. 2000; 275: 10370-10378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), collagen XI alone produced turbidity curves without lag periods and with plateau levels linearly depending on concentration (see also Fig. 5). Unlike collagen I, the protein formed long flexible filaments immediately after reduction of the salinity. In the electron microscope, the filaments displayed a uniform width of 26.34 ± 1.9 nm, but no banding pattern (Fig. 6B). They were subsequently compacted into fibrils of a similarly homogeneous width (24.6 ± 1.6 nm, Fig. 3C) and a weak banding pattern (inset in Fig. 3C). Next, mixtures of collagens I and XI were subjected to in vitro fibrillogenesis. In a given set of experiments, the concentration of either collagen I or XI was kept cons