Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

Thermostability gradient in the collagen triple helix reveals its multi-domain structure

A triple-helical conformation and stability at physiological temperature are critical for the mechanical and biological functions of the fibril-forming collagens. Here, we characterized the role of consecutive domains of collagen II in stabilizing the triple helix. Analysis of melting temperatures of genetically engineered collagen-like proteins consisting of tandem repeats of the D1, D2, D3 or D4 collagen II periods revealed the presence of a gradient of thermostability along the collagen molecule with thermolabile N-terminal domains and thermostable C-terminal domains. These results imply a multi-domain character of the collagen triple helix. Assays of thermostabilities of the Arg75Cys and Arg789Cys collagen II mutants suggest that, in contrast to the thermostable domains, the thermolabile domains are able to accommodate amino acid substitutions without altering the thermostability of the entire collagen molecule.     

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D=67nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.     

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.     

Morphology of sheet-like assemblies of pN-collagen, pC-collagen and procollagen studied by scanning transmission electron microscopy mass measurements

At high concentrations, type I pN-collagen, pC-collagen and procollagen (the first 2 generated from procollagen by enzymic cleavage of C-propeptides and N-propeptides, respectively) can all be made to assemble in vitro into thin D-periodic sheets or tapes. Scanning transmission electron microscopy mass measurements show that the pN-collagen sheets and procollagen tapes have a mass per unit area corresponding to that of approximately 6.8 monolayers of close-packed molecules. pN-collagen sheets are extensive and remarkably uniform in mass thickness (fractional S.D. 0.035); procollagen tapes are neither as extensive nor as uniform in thickness. The mean thickness of pC-collagen tapes is less and the variability is greater. In pN-collagen sheets, the overlap: gap mass contrast in a D-period is increased from 5:4 (the ratio in a native collagen fibril) to 6:4, showing that the N-propeptides do not project into the gap but are folded back over the overlap zone. Assuming all N-propeptides to be constrained to the two surfaces of a sheet, their surface density can be found from the mass thickness of the sheet. In a lateral direction (i.e. normal to the axial direction where the spacing is D-periodic), the N-propeptide domains are calculated to be spaced, centre to centre, by 2.23 (+/- 0.1) nm on both surfaces. This value (approx. 1.5 x the triple-helix diameter) implies close-packing laterally with adjacent domains in contact. Sheet formation and the "surface-seeking" behaviour of propeptides can be understood in terms of the dual character of the molecules, evident from solubility data, with propeptides possessing interaction properties very different from those displayed by the rest of the molecule. The form and stability of sheets (and of first-formed fibrils assembling in vivo) could, it is suggested, depend on the partially fluid-like nature of lateral contacts between collagen molecules.     

Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.     

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.     

D-periodic assemblies of type I procollagen

The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.

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-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. 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.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Two type XII-like collagens localize to the surface of banded collagen fibrils

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.     

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.     

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

In Situ D-periodic Molecular Structure of Type II Collagen

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.     

Linear aggregation and the turbidimetric lag phase: type I collagen fibrillogenesis in vitro.

Using classical light scattering theory it is shown that the turbidimetric lag phase is a property of macromolecular systems that form linear aggregates during the initial period of self-assembly. This analysis and previous observations on Type I collagen suggests that initiation occurs by the conversion of single molecules into linear dimers in which neighboring molecules are staggered by 4·0 D (D = 67 nm). Linear.growth of dimers occurs by 4 D addition of either single molecules or linear aggregates until the aggregate is about 60 to 70 molecules long. Linear growth appears to involve charged binding sites in the amino and carboxy termini. The same sites have been shown to be involved in crosslink formation and in the attachment of disaccharides.Lateral growth occurs via the rapid formation of several discretely sized intermediates which lead to the formation of narrow fibrils ～ 25 nm wide. Narrow fibrils individually wrap around each other increasing the diameter to 100 nm or greater.     

Fibril-forming collagens in lamprey

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.