David J.S. Hulmes

RADIAL PACKING, ORDER, AND DISORDER IN COLLAGEN FIBRILS

Collagen fibrils resemble smectic, liquid crystals in being highly ordered axially but relatively disordered laterally. In some connective tissues, x-ray diffraction reveals three-dimensional crystallinity in the molecular packing within fibrils, although the continued presence of diffuse scatter indicates significant underlying disorder. In addition, several observations from electron microscopy suggest that the molecular packing is organized concentrically about the fibril core. In the present work, theoretical equatorial x-ray diffraction patterns for a number of models for collagen molecular packing are calculated and compared with the experimental data from tendon fibrils. None of the models suggested previously can account for both the crystalline Bragg peaks and the underlying diffuse scatter. In addition, models in which any of the nearest-neighbor, intermolecular vectors are perpendicular to the radial direction are inconsistent with the observed radial orientation of the principal approximately 4 nm Bragg spacing. Both multiple-start spiral and concentric ring models are devised in which one of the nearest-neighbor vectors is along the radial direction. These models are consistent with the radial orientation of the approximately 4 nm spacing, and energy minimization results in radially oriented crystalline domains separated by disordered grain boundaries. Theoretical x-ray diffraction patterns show a combination of sharp Bragg peaks and underlying diffuse scatter. Close agreement with the observed equatorial diffraction pattern is obtained. The concentric ring model is consistent with the observation that the diameters of collagen fibrils are restricted to discrete values.

Collagen–mineral axial relationship in calcified turkey leg tendon by X-ray and neutron diffraction

The axial relationship between the collagen and mineral components in calcified turkey leg tendon was investigated using low-angle X-ray and neutron diffraction. The results indicate that the mineral is arranged with the same axial periodicity as the collagen and occupies the gap region of the collagen structure.

Interpretation of the meridional X-ray diffraction pattern from collagen fibres in terms of the known amino acid sequence.

A brief review is given of attempts to interpret the low-angle meridional X-ray diffraction patterns of tendon to yield the axially projected electron density of collagen fibrils. Both X-ray/electron microscope correlations and more conventional crystallographic methods have been used. For wet tendon, there have been two fundamental assumptions; either that those parts of the fibril which are positively stained in electron micrographs are intrinsically electron dense, or that the electron density is centrosymmetric. The validity of these assumptions is tested by synthesising models for the electron density based on the known amino acid sequence of collagen and the Hodge-Petruska scheme. Allowance is made for the effect of the water environment. These models are then Fourier transformed to give a set of calculated intensities for comparison with the observed low-angle meridional X-ray data. The calculated intensities are very sensitive to the conformation of the non-triple-helical terminal peptides (telopeptides) and to the D period. Difference Fourier syntheses are used to refine a model which gives the best agreement with the observed data whilst being both physically and chemically acceptable. The calculated phases of this model (model E) are then combined with the observed amplitudes to give a Fourier synthesis of the axially projected electron density in wet collagen fibrils. The implications of this result for the study of various metabolic and pathological situations in connective tissue are discussed.

Collagen polymorphism: Its origins in the amino acid sequence☆

Abstract The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 A ( D ) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 A periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.

Electron microscopy of the collagen fibril

Collagen is identified by those properties that stem from the predominantly triple-chain helical structure of its molecules. A prerequisite for the formation of this triple helix is a Gly-X-Y repeating tripeptide unit in the amino acid sequence of the three chains, where X and Y can be any amino acids but are often the imino acids proline and hydroxyproline. This sequence, with glycine in every third position and with an unusual abundance of hydroxyproline, forms the basis for the chemical identification of collagen (for review, see 1). An unambiguous physical identification is provided by X-ray diffraction; the helix parameters established by high-angle X-ray scattering are unique to collagen (2).

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.

Tyrosine-rich acidic matrix protein (TRAMP) is a tyrosine-sulphated and widely distributed protein of the extracellular matrix

Tyrosine‐rich acidic matrix protein (TRAMP; 22 kDa extracellular matrix protein; dermatopontin) is a protein that co‐purifies with lysyl oxidase and with dermatan sulphate proteoglycans, with possible functions in cell—matrix interactions and matrix assembly. Using a rabbit polyclonal antiserum raised against porcine TRAMP, which cross‐reacts with both the human and murine forms of the protein, we show by immunoblotting that TRAMP has a widespread tissue distribution, including skin, skeletal muscle, heart, lung, kidney, cartilage and bone. In cultures of human skin fibroblasts, TRAMP incorporates both [35S]sulphate and [3H]tyrosine and is secreted into the medium, as shown by immunoprecipitation. Amino acid analysis of immunoprecipitated TRAMP demonstrates that many of the tyrosine residues in TRAMP are sulphated.

Extracellular and cell surface proteases in wound healing: new players are still emerging

Tissue remodelling results from the concerted action of numerous extracellular and cell surface proteases. These act to synchronize the synthesis and degradation of the extracellular matrix with the control of cytokine activity and cell signalling in order to create appropriate environments for cell proliferation, migration and differentiation. Wound healing is a complex example of tissue remodelling that includes several steps occurring either concomitantly or successively during the process of repair: haemostasis, inflammation, angiogenesis, re-epithelialisation, granulation tissue formation, wound contraction and matrix remodelling. The main extracellular and cell surface proteases involved in wound healing are serine proteases, especially plasmin, and metalloproteases of the metzincin family (MMPs, ADAM(TS)s, tolloids, meprins, pappalysins) with cysteine proteases playing less prominent roles. Several regulatory proteins and hundreds of substrates have been identified for these proteases, either in vitro or in vivo. The aim of this review is not to present an exhaustive list of proteases and related molecules but to give an overview of the proteolytic events that are potentially relevant during tissue repair. New developments aimed at approaching a more integrative view of all the molecular events involved in tissue remodelling are also discussed.

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold CONSERVED AND SPECIFIC FEATURES OF THE CUB1 DOMAIN CONTRIBUTE TO THE MOLECULAR BASIS OF PROCOLLAGEN C-PROTEINASE ENHANCER-1 ACTIVITY

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.