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Noncollagenous Cartilage Proteins Current Status of an Emerging Research Field
Collagen ReI. Res. Vol. 411984, pp. 219-229
Noncollagenous Cartilage Proteins Current Status of an Emerging Research Field MATS PAULSSONl and DICK HEINEGARD2 1
2
Department of Connective Tissue Research, Max-Planck-Institut fiir Biochemie, D-8033 Martinsried bei Miinchen, Federal Republic of Germany and Department of Physiological Chemistry, University of Lund, P.O.Box 750 5-220 07 Lund, Sweden.
Key words: cartilage, extracellular matrix, glycoproteins and noncollagenous proteins.
Introduction Cartilage extracellular matrix is an eminent model for the study of how the physical properties of a connective tissue depend on the structure and interactions of its constituent macromolecules. Therefore, much effort has been directed towards the structural characterization of cartilage proteoglycans and type II collagen (for review see Heinegard and Paulsson, 1984; Bornstein and Traub, 1979). According to the present understanding, the proteoglycans because of their high charge density provide resistance to compression of the tissue (Kempson, 1980). Collagen fibers keep the proteoglycan aggregates fixed in the matrix by physical entrapment and do in addition contribute tensile strength. Attempts to explain all characteristics of cartilage extracellular matrix only from the properties of these major molecular species do, however, suffer from oversimplification. Already from the work of Marner in the late 19th century (Marner, 1888, 1889), it is known that cartilage in addition contains a considerable fraction of noncollagenous proteins, which he termed the "albumoid". Later work by Szirmai and collaborators (Szirmai et aI., 1967) indicated that between 15 and 50 0J0 of the dry weight of horse nasal septum cartilage consists of noncollagenous protein, with the highest amounts present in the periphery of the tissue and at old age of the animal (Fig. 1). Proteoglycan core protein can only constitute a small fraction at the total noncollagenous protein (Szirmai, 1969). Even if those figures might be an overestimate, noncollagenous proteins are major constituents of cartilage and could contribute to the mechanical properties of the tissue as well as play a role in skeletal growth. Despite this, very few publications have concerned these proteins, with the exception of the link proteins, which because of their role in proteoglycan aggregation have received some attention.
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In the following we will summarize the rather scarce information available on cartilage noncollagenous proteins, with special emphasis on such aspects that raise questions for the future. Link proteins The first class of non collagenous proteins isolated from cartilage and studied using modern biochemical methodology were the link proteins. When the guanidinium chloride extraction - CsCI density gradient centrifugation scheme for the purification of cartilage proteoglycans was developed in the late 1960's, it was found that a factor able to mediate aggregation of proteoglycans was present in the upper, protein rich fraction of dissociative (i. e. guanidinium chloride containing) CsCI gradients of previously purified proteoglycan aggregates (Hascall and Sajdera, 1969). Later it was discovered that the active agent is not a protein, but hyaluronic acid present in upper fraction from the CsClgradient, which was in these early days cut to represent as much as 3/5 of the gradient (Hardingham and Muir, 1972, 1974). The link proteins in the low buoyancy fraction do, however, also participate in proteoglycan aggregation
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(Gregory, 1973; Heinegard and Hascall, 1974; Hardingham, 1979) by binding both to the proteoglycan core protein (Heinegard and Hascall, 1979; Franzen et aI., 1981) and to the hyaluronic acid (Hascall and Heinegard, 1974; Tengblad, 1981). They thereby stabilize the already strong bond between those components (Fig. 2; Hascall and Heinegard, 1974; Hardingham, 1979; Franzen et aI., 1981). The link proteins are microheterogeneous and three components in the molecular weight range of 40-50,000 daltons have been identified (Keiser et aI., 1972; Baker and Caterson, 1977). The two major components, Mr 46,000 and 40,500, respectively, have been purified to homogeneity and appear closely related both shown by compositional analysis and by peptide mapping (Baker and Caterson, 1979; Bonnet et aI., 1978). The third, smaller component that is also resolved by electrophoresis appears to be derived from the other two by proteolytic modification (Roughley et aI., 1982), perhaps occurring as a physiological phenomenon. Recent observations indicate that the link proteins may have other functions in addition to the participation in cartilage proteoglycan aggregation. In studies of localization by immunoelectronmicroscopy Poole et aI., (1982) observed that a portion of the link protein in articular cartilage is closely associated with collagen fibers. Subsequently, an interaction between link protein and collagen, in particular types I and III, was demonstrated in solid-phase binding assays (Chandrasekhar et aI., 1983). It is still uncertain whether link protein-collagen interactions occur in vivo, but the possibility is interesting, especially taken together with the recent demonstration of link protein in tissues other than cartilage, such as aortic wall (Gardell et aI., 1980) and several structures in the eye (Poole et ai., 1982).
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M. Paulsson and D. Heinegard
Chondronectin Chondronectin is another noncollagenous protein that appears to be present in cartilage. It was first identified in serum as a factor that mediates the attachment of chondrocytes to substrates of type II collagen (Hewitt et aI., 1980), in analogy with the fibronectin-mediated attachment of fibroblasts. By the use of immunofluorescence microscopy, chondronectin has been demonstrated in the vicinity of chondrocytes in cartilage sections and at the surface of cultured chon~ drocytes (Hewitt et aI., 1982). Chondronectin has only been purified from serum and was found to be a glycoprotein consisting of subunits with an apparent molecular weight of 70,000 (Hewitt et aI., 1982). As the intact protein has been estimated to have a molecular weight of about 180,000 daltons, it is likely to consist of two or three such subunits. Some aspects of chondronectin have not yet been explored. It does not appear to be a major component of cartilage extracellular matrix, but rather a minor pericellular protein (Hewitt et ai., 1982). Is it likely then that the relatively large amounts in serum, 2.5-5 ,ug/ml, are derived from cartilage? Even though cartilage extracts contain a factor similar by several criteria (Hewitt et ai., 1982), this protein is not necessarily identical with serum chondronectin, which could be derived from other sources. A similar case has been shown for plasma fibronectin, where the major portion appears to be secreted by hepatocytes in a manner similar to that of many other serum proteins (Tamkun and Hynes, 1983). 31 kDa collagen-binding glycoprotein An alternative mechanism for chondrocyte-matrix attachment is suggested by the work of Mollenhauer and von der Mark (1983) who identified and purified a collagen-binding glycoprotein from chick chondrocyte plasma membranes. This 31 kDa protein appears to be an integral component of the plasma membrane, as indicated by its insertion into lecithin vesicles. It binds to several types of collagen, probably by one end of the collagen molecule. The protein would then be able to provide a direct link between the chondrocyte and the collagen in the cellular environment. It appears specific for the chondrocyte phenotype as it can only be detected on chondrocytes and to a lesser extent on embryonic limb bud cells. The composition of the 31 kDa protein shows some interesting features. The content of glycine is high (183.5 res./lOOO res.) and the molecule also contains some hydroxy lysine but no hydroxyproline. It contains 30 % carbohydrate out of which two thirds chromatographed as fucose on gas chromatography (Mollenhauer and von der Mark, 1983). Despite the high carbohydrate content the protein is poorly soluble in aqueous buffers and requires detergents for solubilization and disaggregation. Taken together, this would indicate a highly specialized structure, where one region is hydrophobic and suited for insertion into lipid bilayers, while another region contains large amounts of carbohydrate and is presumably exposed to the extracellular space. The presence of cell surface receptors for connective tissue macromolecules may be taken to imply the presence of a specialized pericellular region of the extracellular matrix that could provide feed-back information to the cells on
Noncollagenous Cartilage Proteins
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the state of the matrix and in which some events in the processing of newly secreted macromolecules might take place. It has also been shown that cartilage proteoglycans bind to the chondrocytes by saturable interaction with cell surface hyaluronic acid (Sommarin and Heinegard, 1983). A morphological correlate to such a biochemically defined pericellular region could be the territorial matrix, which is seen as an intensely basophilic zone surrounding the chondrocytes when certain stains are used (Szirmai, 1969). Also immunofluorescence microscopy data using antibodies directed against cartilage matrix antigens indicates the presence of a pericellular zone of different composition or organization than the rest of the matrix (Poole et aI., 1980; Cederholm, Paulsson and Heinegard, unpublished results). 69 kDa foetal cartilage matrix protein This recently described protein (Choi et aI., 1983), has a strong affinity for hydroxylapatite and appears to be enriched in those areas of the epiphyseal cartilage where the first mineral is deposited. It appears then that the 69 kDa protein has a role in the mineralization which occurs during endochondral ossification. The 69 kDa protein was first recognized because of its abundance in third trimester foetal calf epiphyseal cartilage. It was purified from this source using its 35 kDa subunits as a marker. The purified protein proved to be a dimer which yields the subunits first after reduction of disulphide bonds. It is a glycoprotein containing 4 % carbohydrate, probably in the form of high mannosetype oligosaccharides. Antibodies raised against the protein were used to demonstrate that it is distinct from other known connective tissue proteins and that it is present also in some adult bovine cartilages. The work on the 69 kDa protein demonstrates one way to gain insight into the functions of different cartilage noncollagenous proteins. By screening the relative amounts of a number of proteins in a cartilage undergoing dynamic changes a particular protein could be singled out, the occurrence of which correlated with a specific event. Perhaps the same approach can be used for other developing cartilages as well as in the study of disease in cartilage. 148 kDa cartilage matrix protein Our own investigations of cartilage noncollagenous proteins began with the observation that proteoglycans aggregates isolated from bovine tracheal or nasal cartilages by methods avoiding the use of denaturing agents in addition to the link proteins contained one particular protein which cofractionated with the proteoglycans (Paulsson and Heinegard, 1979) on both molecular sieve and ion exchange chromatography as well as in rate-zonal centrifugation. Therefore it appeared likely that there is an interaction between this cartilage matrix protein and proteoglycans. The matrix protein was purified from guanidinium chloride extracts of bovine tracheal cartilage (Paulsson and Heinegard, 1981). The molecular weights of the intact protein and of the subunits obtained after reduction and alkylation were
224
M. Pauls son and D. Heinegard
determined by sedimentation equilibrium centrifugation in 6 M guanidinium chloride and were found to be 148,000 and 52,000 daltons, respectively, showing that the protein is composed of three subunits of similar size. Chemical analysis of the 148 kDa protein showed that hydroxyproline is absent, demonstrating the noncollagenous nature of the protein. It contains 3.9 Ofo carbohydrate, mainly in the form of N-linked oligosaccharides with a high mannose content. Antibodies raised against the purified 148 kDa protein were used to develop a radioimmunoassay (Pauls son and Heinegard, 1982). No antigenic relationship between the 148 kDa protein and previously known connective tissue proteins could be detected. The 148 kDa protein was also shown to be specific for cartilage, by the analysis of guandinium chloride extracts of a large number of bovine tissues. The largest quantities of the 148 kDa protein were found in extracts of tracheal cartilage, where this protein alone contributed at least 4 0/0 of the tissue dry weight. The protein was also present in extracts of nasal septum, xiphisternal, auricular and epiphyseal cartilage, but in gradually lower quantities. Surprisingly, it could be detected neither in extracts of articular cartilage nor of the intervertebral disc. The distribution of the 148 kDa protein was confirmed by SDS-PAGE (Fig. 3). Electrophoresis also gave some information on the distribution of other proteins among cartilage tissues. The link proteins and a 36 kDa protein were present in all cartilages (Fig. 3). This 36 kDa protein is distinct from the 35 kDa subunits of the 69 kDa protein studied by Choi et a1. (1983) as it occurs as a monomer also under non-reducing conditions. Extracts of articular cartilage and of intervertebral disc, while lacking the 148 kDa protein, contained other predominant proteins that are less abundant in other cartilages. Some noncollagenous proteins, then, such as the link proteins and the 36 kDa protein are probably common to all cartilages, while others, like the 148 kDa protein are subject to variation and might be markers of subdifferentiation among cartilages. Both the 148 kDa protein and the 36 kDa protein are major biosynthesis products in organ cultures of bovine tracheal cartilage, each incorporating considerably more [3H]-leucine than does the proteoglycan core protein (Paulsson et aI., 1983). The 148 kDa protein has a very slow, turnover, indicative of a structural function, while the 36 kDa protein is eliminated more rapidly. The major antigenic determinant in the 148 kDa protein is located in a region of the molecule that is highly resistant to proteolytic digestion (Paulsson et aI., 1984). Therefore, radioimmunoassay of trypsin digests could be used to demonstrate that a substantial portion of the protein was not extracted even with the 4-5 M guanidinium chloride used. Using a sequence of guanidinium chloride extraction followed by trypsin digestion of the residue in the analysis of tracheal cartilage from steers of different ages, it could be shown that comparatively small amounts of the 148 kDa protein were present at birth (Fig. 4 a). The guanidinium chloride-soluble as well as the trypsin-soluble pools of the protein then increase markedly with age, to reach maxima at 3-4 years and 6-10 years, respectively. The ratio between the trypsin-soluble and guanidinium chloridesoluble pools increases continuously (Fig. 4 b; Pauls son et aI., 1984). An attractive interpretation is that the guanidinium chloride-soluble pool of 148 kDa protein is a transient one, consisting of comparatively recently secreted molecules that will eventually be laid down in a manner that renders them insoluble in
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guanidinium chloride. The mechanism by which the 148 kDa protein becomes insoluble is not known, but it is possible that in analogy with collagen it may depend on the formation of covalent crosslinks. The laying down of an insoluble form of the 148 kDa protein could also be taken to indicate that this protein mainly serves a structural function in the matrix. Another observation, relevant in this context, is that the 148 kDa protein is mainly detected in the truly interterritorial matrix as shown by immunofluorescence microscopy of bovine tracheal cartilage (Cederholm, Paulsson and Heinegard, unpublished results). Concluding remarks As discussed above, some knowledge is emerging about single non collagenous proteins. Most of the proteins present in cartilage matrix have not yet been characterized, though, and the data on their function is limited. It should be
Noncollagenous Cartilage Proteins
227
remembered that the cartilage noncollagenous proteins probably are a very heterogenous group with regard to function. Some may play a structural role, as the link proteins and as we believe is the case with the 148 kDa cartilage protein. Other might be there to mediate interactions between the chondrocytes and the extracellular matrix, for example the 31 kDa collagen-binding protein and chondronectin. A third group will be enzymes involved in matrix formation and degradation as well as proteins serving other kinds catalytic or regulatory functions in the matrix. One representative of the latter group would then be the 69 kDa foetal cartilage protein with its apparent role in the mineralization process. To add to the complexity it appears that different cartilages from the same species differ considerably in what proteins they contain (Fig. 3). This variation is greater than that seen among proteoglycans and collagen, which might indicate that the noncollagenous proteins are particularly important for the subdifferentiation among cartilages. Such proteins could for example influence the mechanical properties of a tissue by affecting the organization of the collagen and proteoglycans or regulate mineral deposition and thereby determine if a certain cartilage will become calcified or not. Further insight might be gained from studies of the exact localization of the different proteins in the matrix. Available data indicate that there are two separate extracellular compartments in cartilage, the territorial or pericellular matrix and the interterritorial matrix. In addition, the border between these compartments is probably formed by a specialized structure, the "basket" referred to by Szirmai (1969) and other authors. The compartments may share many molecular constituents, but some molecules might occur only in one. This subdivision within a cartilage matrix has not yet received enough attention from biochemists, but might be of large importance for the understanding of molecular events occurring in the matrix. Acknowledgements Our own original work was supported by grants from the Swedish Medical Research Council (5668), Konung Gustaf V:s 80-arsfond, Alfred Osterlunds Stiftelse, Kocks Stiftelser and the Medical Faculty, University of Lund. M. P. is presently the recipient of a Long Term Fellowship from the European Molecular Biology Organization.
References Baker, J R. and Caterson, B.: The purification and cyanogen bromide cleavage of the link proteins from cartilage proteoglycan. Biochem. Biophys. Res. Comm. 77: 1-10, 1977. Baker, J. R. and Caterson, B.: The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage. J. Bioi. Chem. 254: 2387-2393, 1979. Bonnet, F., Perin, J-P. and Jolles, P.: Isolation and chemical characterization of two distinct link proteins from bovine nasal cartilage proteoglycan complex. Biochim. Biophys. Acta 532: 242-248, 1978. Bornstein, P. and Traub, W.: The chemistry and biology of collagen. In: The Proteins, Vol. IV, ed. by H. Neurath and R. Hill, Academic Press, New York 1979, pp. 411-632.
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Chandrasekhar, S., Kleinman, H. K. and Hassel, J. R.: Interaction of link protein with collagen. j. BioI. Chem. 258: 6226-6231, 1983. Choi, H. U., Tang, L.-H., Johnson, T. L., Pal, S., Rosenberg, L. c., Reiner, A and Poole, A. R.: Isolation and characterization of a 35,000 molecular weight subunit fetal cartilage matrix protein. J. Bioi. Chem. 258: 655-661, 1983. Franzen, A., Bjarnsson, S. and Heinegard, D.: Cartilage proteoglycan aggregate formation. Role of link protein. Biochem. j. 197: 669-674, 1981. Gardell, S., Baker, J., Caterson, B., Heinegard, D. and Roden, L.: Link protein and a hyaluronic acid-binding region as components of aorta proteoglycan. Biochem. Biophys. Res. Comm. 95: 1823-1831, 1980. Gregory, J. D.: Multiple aggregation factors in cartilage proteoglycan. Biochem. j. 133: 383-386, 1973. Hardingham, T. E.: The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem. j. 177: 237-247, 1979. Hardingham, T. E. and Muir, H.: The specific interaction of hyaluronic acid with cartilage proteoglycans. Biochim. Biophys. Acta 279: 401-405, 1972. Hardingham, T. E. and Muir, H.: Hyaluronic acid in cartilage and proteoglycan aggregation. Biochem. j. 139: 565-581,1974. Hascall, V. C. and Heinegard, D. K.: Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. j. BioI. Chem. 249:4242-4249,1974. Hascall, V. C. and Sajdera, S. W.: Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. j. BioI. Chem. 244: 2384-2396, 1969. Heinegard, D. K. and Hascall, V. c.: Aggregation of cartilage proteoglycans. III. Characteristics of the proteins isolated from trypsin digests of aggregates. j. Bioi. Chem. 249: 4250-4256, 1974. Heinegard, D. K. and Hascall, V. c.: The effects of dansylation and acetylation on the interaction between hyaluronic acid and the hyaluronic acid-binding region of cartilage proteoglycans. j. Bioi. Chem. 254: 921-926, 1979. Heinegard, D. and Paulsson, M.: Structure and metabolism of proteoglycans. In: Extracellular matrix biochemistry, ed. by Piez, K. and Reddi, A. H., Elsvier-North Holland, New York, 1984, pp. 277-328. Hewitt, A. T., Kleinman, H. K., Pennypacker, J. P. and Martin, G. R.: Identification of and adhesion factor for chondrocytes. Proc. Natl. Acad. Sci. USA. 77: 385-388, 1980. Hewitt, A. T., Varner, H. H., Silver, M. H., Dessau, W., Wilkes, C. M. and Martin, G. R.: The isolation and partial characterization of chondronectin, an attachment factor for chondrocytes. J. Bioi. Chem. 257: 2330-2334, 1982. Keiser, H., Shulman, H. J. and Sandson, J. I.: Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies. Biochem. j. 126: 163-169, 1972. Kempson, C. E.: The mechanical properties of articular cartilage. In: The joints and Synovial Fluid, II, ed. by Sokoloff, L. Acacemic Press, New York, 1980, pp. 177-238. Mollenhauer, J. and von der Mark, K.: Isolation and characterization of a collagenbinding glycoprotein from chondrocyte membranes. EMBO journal 2: 45-50, 1983. Marner, c. T.: Histochemische Beobachtungen tiber die hyaline Grundsubstanz des Trachealknorpels. Hoppe-Seylers Z. Physiol. Chem. 12: 396-404, 1888. Marner, c. T.: Chemische Studien tiber den Trachealknorpel. Skand. Arch. Physiol. 1: 210-243, 1889. Paulsson, M. and Heinegard, D.: Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage. Biochem. j. 183: 539-545, 1979. Pauls son, M. and Heinegard, D.: Purification and structural characterization of a cartilage matrix protein. Biochem. J. 197: 367-375, 1981.
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Paulsson, M. and Heinegard, D.: Radioimmunoassay of the 148-kilodalton cartilage protein. Distribution of the protein among bovine tissues. Biochem. J. 207: 207-213, 1982. Paulsson, M., Inerot, S. and Heinegard, D.: Variation in quantity and extractability of the 148-kilodalton protein with age. Biochem. J., In press, 1984. Paulsson, M., Sommarin, Y. and Heinegard, D.: Metabolism of cartilage proteins in cultured tissue sections. Biochem. J. 212: 659-667, 1983. Poole, A. R., Pidoux, 1., Reiner, A., Coster, L. and Hassel, J. R.: Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein. J. Cell. Bioi. 93: 910-920, 1982. Poole, A. R., Pidoux, 1., Reiner, A., Tang, L.-H., Choi, H. and Rosenberg, L.: Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study. J. Histochem. Cytochem. 28: 621-635, 1980. Poole, A. R., Pidoux, I., Reiner, A. and Rosenberg, L.: An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J. Cell. Bioi. 93: 921-937, 1982. Roughley, P. J., Poole, A. R. and Mort, J. S.: The heterogeneity of link proteins isolated from human articular cartilage proteoglycan aggregates. J. Bioi. Chem. 257: 1190811914, 1982. Sommarin, Y. and Heinegard, D.: Specific interaction between cartilage proteoglycans and hyaluronic acid at the chondrocyte cell surface. Biochem. J. 214: 777-784, 1983. Szirmai, J. A.: Structure of cartilage. In: Aging of connective and skeletal tissue, ed. by Engel, A. and Larsson, T., Nordiska Bokhandelns Foriag, Stockholm, 1969, pp. 163-184. Szirmai, J. A., van Boven-De Tyssonsk, E. and Garden, S.: Microchemical analysis of glycosaminoglycans, collagen, total protein and water in histological layers of nasal septum cartilage. Biochim. Biophys. Acta 136: 331-350, 1967. Tamkun, J. W. and Hynes, R. 0.: Plasma fibronectin is synthesized and secreted by hepatocytes. J. Bioi. Chem. 258: 4641-4647, 1983. Tengblad, A.: A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronic acid substituted Sepharose gel. Biochem. J. 199: 297-305, 1981. Dick Heinegard, Department of Physiological Chemistry, University of Lund P. O. Box 750, S-220 07 Lund, Sweden.
Noncollagenous Cartilage Proteins Current Status of an Emerging Research Field MATS PAULSSONl and DICK HEINEGARD2 1
2
Department of Connective Tissue Research, Max-Planck-Institut fiir Biochemie, D-8033 Martinsried bei Miinchen, Federal Republic of Germany and Department of Physiological Chemistry, University of Lund, P.O.Box 750 5-220 07 Lund, Sweden.
Key words: cartilage, extracellular matrix, glycoproteins and noncollagenous proteins.
Introduction Cartilage extracellular matrix is an eminent model for the study of how the physical properties of a connective tissue depend on the structure and interactions of its constituent macromolecules. Therefore, much effort has been directed towards the structural characterization of cartilage proteoglycans and type II collagen (for review see Heinegard and Paulsson, 1984; Bornstein and Traub, 1979). According to the present understanding, the proteoglycans because of their high charge density provide resistance to compression of the tissue (Kempson, 1980). Collagen fibers keep the proteoglycan aggregates fixed in the matrix by physical entrapment and do in addition contribute tensile strength. Attempts to explain all characteristics of cartilage extracellular matrix only from the properties of these major molecular species do, however, suffer from oversimplification. Already from the work of Marner in the late 19th century (Marner, 1888, 1889), it is known that cartilage in addition contains a considerable fraction of noncollagenous proteins, which he termed the "albumoid". Later work by Szirmai and collaborators (Szirmai et aI., 1967) indicated that between 15 and 50 0J0 of the dry weight of horse nasal septum cartilage consists of noncollagenous protein, with the highest amounts present in the periphery of the tissue and at old age of the animal (Fig. 1). Proteoglycan core protein can only constitute a small fraction at the total noncollagenous protein (Szirmai, 1969). Even if those figures might be an overestimate, noncollagenous proteins are major constituents of cartilage and could contribute to the mechanical properties of the tissue as well as play a role in skeletal growth. Despite this, very few publications have concerned these proteins, with the exception of the link proteins, which because of their role in proteoglycan aggregation have received some attention.
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In the following we will summarize the rather scarce information available on cartilage noncollagenous proteins, with special emphasis on such aspects that raise questions for the future. Link proteins The first class of non collagenous proteins isolated from cartilage and studied using modern biochemical methodology were the link proteins. When the guanidinium chloride extraction - CsCI density gradient centrifugation scheme for the purification of cartilage proteoglycans was developed in the late 1960's, it was found that a factor able to mediate aggregation of proteoglycans was present in the upper, protein rich fraction of dissociative (i. e. guanidinium chloride containing) CsCI gradients of previously purified proteoglycan aggregates (Hascall and Sajdera, 1969). Later it was discovered that the active agent is not a protein, but hyaluronic acid present in upper fraction from the CsClgradient, which was in these early days cut to represent as much as 3/5 of the gradient (Hardingham and Muir, 1972, 1974). The link proteins in the low buoyancy fraction do, however, also participate in proteoglycan aggregation
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(Gregory, 1973; Heinegard and Hascall, 1974; Hardingham, 1979) by binding both to the proteoglycan core protein (Heinegard and Hascall, 1979; Franzen et aI., 1981) and to the hyaluronic acid (Hascall and Heinegard, 1974; Tengblad, 1981). They thereby stabilize the already strong bond between those components (Fig. 2; Hascall and Heinegard, 1974; Hardingham, 1979; Franzen et aI., 1981). The link proteins are microheterogeneous and three components in the molecular weight range of 40-50,000 daltons have been identified (Keiser et aI., 1972; Baker and Caterson, 1977). The two major components, Mr 46,000 and 40,500, respectively, have been purified to homogeneity and appear closely related both shown by compositional analysis and by peptide mapping (Baker and Caterson, 1979; Bonnet et aI., 1978). The third, smaller component that is also resolved by electrophoresis appears to be derived from the other two by proteolytic modification (Roughley et aI., 1982), perhaps occurring as a physiological phenomenon. Recent observations indicate that the link proteins may have other functions in addition to the participation in cartilage proteoglycan aggregation. In studies of localization by immunoelectronmicroscopy Poole et aI., (1982) observed that a portion of the link protein in articular cartilage is closely associated with collagen fibers. Subsequently, an interaction between link protein and collagen, in particular types I and III, was demonstrated in solid-phase binding assays (Chandrasekhar et aI., 1983). It is still uncertain whether link protein-collagen interactions occur in vivo, but the possibility is interesting, especially taken together with the recent demonstration of link protein in tissues other than cartilage, such as aortic wall (Gardell et aI., 1980) and several structures in the eye (Poole et ai., 1982).
222
M. Paulsson and D. Heinegard
Chondronectin Chondronectin is another noncollagenous protein that appears to be present in cartilage. It was first identified in serum as a factor that mediates the attachment of chondrocytes to substrates of type II collagen (Hewitt et aI., 1980), in analogy with the fibronectin-mediated attachment of fibroblasts. By the use of immunofluorescence microscopy, chondronectin has been demonstrated in the vicinity of chondrocytes in cartilage sections and at the surface of cultured chon~ drocytes (Hewitt et aI., 1982). Chondronectin has only been purified from serum and was found to be a glycoprotein consisting of subunits with an apparent molecular weight of 70,000 (Hewitt et aI., 1982). As the intact protein has been estimated to have a molecular weight of about 180,000 daltons, it is likely to consist of two or three such subunits. Some aspects of chondronectin have not yet been explored. It does not appear to be a major component of cartilage extracellular matrix, but rather a minor pericellular protein (Hewitt et ai., 1982). Is it likely then that the relatively large amounts in serum, 2.5-5 ,ug/ml, are derived from cartilage? Even though cartilage extracts contain a factor similar by several criteria (Hewitt et ai., 1982), this protein is not necessarily identical with serum chondronectin, which could be derived from other sources. A similar case has been shown for plasma fibronectin, where the major portion appears to be secreted by hepatocytes in a manner similar to that of many other serum proteins (Tamkun and Hynes, 1983). 31 kDa collagen-binding glycoprotein An alternative mechanism for chondrocyte-matrix attachment is suggested by the work of Mollenhauer and von der Mark (1983) who identified and purified a collagen-binding glycoprotein from chick chondrocyte plasma membranes. This 31 kDa protein appears to be an integral component of the plasma membrane, as indicated by its insertion into lecithin vesicles. It binds to several types of collagen, probably by one end of the collagen molecule. The protein would then be able to provide a direct link between the chondrocyte and the collagen in the cellular environment. It appears specific for the chondrocyte phenotype as it can only be detected on chondrocytes and to a lesser extent on embryonic limb bud cells. The composition of the 31 kDa protein shows some interesting features. The content of glycine is high (183.5 res./lOOO res.) and the molecule also contains some hydroxy lysine but no hydroxyproline. It contains 30 % carbohydrate out of which two thirds chromatographed as fucose on gas chromatography (Mollenhauer and von der Mark, 1983). Despite the high carbohydrate content the protein is poorly soluble in aqueous buffers and requires detergents for solubilization and disaggregation. Taken together, this would indicate a highly specialized structure, where one region is hydrophobic and suited for insertion into lipid bilayers, while another region contains large amounts of carbohydrate and is presumably exposed to the extracellular space. The presence of cell surface receptors for connective tissue macromolecules may be taken to imply the presence of a specialized pericellular region of the extracellular matrix that could provide feed-back information to the cells on
Noncollagenous Cartilage Proteins
223
the state of the matrix and in which some events in the processing of newly secreted macromolecules might take place. It has also been shown that cartilage proteoglycans bind to the chondrocytes by saturable interaction with cell surface hyaluronic acid (Sommarin and Heinegard, 1983). A morphological correlate to such a biochemically defined pericellular region could be the territorial matrix, which is seen as an intensely basophilic zone surrounding the chondrocytes when certain stains are used (Szirmai, 1969). Also immunofluorescence microscopy data using antibodies directed against cartilage matrix antigens indicates the presence of a pericellular zone of different composition or organization than the rest of the matrix (Poole et aI., 1980; Cederholm, Paulsson and Heinegard, unpublished results). 69 kDa foetal cartilage matrix protein This recently described protein (Choi et aI., 1983), has a strong affinity for hydroxylapatite and appears to be enriched in those areas of the epiphyseal cartilage where the first mineral is deposited. It appears then that the 69 kDa protein has a role in the mineralization which occurs during endochondral ossification. The 69 kDa protein was first recognized because of its abundance in third trimester foetal calf epiphyseal cartilage. It was purified from this source using its 35 kDa subunits as a marker. The purified protein proved to be a dimer which yields the subunits first after reduction of disulphide bonds. It is a glycoprotein containing 4 % carbohydrate, probably in the form of high mannosetype oligosaccharides. Antibodies raised against the protein were used to demonstrate that it is distinct from other known connective tissue proteins and that it is present also in some adult bovine cartilages. The work on the 69 kDa protein demonstrates one way to gain insight into the functions of different cartilage noncollagenous proteins. By screening the relative amounts of a number of proteins in a cartilage undergoing dynamic changes a particular protein could be singled out, the occurrence of which correlated with a specific event. Perhaps the same approach can be used for other developing cartilages as well as in the study of disease in cartilage. 148 kDa cartilage matrix protein Our own investigations of cartilage noncollagenous proteins began with the observation that proteoglycans aggregates isolated from bovine tracheal or nasal cartilages by methods avoiding the use of denaturing agents in addition to the link proteins contained one particular protein which cofractionated with the proteoglycans (Paulsson and Heinegard, 1979) on both molecular sieve and ion exchange chromatography as well as in rate-zonal centrifugation. Therefore it appeared likely that there is an interaction between this cartilage matrix protein and proteoglycans. The matrix protein was purified from guanidinium chloride extracts of bovine tracheal cartilage (Paulsson and Heinegard, 1981). The molecular weights of the intact protein and of the subunits obtained after reduction and alkylation were
224
M. Pauls son and D. Heinegard
determined by sedimentation equilibrium centrifugation in 6 M guanidinium chloride and were found to be 148,000 and 52,000 daltons, respectively, showing that the protein is composed of three subunits of similar size. Chemical analysis of the 148 kDa protein showed that hydroxyproline is absent, demonstrating the noncollagenous nature of the protein. It contains 3.9 Ofo carbohydrate, mainly in the form of N-linked oligosaccharides with a high mannose content. Antibodies raised against the purified 148 kDa protein were used to develop a radioimmunoassay (Pauls son and Heinegard, 1982). No antigenic relationship between the 148 kDa protein and previously known connective tissue proteins could be detected. The 148 kDa protein was also shown to be specific for cartilage, by the analysis of guandinium chloride extracts of a large number of bovine tissues. The largest quantities of the 148 kDa protein were found in extracts of tracheal cartilage, where this protein alone contributed at least 4 0/0 of the tissue dry weight. The protein was also present in extracts of nasal septum, xiphisternal, auricular and epiphyseal cartilage, but in gradually lower quantities. Surprisingly, it could be detected neither in extracts of articular cartilage nor of the intervertebral disc. The distribution of the 148 kDa protein was confirmed by SDS-PAGE (Fig. 3). Electrophoresis also gave some information on the distribution of other proteins among cartilage tissues. The link proteins and a 36 kDa protein were present in all cartilages (Fig. 3). This 36 kDa protein is distinct from the 35 kDa subunits of the 69 kDa protein studied by Choi et a1. (1983) as it occurs as a monomer also under non-reducing conditions. Extracts of articular cartilage and of intervertebral disc, while lacking the 148 kDa protein, contained other predominant proteins that are less abundant in other cartilages. Some noncollagenous proteins, then, such as the link proteins and the 36 kDa protein are probably common to all cartilages, while others, like the 148 kDa protein are subject to variation and might be markers of subdifferentiation among cartilages. Both the 148 kDa protein and the 36 kDa protein are major biosynthesis products in organ cultures of bovine tracheal cartilage, each incorporating considerably more [3H]-leucine than does the proteoglycan core protein (Paulsson et aI., 1983). The 148 kDa protein has a very slow, turnover, indicative of a structural function, while the 36 kDa protein is eliminated more rapidly. The major antigenic determinant in the 148 kDa protein is located in a region of the molecule that is highly resistant to proteolytic digestion (Paulsson et aI., 1984). Therefore, radioimmunoassay of trypsin digests could be used to demonstrate that a substantial portion of the protein was not extracted even with the 4-5 M guanidinium chloride used. Using a sequence of guanidinium chloride extraction followed by trypsin digestion of the residue in the analysis of tracheal cartilage from steers of different ages, it could be shown that comparatively small amounts of the 148 kDa protein were present at birth (Fig. 4 a). The guanidinium chloride-soluble as well as the trypsin-soluble pools of the protein then increase markedly with age, to reach maxima at 3-4 years and 6-10 years, respectively. The ratio between the trypsin-soluble and guanidinium chloridesoluble pools increases continuously (Fig. 4 b; Pauls son et aI., 1984). An attractive interpretation is that the guanidinium chloride-soluble pool of 148 kDa protein is a transient one, consisting of comparatively recently secreted molecules that will eventually be laid down in a manner that renders them insoluble in
Noncollagenous Cartilage Proteins
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226
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guanidinium chloride. The mechanism by which the 148 kDa protein becomes insoluble is not known, but it is possible that in analogy with collagen it may depend on the formation of covalent crosslinks. The laying down of an insoluble form of the 148 kDa protein could also be taken to indicate that this protein mainly serves a structural function in the matrix. Another observation, relevant in this context, is that the 148 kDa protein is mainly detected in the truly interterritorial matrix as shown by immunofluorescence microscopy of bovine tracheal cartilage (Cederholm, Paulsson and Heinegard, unpublished results). Concluding remarks As discussed above, some knowledge is emerging about single non collagenous proteins. Most of the proteins present in cartilage matrix have not yet been characterized, though, and the data on their function is limited. It should be
Noncollagenous Cartilage Proteins
227
remembered that the cartilage noncollagenous proteins probably are a very heterogenous group with regard to function. Some may play a structural role, as the link proteins and as we believe is the case with the 148 kDa cartilage protein. Other might be there to mediate interactions between the chondrocytes and the extracellular matrix, for example the 31 kDa collagen-binding protein and chondronectin. A third group will be enzymes involved in matrix formation and degradation as well as proteins serving other kinds catalytic or regulatory functions in the matrix. One representative of the latter group would then be the 69 kDa foetal cartilage protein with its apparent role in the mineralization process. To add to the complexity it appears that different cartilages from the same species differ considerably in what proteins they contain (Fig. 3). This variation is greater than that seen among proteoglycans and collagen, which might indicate that the noncollagenous proteins are particularly important for the subdifferentiation among cartilages. Such proteins could for example influence the mechanical properties of a tissue by affecting the organization of the collagen and proteoglycans or regulate mineral deposition and thereby determine if a certain cartilage will become calcified or not. Further insight might be gained from studies of the exact localization of the different proteins in the matrix. Available data indicate that there are two separate extracellular compartments in cartilage, the territorial or pericellular matrix and the interterritorial matrix. In addition, the border between these compartments is probably formed by a specialized structure, the "basket" referred to by Szirmai (1969) and other authors. The compartments may share many molecular constituents, but some molecules might occur only in one. This subdivision within a cartilage matrix has not yet received enough attention from biochemists, but might be of large importance for the understanding of molecular events occurring in the matrix. Acknowledgements Our own original work was supported by grants from the Swedish Medical Research Council (5668), Konung Gustaf V:s 80-arsfond, Alfred Osterlunds Stiftelse, Kocks Stiftelser and the Medical Faculty, University of Lund. M. P. is presently the recipient of a Long Term Fellowship from the European Molecular Biology Organization.
References Baker, J R. and Caterson, B.: The purification and cyanogen bromide cleavage of the link proteins from cartilage proteoglycan. Biochem. Biophys. Res. Comm. 77: 1-10, 1977. Baker, J. R. and Caterson, B.: The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage. J. Bioi. Chem. 254: 2387-2393, 1979. Bonnet, F., Perin, J-P. and Jolles, P.: Isolation and chemical characterization of two distinct link proteins from bovine nasal cartilage proteoglycan complex. Biochim. Biophys. Acta 532: 242-248, 1978. Bornstein, P. and Traub, W.: The chemistry and biology of collagen. In: The Proteins, Vol. IV, ed. by H. Neurath and R. Hill, Academic Press, New York 1979, pp. 411-632.
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Chandrasekhar, S., Kleinman, H. K. and Hassel, J. R.: Interaction of link protein with collagen. j. BioI. Chem. 258: 6226-6231, 1983. Choi, H. U., Tang, L.-H., Johnson, T. L., Pal, S., Rosenberg, L. c., Reiner, A and Poole, A. R.: Isolation and characterization of a 35,000 molecular weight subunit fetal cartilage matrix protein. J. Bioi. Chem. 258: 655-661, 1983. Franzen, A., Bjarnsson, S. and Heinegard, D.: Cartilage proteoglycan aggregate formation. Role of link protein. Biochem. j. 197: 669-674, 1981. Gardell, S., Baker, J., Caterson, B., Heinegard, D. and Roden, L.: Link protein and a hyaluronic acid-binding region as components of aorta proteoglycan. Biochem. Biophys. Res. Comm. 95: 1823-1831, 1980. Gregory, J. D.: Multiple aggregation factors in cartilage proteoglycan. Biochem. j. 133: 383-386, 1973. Hardingham, T. E.: The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem. j. 177: 237-247, 1979. Hardingham, T. E. and Muir, H.: The specific interaction of hyaluronic acid with cartilage proteoglycans. Biochim. Biophys. Acta 279: 401-405, 1972. Hardingham, T. E. and Muir, H.: Hyaluronic acid in cartilage and proteoglycan aggregation. Biochem. j. 139: 565-581,1974. Hascall, V. C. and Heinegard, D. K.: Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. j. BioI. Chem. 249:4242-4249,1974. Hascall, V. C. and Sajdera, S. W.: Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. j. BioI. Chem. 244: 2384-2396, 1969. Heinegard, D. K. and Hascall, V. c.: Aggregation of cartilage proteoglycans. III. Characteristics of the proteins isolated from trypsin digests of aggregates. j. Bioi. Chem. 249: 4250-4256, 1974. Heinegard, D. K. and Hascall, V. c.: The effects of dansylation and acetylation on the interaction between hyaluronic acid and the hyaluronic acid-binding region of cartilage proteoglycans. j. Bioi. Chem. 254: 921-926, 1979. Heinegard, D. and Paulsson, M.: Structure and metabolism of proteoglycans. In: Extracellular matrix biochemistry, ed. by Piez, K. and Reddi, A. H., Elsvier-North Holland, New York, 1984, pp. 277-328. Hewitt, A. T., Kleinman, H. K., Pennypacker, J. P. and Martin, G. R.: Identification of and adhesion factor for chondrocytes. Proc. Natl. Acad. Sci. USA. 77: 385-388, 1980. Hewitt, A. T., Varner, H. H., Silver, M. H., Dessau, W., Wilkes, C. M. and Martin, G. R.: The isolation and partial characterization of chondronectin, an attachment factor for chondrocytes. J. Bioi. Chem. 257: 2330-2334, 1982. Keiser, H., Shulman, H. J. and Sandson, J. I.: Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies. Biochem. j. 126: 163-169, 1972. Kempson, C. E.: The mechanical properties of articular cartilage. In: The joints and Synovial Fluid, II, ed. by Sokoloff, L. Acacemic Press, New York, 1980, pp. 177-238. Mollenhauer, J. and von der Mark, K.: Isolation and characterization of a collagenbinding glycoprotein from chondrocyte membranes. EMBO journal 2: 45-50, 1983. Marner, c. T.: Histochemische Beobachtungen tiber die hyaline Grundsubstanz des Trachealknorpels. Hoppe-Seylers Z. Physiol. Chem. 12: 396-404, 1888. Marner, c. T.: Chemische Studien tiber den Trachealknorpel. Skand. Arch. Physiol. 1: 210-243, 1889. Paulsson, M. and Heinegard, D.: Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage. Biochem. j. 183: 539-545, 1979. Pauls son, M. and Heinegard, D.: Purification and structural characterization of a cartilage matrix protein. Biochem. J. 197: 367-375, 1981.
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Paulsson, M. and Heinegard, D.: Radioimmunoassay of the 148-kilodalton cartilage protein. Distribution of the protein among bovine tissues. Biochem. J. 207: 207-213, 1982. Paulsson, M., Inerot, S. and Heinegard, D.: Variation in quantity and extractability of the 148-kilodalton protein with age. Biochem. J., In press, 1984. Paulsson, M., Sommarin, Y. and Heinegard, D.: Metabolism of cartilage proteins in cultured tissue sections. Biochem. J. 212: 659-667, 1983. Poole, A. R., Pidoux, 1., Reiner, A., Coster, L. and Hassel, J. R.: Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein. J. Cell. Bioi. 93: 910-920, 1982. Poole, A. R., Pidoux, 1., Reiner, A., Tang, L.-H., Choi, H. and Rosenberg, L.: Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study. J. Histochem. Cytochem. 28: 621-635, 1980. Poole, A. R., Pidoux, I., Reiner, A. and Rosenberg, L.: An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J. Cell. Bioi. 93: 921-937, 1982. Roughley, P. J., Poole, A. R. and Mort, J. S.: The heterogeneity of link proteins isolated from human articular cartilage proteoglycan aggregates. J. Bioi. Chem. 257: 1190811914, 1982. Sommarin, Y. and Heinegard, D.: Specific interaction between cartilage proteoglycans and hyaluronic acid at the chondrocyte cell surface. Biochem. J. 214: 777-784, 1983. Szirmai, J. A.: Structure of cartilage. In: Aging of connective and skeletal tissue, ed. by Engel, A. and Larsson, T., Nordiska Bokhandelns Foriag, Stockholm, 1969, pp. 163-184. Szirmai, J. A., van Boven-De Tyssonsk, E. and Garden, S.: Microchemical analysis of glycosaminoglycans, collagen, total protein and water in histological layers of nasal septum cartilage. Biochim. Biophys. Acta 136: 331-350, 1967. Tamkun, J. W. and Hynes, R. 0.: Plasma fibronectin is synthesized and secreted by hepatocytes. J. Bioi. Chem. 258: 4641-4647, 1983. Tengblad, A.: A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronic acid substituted Sepharose gel. Biochem. J. 199: 297-305, 1981. Dick Heinegard, Department of Physiological Chemistry, University of Lund P. O. Box 750, S-220 07 Lund, Sweden.