Synopsis
The proteoglycans of cartilage are complex molecules in which chondroitin sulphate and keratan sulphate chains are covalently linked to a protein core, forming a polydisperse population of proteoglycan monomers. By interaction with hyaluronic acid and link proteins, the monomers form large macromolecular complexes.In vivo the proteoglycans mainly occur in such aggregates. In the electron microscope, the cartilaginous matrix can be seen to be made up of thin collagen fibrils and polygonal granules about 10–50 nm in diameter. Addition of the polyvalent cationic dye Ruthenium Red to glutaraldehyde and osmium tetroxide fixatives yields a dense selective staining of the matrix granules. Following a short digestion of cartilage slices with either of the chondroitin sulphate-degrading enzymes hyaluronidase and chondroitinase or with the proteolytic enzyme papain, the matrix granules were few in number or completely absent and the proteoglycan content, measured as hexosamine, decreased by up to 90%. Similarly, extraction of the cartilage with 4 M guanidine-HCl removed all matrix granules and most of the proteoglycans. From these findings, it can be concluded that the matrix granules represent proteoglycans, most probably in aggregate form, and that Ruthenium Red staining may be used to study the distribution of these macromolecules in thin sections. As a complement to chemical studies on proteoglycan structure, it is also possible to observe and measure individual molecules in the electron microscope after spreading them into a monomolecular layer with cytochromec. This technique has been applied in investigations on proteogly cans isolated from bovine nasal cartilage and other hyaline cartilages. The molecules in the monomer fractions appeared as an extended central core filament to which about 25–30 side-chain filaments were attached at various intervals. The core filament, averaging about 300 nm in length, was interpreted as representing the polysaccharide-binding part of the protein core and the side-chain filaments, averaging about 45 nm in length, as representing the clusters of chondroitin sulphate chains. Statistical treatment of the collected data indicated that no distinct subpopulations existed within the monomer fractions. The electron microscopic results correlated well with chemical data for the corresponding fractions and together with recent observations on various aggregate fractions strongly support present concepts of proteoglycan structure.
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References
Anderson, H. C. &Sajdera, S. W. (1971). The fine structure of bovine nasal cartilage. Extraction as a technique to study proteoglycans and collagen in cartilage matrix.J. Cell Biol. 49, 650–63.
Balazs, E. A. (1970).Chemistry and Molecular Biology of the Intercellular Matrix Glycosaminoglycans and Proteoglycans, Vol. 2 New York: Academic Press.
Hascall, V. C. &Heinegård, D. (1975). The structure of cartilage proteoglycans. In:Extracellular Matrix Influences on Gene Expression (eds. H. C. Slavkin & R. C. Greulich), pp. 423–33. New York: Academic Press.
Hascall, V. C. &Sajdera, S. W. (1969). Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates.J. biol. Chem. 244, 2384–96.
Hascall, V. C. &Sajdera, S. W. (1970). Physical properties and polydispersity of proteoglycan from bovine nasal cartilage.J. biol. Chem. 245, 4920–30.
Heinegård, D. &Hascall, V. C. (1974a). Aggregation of cartilage proteoglycans. III. Characteristics of the proteins isolated from trypsin digests of aggregates.J. biol. Chem. 249, 4250–6.
Heinegård, D. &Hascall, V. C. (1974b). Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans.Archs. Biochem. Biophys. 165, 427–41.
Kleinschmidt, A. K. &Zahn, R. K. (1959). Über Desoxyribonukleinsäure-Molekylen in Protein-Mischfilmen.Z. Naturf. Teil B 146, 770–9.
Lang, D. &Mitani, M. (1970). Simplified quantitative electron microscopy of biopolymers.Biopolymers 9, 373–9.
Lohmander, S., Heinegård, D. & Thyberg, J. (1976). Proteoglycan aggregates. Electron microscopic studies of native and fragmented molecules.Arch. Biochim. Physiol. 84, suppl. XII.
Lohmander, S. &Thyberg, J. (1975). Chemical and electron microscopic studies on proteoglycans of guinea pig costal cartilage. In:Protides of the Biological Fluids 22nd Colloquium (ed. H. Peeters), pp. 193–9. Oxford and New York: Pergamon Press.
Luft, J. H. (1971). Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action.Anat. Rec. 171, 347–68.
Matukas, V. J., Panner, B. J. &Orbison, J. L. (1967). Studies on ultrastructural identification and distribution of protein-polysaccharide in cartilage matrix.J. Cell Biol. 32, 365–77.
Moskalewski, S., Thyberg, J., Lohmander, S. &Friberg, U. (1975). Influence of colchicine and vinblastine on the Golgi complex and matrix deposition in chondrocyte aggregates. An ultrastructural study.Expl. Cell. Res. 95, 440–54.
Rosenberg, L., Hellman, W. &Kleinschmidt, A. K. (1970). Macromolecular models of proteinpolysaccharides from bovine nasal cartilage based on electron microscopic studies.J. biol. Chem. 245, 4123–30.
Sajdera, S. W. &Hascall, V. C. (1969). Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures.J. biol. Chem. 244, 77–87.
Slavkin, H. C. &Greulich, R. C. (1975).Extracellular Matrix Influences on Gene Expression. New York: Academic Press.
Thyberg, J. &Friberg, U. (1971). Ultrastructure of the epiphyseal plate of the normal guinea pig.Z. Zellforsch. mikrosk. Anat. 122, 254–72.
Thyberg, J., Lohmander, S. &Friberg, U. (1973a). Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage.J. Ultrastruct. Res. 45, 407–27.
Thyberg, J., Lohmander, S. &Heinegård, D. (1975). Proteoglycans of hyaline cartilage. Electron microscopic studies on isolated molecules.Biochem. J. 151, 157–66.
Thyberg, J., Nilsson, S. &Friberg, U. (1973b). Electron microscopic studies on guinea pig rib cartilage. Structural heterogeneity and effects of extraction with guanidine HCl.Z. Zellforsch. mikrosk. Anat. 146, 83–102.
Wasteson, Å. (1971). Properties of fractionated chondroitin sulphate from ox nasal septa.Biochem. J. 122, 477–85.
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Thyberg, J. Electron microscopy of cartilage proteoglycans. Histochem J 9, 259–266 (1977). https://doi.org/10.1007/BF01004761
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DOI: https://doi.org/10.1007/BF01004761