Connective tissue polarity unraveled by a markov-chain mechanism of collagen fibril segment self-assembly

The well-established occurrence of pyroelectricity (Lang, 1966) in tissues of living organisms has found a first explanation by a Markov-chain mechanism taking place during collagen fibril self-assembly in extracytoplasmic channels. Recently reported biochemical findings on the longitudinal fusion reactivity of small fibril segments (which undergo C-, N- and C-, C- but not N-, N-terminal fusions; see Graham et al., 2000; Kadler et al., 1996) may provide a mechanism by which a difference in the fusion probabilities P(CC), P(NN) drives the self-assembly into partial macroscopic polar order. In principle, a Markov-chain growth process can lower the noncentrosymmetric infinity 2 symmetry describing dielectric properties of a growing limb (as managed by fibroblasts) into the polar infinity group. It is proposed that macroscopically polar properties enter the biological world by a stochastic mechanism of unidirectional growth. Polarity formation in organisms shows similarity to effects reported for molecular crystals (Hulliger et al., 2002).     

The echinoderm collagen fibril: a hero in the connective tissue research of the 1990s

Collagen fibrils are some of the most-abundant and important extracellular structures in our bodies, yet we are unsure of their shape and size. This is largely due to an inherent difficulty in isolating them from their surrounding tissues. Echinoderms have collagenous tissues that are similar to ours in many ways, yet they can be manipulated to easily relinquish their collagen fibrils, providing an excellent opportunity to study native fibrillar structure. In the early 1990s, they were found to defy the commonly accepted fibrillar model of the time in that they were much shorter, they were shaped like double-ended spindles, and their centers exhibited a reversal in molecular polarity. Realization of these features helped to reform the questions that were being asked about vertebrate fibrils, shifting the focus toward shape and size. Since then, researchers working with both groups (echinoderms and vertebrates) have worked together to find the structure of native fibrils. This information will be fundamental in understanding what holds collagenous tissues together at the fibrillar level, and could have important implications for people with Ehlers-Danlos syndrome.     

ColBuilder: A server to build collagen fibril models

Type I collagen is the main structural component of many tissues in the human body. It provides excellent mechanical properties to connective tissue and acts as a protein interaction hub. There is thus a wide interest in understanding the properties and diverse functions of type I collagen at the molecular level. A precondition is an atomistic collagen I structure as it occurs in native tissue. To this end, we built full-atom models of cross-linked collagen fibrils by integrating the low-resolution structure of collagen fibril available from x-ray fiber diffraction with high-resolution structures of short collagen-like peptides from x-ray crystallography and mass spectrometry data. We created a Web resource of collagen models for 20 different species with a large variety of cross-link types and localization within the fibril to facilitate structure-based analyses and simulations of type I collagen in health and disease. To easily enable simulations, we provide parameters of the modeled cross-links for an Amber force field. The repository of collagen models is available at https://colbuilder.h-its.org.     

Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices

In nested collagen matrices, human fibroblasts migrate from cell-containing dermal equivalents into surrounding cell-free outer matrices. Time-lapse microscopy showed that in addition to cell migration, collagen fibril flow occurred in the outer matrix toward the interface with the dermal equivalent. Features of this flow suggested that it depends on the same cell motile machinery that normally results in cell migration. Collagen fibril flow was capable of producing large-scale tissue translocation as shown by closure of a approximately 1-mm gap between paired dermal equivalents in floating, nested collagen matrices. Our findings demonstrate that when fibroblasts interact with collagen matrices, tractional force exerted by the cells can couple to matrix translocation as well as to cell migration.     

Lipid-apolipoprotein interactions in amyloid fibril formation and relevance to atherosclerosis

The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-β structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma.     

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.     

Confined compression of a tissue-equivalent: collagen fibril and cell alignment in response to anisotropic strain

A method to impose and measure a one dimensional strain field via confined compression of a tissue-equivalent and measure the resulting cell and collagen fibril alignment was developed Strain was determined locally by the displacement of polystyrene beads dispersed and entrapped within the network of collagen fibrils along with the cells, and it was correlated to the spatial variation of collagen network birefringence and concentration. Alignment of fibroblasts and smooth muscle cells was determined based on the long axis of elongated cells. Cell and collagen network alignment were observed normal to the direction of compression after a step strain and increased monotonically up to 50% strain. These results were independent of time after straining over 24 hr despite continued cell motility after responding instantly to the step strain with a change in alignment by deforming/convecting with the strained network. Since the time course of cell alignment followed that of strain and not stress which, due to the viscoelastic fluid-like nature of the network relaxes completely within the observation period, these results imply cell alignment in a compacting tissue-equivalent is due to fibril alignment associated with anisotropic network strain. Estimation of a contact guidance sensitivity parameter indicates that both cell types align to a greater extent than the surrounding fibrils.     

Inhibition of collagenase by Cr(III): its relevance to stabilization of collagen

Bacterial collagenase has now been reacted with a select series of Cr(III) complexes and modifications in the activity of chromium-modified collagenase has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. A homologous series of Cr(III) complexes with dimeric, trimeric and tetrameric structures as in 1, 2 and 3 respectively has been investigated for their ability to inhibit the action of collagenase against FALGPA. Whereas competitive and non-competitive modes of inhibition of collagenase are expressed by 1, (dimer) and 2, (trimer) respectively, the tetramer, 3, exhibits poor affinity to collagenase and the inhibition of the enzyme activity is uncompetitive. Evidence for different modes of inhibition of collagenase depending on the nature of Cr(III) species has been presented in this work. Circular dichroism and gel electrophoresis data on Cr(III) modified collagenase corroborate the hypothesis that the inhibition of collagenase by the heavy metal ion arises from secondary and quaternary structural changes in the enzyme. The implications of the observed Cr(III) species specific inhibition of collagenase in gaining new insight into the mechanism of stabilization of collagen by Cr(III) are discussed.     

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule’s size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.     

The unfolded protein response and its relevance to connective tissue diseases

The unfolded protein response (UPR) has evolved to counter the stresses that occur in the endoplasmic reticulum (ER) as a result of misfolded proteins. This sophisticated quality control system attempts to restore homeostasis through the action of a number of different pathways that are coordinated in the first instance by the ER stress-senor proteins IRE1, ATF6 and PERK. However, prolonged ER-stress-related UPR can have detrimental effects on cell function and, in the longer term, may induce apoptosis. Connective tissue cells such as fibroblasts, osteoblasts and chondrocytes synthesise and secrete large quantities of proteins and mutations in many of these gene products give rise to heritable disorders of connective tissues. Until recently, these mutant gene products were thought to exert their effect through the assembly of a defective extracellular matrix that ultimately disrupted tissue structure and function. However, it is now becoming clear that ER stress and UPR, because of the expression of a mutant gene product, is not only a feature of, but may be a key mediator in the initiation and progression of a whole range of different connective tissue diseases. This review focuses on ER stress and the UPR that characterises an increasing number of connective tissue diseases and highlights novel therapeutic opportunities that may arise.     

The role of hydrophobic bonding in collagen fibril formation: a quantitative model

A model has been developed for approximating the free energy of collagen fibril formation (ΔF_f) and the equilibrium solubility of collagen under physiological conditions. The model utilizes an expression of Flory for rodlike polymers, with the modification that the “pure” anisotropic phase is defined as a collagen fibril containing about 0.3 g water/g collagen. The model also assumes that χ₁, the polymer–solvent interaction term, is entirely due to hydrophobic effects. χ₁ is estimated from hydrophobic bond energies of amino acid side chains, using the results of Némethy and Scheraga. The temperature dependence of χ₁ is utilized to calculate equilibrium solubilities and ΔF_f as a function of temperature.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

Different architectures of the collagen fibril: morphological aspects and functional implications

Several tissues known to contain collagen fibrils with a ‘helical’ arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appeared to be tissue-specific and fairly constant within the same tissue. No statistical differences, on the contrary, were detectable in the coiling angle which appeared similar in all the tissues and independent of both diameter and age of the fibril. Rat tail tendon was also examined under the same technical conditions and showed collagen fibrils of large and very heterogeneous diameter and with a consistent ‘straight’ arrangement. These data seem to suggest that the ‘helical’ and ‘straight’ arrangements may actually identify different types of collagen fibrils. The authors discuss the possible functional significance of these arrangements and present two hypotheses on the three-dimensional structure of the ‘helical’ fibril.     

Stress transfer in collagen fibrils reinforcing connective tissues: effects of collagen fibril slenderness and relative stiffness

Unlike engineering fibre composite materials which comprise of fibres that are uniform cylindrical in shape, collagen fibrils reinforcing the proteoglycan-rich (PG) gel in the extra-cellular matrices (ECMs) of connective tissues are taper-ended (paraboloidal in shape). In an earlier paper we have discussed how taper of a fibril leads to an axial stress up-take which differs from that of a uniform cylindrical fibre and implications for fibril fracture. The present paper focuses on the influence of fibre aspect ratio, q (slenderness), and Young's modulus (stiffness), relative to that of the gel phase, E(R), on the magnitude of the axial tensile stresses generated within a fibril and wider implications on failure at tissue level. Fibre composite models were evaluated using finite element (FE) and mathematical analyses. When the applied force is low, there is elastic stress transfer between the PG gel and a fibril. FE modelling shows that the stress in a fibril increases with E(R) and q. At higher applied forces, there is plastic stress transfer. Mathematical modelling predicts that the stress in a fibril increases linearly with q. For small q values, fibrils may be regarded as fillers with little ability to provide tensile reinforcement. Large q values lead to high stress in a fibril. Such high stresses are beneficial provided they do not exceed the fracture stress of collagen. Modulus difference regulates the strain energy release density, u, for interfacial rupture; large E(R) not only leads to high stress in a fibril but also insures against interfacial rupture by raising the value of u.