Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.     

Molecular packing in type I collagen fibrils

Previous studies of the X-ray diffraction pattern of the crystalline regions of type I collagen fibrils yielded information on the unit cell parameters and also the orientation of the pseudo-hexagonally packed molecular segments in the overlap region. The absence of Bragg reflections at high angles attributable to the molecular segments in the gap region led to the suggestion that these segments were more mobile than those in the overlap region. We report a study of the low-angle Bragg reflections in a search for information about the nature of the orientation and packing of the molecular segments in the gap region. We conclude that the (m = 0, n = 0) helix layer plane of the molecular segments in the overlap region makes little or no contribution to the Bragg reflections at low angles, and identify three possible origins for the observed low-angle reflections in the electron density contrast associated with: (1) the "hole" created by the missing molecular segment in the gap region; (2) the telopeptides; or (3) the axial regularities in amino acid residues of a particular type, with periodicities of D/5 or D/6. Sufficient information is available to investigate the first two of these possibilities, and the results obtained suggest specific arrangements for the molecular segments in the overlap and gap regions, and specific connectivities between the molecular segments in successive overlap regions. In addition, we have examined the amino acid sequence and identified features related to the mobility of the molecular segments in the gap region and to the regions where it is thought that molecules are kinked.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Cross-linkage sites in type I collagen fibrils studied by neutron diffraction

Cross-links in tendon collagen are essential for the biomechanical strength of healthy tissue. The nature and position of these cross-links has long been a subject for conjecture. We have approached this problem in a non-destructive manner, by studying neutron diffraction from collagen fibrils that have been specifically deuterated by reduction at keto-amine and Schiff base groups with sodium borodeuteride (NaB2H4). The intensities of the first 23 meridional reflections were recorded for both native and reduced tendons. These data were used to calculate the neutron-scattering density profile of the 67 nm (D) repeat of type I collagen fibrils in rat tail tendon. This approach not only succeeds in determining the location of the cross-linkage sites with respect to the fibril structure, as projected onto the fibre axis, but also presents a novel form of the isomorphous derivative solution to the phase problem.     

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.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

Glycation changes the charge distribution of type I collagen fibrils

In aging and diabetes, glycation of collagen molecules leads to the formation of cross-links that could alter the surface charge on collagen fibrils, and hence affect the properties and correct functioning of a number of tissues. The electron-optical stain phosphotungstic acid (PTA) binds to positively charged amino acid side-chains and leads to the characteristic banding pattern of collagen seen in the electron microscope; any change in the charge on these side-chains brought about by glycation will affect the uptake of PTA. We found that, upon glycation, a decrease in stain uptake was observed at up to five regions along the collagen D-period; the greatest decrease in stain uptake was apparent at the c1 band. This reduction in PTA uptake indicates that the binding of fructose leads to an alteration in the surface charge at several sites along the D-period. Not all lysine and arginine residues are involved; there appear to be specific residues that suffer a loss of positive charge.     

Structural changes in human type I collagen fibrils investigated by force spectroscopy

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin, and tendon. Here, we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy (AFM)-based force spectroscopy (FS). The elongation profiles show that in vitro-assembled human type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurring within the fibrils in the 1.5- to 4.5-nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in the extracellular matrix (ECM) remodeling associated with tissue growth and morphogenesis.     

Segregation of type I collagen homo- and heterotrimers in fibrils

Normal type I collagen is a heterotrimer of two α1(I) and one α2(I) chains, but various genetic and environmental factors result in synthesis of homotrimers that consist of three α1(I) chains. The homotrimers completely replace the heterotrimers only in rare recessive disorders. In the general population, they may compose just a small fraction of type I collagen. Nevertheless, they may play a significant role in pathology; for example, synthesis of 10-15% homotrimers due to a polymorphism in the α1(I) gene may contribute to osteoporosis. Homotrimer triple helices have different stability and less efficient fibrillogenesis than heterotrimers. Their fibrils have different mechanical properties. However, very little is known about their molecular interactions and fibrillogenesis in mixtures with normal heterotrimers. Here we studied the kinetics and thermodynamics of fibril formation in such mixtures by combining traditional approaches with 3D confocal imaging of fibrils, in which homo- and heterotrimers were labeled with different fluorescent colors. In a mixture, following a temperature jump from 4 to 32 °C, we observed a rapid increase in turbidity most likely caused by formation of homotrimer aggregates. The aggregates promoted nucleation of homotrimer fibrils that served as seeds for mixed and heterotrimer fibrils. The separation of colors in confocal images indicated segregation of homo- and heterotrimers at a subfibrillar level throughout the process. The fibril color patterns continued to change slowly after the fibrillogenesis appeared to be complete, due to dissociation and reassociation of the pepsin-treated homo- and heterotrimers, but this remixing did not significantly reduce the segregation even after several days. Independent homo- and heterotrimer solubility measurements in mixtures confirmed that the subfibrillar segregation was an equilibrium property of intermolecular interactions and not just a kinetic phenomenon. We argue that the subfibrillar segregation may exacerbate effects of a small fraction of α1(I) homotrimers on formation, properties, and remodeling of collagen fibers.     

The globular domain of the proalpha 1(I) N-propeptide is not required for secretion, processing by procollagen N-proteinase, or fibrillogenesis of type I collagen in mice.

The globular domain in the NH(2)-terminal propeptide (N-propeptide) of the proalpha1(I) chain is largely encoded by exon 2 of the Col1a1 gene and has been implicated in a number of processes that are involved in the biogenesis, maturation, and function of type I collagen. These include intracellular chain association, transcellular transport and secretion, proteolytic processing of the precursor, feedback regulation of synthesis, and control of fibrillogenesis. However, none of these proposed functions has been firmly established. To evaluate the function of this procollagen domain we have used a targeted mutagenesis approach to generate mice that lack exon 2 in the Col1a1 gene. Mouse lines were established on both a mixed 129 OlaHsd/Sv and C57BL/6 background and a pure 129 OlaHsd/Sv background. Adult mice on the mixed background are normal in appearance and are fertile. To the extent that they have been studied, procollagen synthesis, secretion, and proteolytic processing are normal in these mice, and collagen fibrillogenesis is only slightly altered. However, breeding of heterozygous mutant mice on the 129 background generated homozygous mutants at only 64% of the expected frequency. These findings suggest that although the N-propeptide is not essential for collagen biogenesis in mice it may play some essential role during embryonic development.     

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.     

Active negative control of collagen fibrillogenesis in vivo. Intracellular cleavage of the type I procollagen propeptides in tendon fibroblasts without intracellular fibrils

It is established fact that type I collagen spontaneously self-assembles in vitro in the absence of cells or other macromolecules. Whether or not this is the situation in vivo was unknown. Recent evidence shows that intracellular cleavage of procollagen (the soluble precursor of collagen) to collagen can occur in embryonic tendon cells in vivo, and when this occurs, intracellular collagen fibrils are observed. A cause-and-effect relationship between intracellular collagen and intracellular fibrils was not established. Here we show that intracellular cleavage of procollagen to collagen occurs in postnatal murine tendon cells in situ. Pulse-chase analyses showed cleavage of procollagen to collagen via its two propeptide-retained intermediates. Furthermore, immunoelectron microscopy, using an antibody that recognizes the triple helical domain of collagen, shows collagen molecules in large-diameter transport compartments close to the plasma membrane. However, neither intracellular fibrils nor fibripositors (collagen fibril-containing plasma membrane protrusions) were observed. The results show that intracellular collagen occurs in murine tendon in the absence of intracellular fibrillogenesis and fibripositor formation. Furthermore, the results show that murine postnatal tendon cells have a high capacity to prevent intracellular collagen fibrillogenesis.     

Mechanical properties of native and cross-linked type I collagen fibrils

Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 ± 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 ± 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 ± 7 MPa at ambient conditions and to 3.4 ± 0.2 MPa in phosphate-buffered saline.     

Mapping the heparin-binding sites on type I collagen monomers and fibrils

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin- binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell- directed assembly or restructuring of the collagenous extracellular matrix.     

Structural investigations on native collagen type I fibrils using AFM

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.     

Solvent specific persistence length of molecular type I collagen

Type I collagen is a fibril‐forming protein largely responsible for the mechanical stability of body tissues. The tissue level properties of collagen have been studied for decades, and an increasing number of studies have been performed at the fibril scale. However, the mechanical properties of collagen at the molecular scale are not well established. In the study presented herein, the persistence length of pepsin digested bovine type I collagen is extracted from the conformations assumed when deposited from solution onto two‐dimensional surfaces. This persistence length is a measure of the flexibility of the molecule. Comparison of the results for molecules deposited from different solvents allows for the study of the effect of the solutions on the flexibility of the molecule and provides insight into the molecule's behavior in situ. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 329–335, 2014.     

Distinct maturations of N-propeptide domains in fibrillar procollagen molecules involved in the formation of heterotypic fibrils in adult sea urchin collagenous tissues

We have characterized the primary structure of a new sea urchin fibrillar collagen, the 5alpha chain, including nine repeats of the sea urchin fibrillar module in its N-propeptide. By Western blot and immunofluorescence analyses, we have shown that 5alpha is co-localized in adult collagenous ligaments with the 2alpha fibrillar collagen chain and fibrosurfin, two other extracellular matrix proteins possessing sea urchin fibrillar modules. At the ultrastructural level, the 5alpha N-propeptide is detected at the surface of fibrils, suggesting the retention of this domain in mature collagen molecules. Biochemical characterization of pepsinized collagen molecules extracted from the test tissue (the endoskeleton) together with a matrix-assisted laser desorption ionization time-of-flight analysis allowed us to determine that 5alpha is a quantitatively minor fibrillar collagen chain in comparison with the 1alpha and 2alpha chains. Moreover, 5alpha forms heterotrimeric molecules with two 1alpha chains. Hence, as in vertebrates, sea urchin collagen fibrils are made up of quantitatively major and minor fibrillar molecules undergoing distinct maturation of their N-propeptide regions and participating in the formation of heterotypic fibrils.     

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.