Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils
Introduction
In many ways the extracellular matrix (ECM) is much more than the sum of its components. Mutual interactions are as important as the molecule structure in its intricate network of protein fibres (collagen and elastin) held together by carbohydrate strings (proteoglycans and glycoproteins). A wide and lively community of scientists has devoted enormous effort to investigating the structure of ECM macromolecules, whereas the mutual relationships among these molecules have proved more elusive and remain comparatively less understood.
The most prominent players in the ECM are fibrillar collagens (Ottani et al., 2001,2002) and small leucine-rich proteoglycans (SLRPs) (Hocking et al., 1998, Iozzo, 1999, McEwan et al., 2006). All are highly conserved molecules found in a comparable form in every species from the Echinoderms onwards. In particular, SLRPs seem to be involved in many aspects of collagen fibril formation, spatial layout and mechanical coupling. Among other things, proteoglycans are thought to down-regulate collagen fibrillogenesis, decrease (Neame et al., 2000, Douglas et al., 2006) or increase (Salchert et al., 2005, Rühland et al., 2007) fibril diameter, impose a long-range order on tissue macromolecules (Scott, 1992, Hocking et al., 1998, Chakravarti et al., 2000), and contribute to the functional properties of tissues by providing interfibrillar mechanical coupling (Cribb and Scott, 1995, Redaelli et al., 2003, Vesentini et al., 2005, Liu et al., 2005, Liao and Vesely, 2007). SLRPs’ tiny protein core is reported to bind on specific locations of the collagen fibril surface (Scott and Haigh, 1988, Schönherr et al., 1995a, Schönherr et al., 1995b, Kalamajski et al., 2007), while their carbohydrate side chains extend in the interfibrillar space (Iozzo, 1999) where they interact forming antiparallel dimers (‘shape modules’, Scott, 1995). This is, so far, the only structural hypothesis existing for the glycosaminoglycan side chain; there is no other competing paradigm. Other, non-structural, roles concern the binding and storage of growth factors (Ferdous et al., 2007) and other molecules.
A major difficulty of this research field is that ECM macromolecules form a complex, ill-defined set of mutual interactions which are mainly non-covalent: proteins and glycoconjugates are mostly bound by hydrophilic/hydrophobic and electrostatic interactions. Thermodynamics is the driving force of these weak bonds, and the complex treatments needed by some ultrastructural investigation techniques risk destroying their very object. These conditions result in frequent inconsistencies in the scientific literature, where the same molecule is often credited with opposite effects in different papers. Another problem is that any missing or defective molecular species in vivo is likely to be compensated by the overexpression of some other type.
In an attempt to gain fresh data on the collagen-proteoglycan interaction we reconstituted collagen fibrils in the presence of proteoglycans (PGs) or glycosaminoglycans (GAGs) under controlled conditions in a very simple cell-free environment. We limited our research to dermatan sulphate and chondroitin sulphate glycoconjugates because these are universally present in every ECM of connective tissues, at variance with keratan sulphate and heparan sulphate-containing proteoglycans which are more restricted in distribution and possibly in function. Our engineered ECMs were observed by high-resolution scanning electron microscopy (SEM) after the simplest possible pre-treatment.
Section snippets
Materials and methods
Acid-soluble type I collagen was extracted from delipidized calf skin with 0.15 M NaCl and 50 mM Tris–HCl, pH 7.4, containing protease inhibitors. The residue was extracted twice with 0.5 M acetic acid. The material dissolved by the acidic solvent was precipitated with NaCl (2 M final concentration), collected by centrifugation, redissolved in 0.5 M acetic acid, and subjected to a second NaCl precipitation. The final precipitate was dissolved in 0.1 M acetic acid, dialyzed exhaustively against the
Controls
Control fibrils of pure acid-soluble type I collagen showed a clear cross-banding and an average diameter of 50.6 ± 8.5 nm, and formed a spatially uniform, felt-like network. The fibrils often fused laterally, this process leading to the formation of large irregular aggregates (Fig. 1). Treatment of these specimens with Cupromeronic Blue produced no visible effect, and no fibril-bound particles or interfibrillar bridges were ever observed (Fig. 2).
Proteoglycans
The presence of decorin from the start of fibril
Discussion
The control specimen showed that our technique, which requires a relatively simple and bland treatment of the specimens, provides pictures of aspect and quality consistent with other high-resolution techniques (see Raspanti et al., 2000, or Ottani et al., 2002). The absence of fibril-bound particles in the control specimens treated with Cupromeronic Blue confirms the purity of our collagen. At the same time the similarity of these fibrils with those of untreated specimens confirms that copper
Acknowledgments
The authors thank the Centre for Large Instruments for the Biomedical Research of Insubria University for making available their high-resolution scanning electron microscope, and Dr. Barbara Bartolini for the HPLC analysis. This work was supported by FAR grants from the University of Pavia and Insubria University.
References (43)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem.
(1976)- et al.
Decorin-transforming growth factor-interaction regulates matrix organization and mechanical characteristics of three-dimensional collagen matrices
J. Biol. Chem.
(2007) - et al.
Identification of collagen fibril fusion during vertebrate tendon morphogenesis. The process relies on unipolar fibrils and is regulated by collagen–proteoglycan interaction
J. Mol. Biol.
(2000) - et al.
Leucine-rich repeat glycoproteins of the extracellular matrix
Matrix Biol.
(1998) The biology of the small leucine-rich proteoglyans
J. Biol. Chem.
(1999)- et al.
The decorin sequence SYIRIADTNIT binds collagen type I
J. Biol. Chem.
(2007) - et al.
Decorin binds near the C-terminus of type I collagen
J. Biol. Chem.
(2000) - et al.
Skewness angle of interfibrillar proteoglycans increases with applied load on mitral valve chordae tendineae
J. Biomech.
(2007) - et al.
Direct measurement of the rupture force of single pair of decorin interactions
Biochem. Biophys. Res. Co.
(2005) - et al.
Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans
J. Struct. Biol.
(2006)