John E. Scott

Elasticity in extracellular matrix ‘shape modules’ of tendon, cartilage, etc. A sliding proteoglycan‐filament model

Connective tissues (CTs), which define bodily shape, must respond quickly, robustly and reversibly to deformations caused by internal and external stresses. Fibrillar (elastin, collagen) elasticity under tension depends on molecular and supramolecular mechanisms. A second intra‐/inter‐molecular pair, involving proteoglycans (PGs), is proposed to cope with compressive stresses. PG interfibrillar bridges (‘shape modules’), supramolecular structures ubiquitously distributed throughout CT extracellular matrices (ECMs), are examined for potential elastic properties. l‐iduronate residues in shape module decoran PGs are suggested to be molecular springs, cycling through alternative conformations. On a larger scale, anionic glycosaminoglycan (AGAG) interfibrillar bridges in shape modules are postulated to take part in a sliding filament (dashpot‐like) process, which converts local compressions into disseminated tensile strains. The elasticity of fibrils and AGAGs, manifest at molecular and larger‐scale levels, provides a graduated and smooth response to stresses of varying degrees. NMR and rheo NMR, computer modelling, electron histochemical, biophysical and chemical morphological evidence for the proposals is reviewed.

The structure of interfibrillar proteoglycan bridges (shape modules') in extracellular matrix of fibrous connective tissues and their stability in various chemical environments.

Collagen fibrils in extracellular matrices of connective tissues (tendon, cornea, etc.) are bridged and linked by the anionic glycosaminoglycans (AGAGs) of the small proteoglycans (decoron, etc.). It was proposed that these bridges and ties maintain the collagen fibril dispositions in relation to each other, helping to define tissue shape, and hence called shape modules. This investigation describes chemical and physicochemical conditions in which these structures are stable and what treatments cause their disruption. The effects on fixed and unfixed sections of tendon, cornea, lung and ear from rat, mouse and rabbit of pH, electrolyte concentration, EDTA, mercaptoethanol, hydrogen peroxide, free radicals, periodate, acetylation, urea, nonionic detergent and organic solvents were assessed by staining with Cupromeronic blue or Alcec blue in CEC techniques to localise AGAG bridges or their disintegration products. Ca2+ was not involved in the structures, oxidation/reduction had no effect and Triton X100, a nonionic detergent did not damage them. They were stable between pH 4.5 and 9.5. Periodate as a glycol‐cleaving reagent did not affect them. High concentrations of urea (>2.0 m) and MgCl2 (0.5 m) disrupted the tissues. The combination of Triton and urea at concentrations too low to cause damage separately was disruptive. Free radicals in periodate solutions were damaging. Organic solvents caused collapse and rearrangements of the AGAG filaments. Acetylation caused considerable disruption of shape modules. Dermochondan but not keratan sulphate AGAGs were removed by treatment with NaOH. After fixing with glutaraldehyde only free radical and NaOH treatments were severely disruptive of shape modules. The results are compatible with a previously proposed structure for the shape modules, stabilised by hydrophobic and hydrogen bonding.

Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e1 bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

Secondary structure in glycosaminoglycuronans: N.m.r. spectra in dimethyl sulphoxide of disaccharides related to hyaluronic acid and chondroitin sulphate

1H-N.m.r.spectra for solutions in dimethyl sulphoxide-d6 of disaccharides related to hyaluronate and chondroitin sulphate are compared with those of their methylated derivatives. All resonances, including those of HO and HN groups, have been assigned. The temperature and concentration dependences suggest that HO-4 of the hexosamine residue in hyalobiouronate (but not that in chondrosinate) is hydrogen-bonded to O-5 of the uronic acid residue. The resonance of HO-2 of the uronate residue of chondrosinate also shows anomalies that may arise from intra-residue hydrogen-bonding. These findings confirm the existence of some features previously suggested to be present in glycosaminoglycuronan polymers. The resonance of HO-4 of the uronate residue in the disaccharides and in sodium (methyl alpha-D-glucopyranosid)uronate behaves as though there was a hydrogen bond between the carboxylate group and HO-4.

Control of collagen fibril diameters in tissues

It is proposed that radial growth of collagen fibrils, which takes place in all connective tissues to varying extents, according to the tensile stresses exerted on them, proceeds mainly by aggregation of protofibrils (approximately 10 nm) and existing fibrils. In young tissues, fibrils are prevented from making frequent intimate contacts which would lead to aggregation by abundant interfibrillar proteoglycan, that keeps the fibrils apart. Collagen fibrils are probably unable to fuse except when the molecules within them are packed in the same sense, i.e. fusing fibrils are parallel. The roughly equal numbers of parallel and antiparallel fibrils seen in several tissues must limit radial fibril growth in older tissues, where proteoglycan is usually less abundant. Possible origins of the balance of fibril polarities, which must be conserved after fibril nucleation on cell or non-cell templates, are analysed. The two controlling factors, ambient proteoglycan and fibril polarity, working against the tendency of fibrils to fuse, account for many features of the observed distributions of collagen fibril diameters in diverse tissues and at different ages.

Cartilage elasticity resides in shape module decoran and aggrecan sumps of damping fluid: implications in osteoarthrosis

Cartilage ultrastructure is based on collagen fibrils tied together by proteoglycans (PGs). Interfibrillar orthogonal PG bridges (‘shape modules’) were located by electron histochemistry using Cupromeronic blue methodology. Their frequency and size, similar to those in tendon, cornea, etc., were compatible with biochemical estimates of tissue decoran (formerly decorin), the PG component of shape module bridges. Digestion by hyaluronanase and chondroitinase AC helped to identify aggrecan and decoran and exemplified the destruction of shape modular organization by glycan‐splitting agents. The anionic glycosaminoglycan (AGAG) of decoran, dermochondan sulphate (DS, formerly dermatan sulphate), contains l‐iduronate, an elastic sugar unit. Chondroitan, keratan (present in aggrecan) and hyaluronan are not similarly elastic but can participate in sliding‐filament reversible deformability. Mechanical properties predicted for the interfibrillar bridges accord with anisotropic stress/strain responses of articular cartilage to compressive or tensile stresses. We propose that fluid from pericellular aggrecan‐rich domains moves under pressure into the interterritorial fibrillar arrays against the elastic resistance of the shape modules, which return the fluid, post‐compression, to its original position. Cartilage is tendon‐like, with the addition of expansile aggrecan‐rich reservoirs of aqueous shock absorber fluid. Rupture or loss of interfibrillar ties would allow expansile PG to force the collagenous matrix apart, imbibing water, increasing swelling and fissuring – characteristic manifestations of osteoarthrosis (OA), a joint disease of major economic importance. Decoran may be a primary target of the OA disease process.

Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.

Oxygen and the connective tissues

Avascular connective tissues (cartilage, discs, cornea) change with maturation and aging, particularly in large animals, where diffusion paths are longest. It is suggested that the changes in such tissues are responses to increasing difficulties in obtaining oxygen. Two almost identical structural polymers are made in these tissues: chondroitin sulphate, which requires large amounts of oxygen for biosynthesis and keratan sulphate, which requires relatively little. The observed balance of these polymers in the tissue is proposed to depend on the control of biosynthesis by the ambient oxygen tension, and/or selective breakdown.

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.

Proteoglycan: collagen interactions in dermatosparactic skin and tendon. An electron histochemical study using cupromeronic blue in a critical electrolyte concentration method.

Proteoglycans (PGs), stained for electron microscopy with Cupromeronic blue, were observed in skin and tendon from normal and dermatosparactic calves. Very frequently they (i.e. dermatan sulphate (DS) PGs) were seen arrayed orthogonally to the collagen fibrils, in the gap zone, usually at the d band, in both diseased and normal tissues. Where UO2(2+) staining showed regular and normal packing of collagen molecules, orthogonally located DS PGs were seen. No qualitative differences between controls and pathological tissues were identified, but quantitatively it appears likely that considerable areas of the surface of dermatosparactic skin collagen fibrils may be without associated PGs.