Abstracts of Communications 567th Meeting of the Biochemical Society University of Durham
6 and 7 January 1977
SECRETIONAL ASPECTS OF GLYCOCONJUGATES: a Colloquium organized by P. W. Kent (Durham) Metabolism of Proteoglycans and Glycosaminoglycans in Cultivated Fibroblasts E. BUDDECKE Institute of Physiological Chemistry, University of Miinster, Miinster, Germany
Proteoglycans and their glycosaminoglycan moieties exhibit chemical and metabolic heterogeneity. The chemical heterogeneity manifests itself in a molecular-weight polydispersity, variation in the degree of sulphation and the occurrence of hybrid glycosaminoglycans. The basic phenomenon of metabolic heterogeneity is a significant difference in the rate of incorporation of [35S]sulphateand 14C radioactivity into the individual glycosaminoglycans. Studies on cultured skin fibroblasts gave evidence that the metabolic heterogeneity of glycosaminoglycans is related not only to different rates of biosynthesis but rather reflects the existence of topographically distinct metabolic pools of glycosaminoglycans; an extracellular, a pericellular (cell-membraneassociated, which can be solubilized by trypsin) and an intracellular pool which is destined for immediate degradation may be distinguished. (1) Synthesis and distribution of glycosaminoglycans into diferent metobolic pools
For a more thorough analysis of the formation, composition and turnover of the extracellular, pericellular and intracellular glycosaminoglycan pools, cell cultures grown from the intima of bovine aorta were used for the following reasons. The arterial tissue cells exhibit ‘fibroblast-like’ morphology, they retain their state of differentiation with regard to the synthesis of glycosaminoglycans, and the distribution pattern of their extracellular glycosaminoglycans has been shown to resemble closely the glycosaminoglycan composition of bovine aortic tissue, thus giving evidence that arterial fibroblasts express their characteristic phenotype even under culture conditions. When the incorporation of [35S]sulphate and [14C]glucosamine into glycosaminoglycans was investigated in cultured cells the newly synthesized glycosaminoglycans were found to be distributed among an extracellular pool, a cell-membrane-associated or pericellular pool and an intracellular pool. From the kinetics of 35S incorporation into the glycosaminoglycan pools it was evident that the newly synthesized glycosaminoglycans are transferred in rather similar proportions into the intracellular, pericellular
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BIOCHEMICAL SOCIETY TRANSACTIONS
and extracellular pools. In separate short-term experiments a linear incorporation of radioactivity into the glycosaminoglycans of all three pools was found for a period up to 4h. The incorporation of [3sS]sulphate into the intracellular and pericellular glycosaminoglycans increases in a hyperbolic manner towards a constant degree of labelling, which reflects an equilibrium between the entry of labelled macromolecules into the pool and their disappearance from it. This metabolic steady state is reached after 8-12h for the intracellular and after about 24h for the pericellular pool. An equilibrium between the secretion of glycosaminoglycans into the culture medium and their pinocytosis and/or degradation was not reached within an incubation period of 6 days. After incubation for 48h, the proportions of radioactivity incorporated into the intracellular, pericellular and extracellular pool was about 1 :3 :10-25. Cultured fibroblasts synthesize chondroitin sulphate, keratan sulphate, heparan sulphate and hyaluronate, all types of glycosaminoglycans being present in all three pools, each pool having a characteristic distribution pattern. Heparan sulphate was localized predominantly in the intracellular and pericellular pools, whereas chondroitin sulphate is mainly found periceIIuIarly and extracellularly. An alkali-soluble glycosaminoglycan pool, tentatively named ‘undercellular’ pool, localized between the cell monolayer and the surface of the culture flask consists mainly of heparan sulphate. (2) Turnover of sulphated glycosaminoglycans The metabolic fate of sulphated glycosaminoglycans localized intracellularly and pericellularly may be followed in prelabelling experiments. After incubation of the cultures in the presence of NaZJSSO4or [l-14C]glucosamine for 3 days, most of the sulphated glycosaminoglycans inside the cells disappear with a half-life of 7h, as computed from the decay of radioactivity over a period of 12h. However, the radioactivity determined after 24h was regularly and significantly higher than expected from the initial kinetics, thus indicating that a smaller portion has an apparent half-life of about 15h. The intracellular glycosaminoglycans are considered to be mainly a part of a degradative pool, as judged from the size distribution of intracellular glycosaminoglycans. Radioautographic studies on the electron-microscope level, however, gave evidence for the existence of a second intracellular glycosaminoglycan pool localized in the cell nuclei. The pericellular glycosaminoglycans leave their pool with a half-life of 12-14h by two different routes: about 60 % disappear as macromolecules into the culture medium and the remainder are pinocytosed and degraded to a large extent.
(3) Uptake and degradation of extracellular 35S-labelledproteoglycans In the course of catabolism the extracellular proteoglycans are taken up by the cells and subjected to (lysosomal) degradation. The uptake of proteoglycans proceeds as adsorptive pinocytosis, which is defined as internalization of proteoglycans adsorbed on to the cell surface. It does not imply quantitative internalization of the adsorbed proteoglycans. The term adsorption is used to describe the integration of extracellular proteoglycans into the pericellular (trypsin-removable) pool. The rate of pinocytosis is defined as the sum of intracellular glycosaminoglycans and their degradation products released into the medium minus the control value. The alcohol-soluble (inorganic sulphate-containing) part of the pinocytosed sulphated proteoglycans represents the amount of degraded proteoglycans. For studies on the pinocytotic uptake and intracellular degradation of proteoglycans, partialIy purified 35S-labelIedproteoglycan fractions were used, rich in heparan sulphate, chondroitin sulphate and dermatan sulphate. When those 3sS-labelled proteoglycans obtained from arterial fibroblast secretions were added to the culture medium
567th MEETING, DURHAM
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of aortic cells, the pinocytosis proceeded with high efficiency. The uptake remained linear with time for at most 24h and then reached a plateau, probably owing to the competition for uptake by newly secreted unlabelled proteoglycans. A considerable amount of added proteoglycans, however, became associated with the cell surface (pericellular pool of glycosaminoglycans) and are not removed by repeated washing with Hanks solution. The association of 35S-labelled proteoglycans with the pericellular pool is time-dependent and requires about 4 h until the steady state is reached, but only a small part of this pool represents material required for immediate pinocytosis. The incorporation of proteoglycans into the pericellular pool cannot be explained by adsorption, since this process is in part temperature-dependent. From the kinetics of adsorption and uptake processes it has to be concluded that binding for integration into the pericellular pool and for pinocytotic uptake appear as separate processes. This is suggested by the observation that the proteoglycan concentration that is required for a half-maximal saturation of the pericellular pool is lower than that for the half-maximal rate of pinocytosis. Adsorptive pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan sulphate-rich proteoglycan. N o competition of uptake between dermatan sulphate-rich and heparan sulphate-rich proteoglycans was observed. Competition experiments suggest that the individual proteoglycans are recognized by different receptors on the cell surface. Structural integrity is one of the prerequisites for optimal pinocytosis. Proteolytic digestion of the protein core or elimination of the polysaccharide chain by alkali results in marked decrease in uptake by arterial fibroblasts.
Macromolecular Interactions and Connective-TissueMetabolism HELEN MUIR Biochemistry Division, Kennedy Institute of Rheumatology, Bute Gardens, Hammersmith, London W6 7 D W, U.K.
The particular characteristics of each type of connective tissue result from differences in the relative proportions of fibrillar proteins and other constituents, principally in the relative proportions of collagen and proteoglycan. The types of collagen and proteoglycan vary in different connective tissues and interactions between them will also affect the properties of the tissue. The situation in cartilage has perhaps been studied most extensively, where the contribution of collagen and proteoglycan to its mechanical properties has been established. Cartilage is a highly specialized form of connective tissue that can withstand compressive forces to a remarkable degree. Articular cartilage is able to distribute stress under impact loading (Freeman & Kempson, 1973) because it is not rigid but stiff, although it contains about 70-75% water (Muir et al., 1969). The fluid pressure within cartilage rises immediately a load is applied, but water is driven out only slowly because proteoglycans, which are highly hydrated compounds, are entrapped in the collagen network and impede the flow of interstitial water. Proteoglycans remain within the cartilage, however (Linn & Sokoloff, 1965), and when the load is released the water is reimbibed (Maroudas, 1973; Freeman & Kempson, 1973). The resilience or compressive stiffness of cartilage is therefore directly correlated with the proteoglycan content (Kempson et al., 1970), whereas tensile stiffness and tensile strength depend on the collagen content (Kempson et al., 1973). The molecular interactions of collagen and proteoglycan are likely to affect this situation. The decreased tensile stiffness of articular cartilage from areas adjacent to osteoarthritic lesions compared with cartilage from unaffected joints (Kempson et al., 1973) may be due to some
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6 and 7 January 1977
SECRETIONAL ASPECTS OF GLYCOCONJUGATES: a Colloquium organized by P. W. Kent (Durham) Metabolism of Proteoglycans and Glycosaminoglycans in Cultivated Fibroblasts E. BUDDECKE Institute of Physiological Chemistry, University of Miinster, Miinster, Germany
Proteoglycans and their glycosaminoglycan moieties exhibit chemical and metabolic heterogeneity. The chemical heterogeneity manifests itself in a molecular-weight polydispersity, variation in the degree of sulphation and the occurrence of hybrid glycosaminoglycans. The basic phenomenon of metabolic heterogeneity is a significant difference in the rate of incorporation of [35S]sulphateand 14C radioactivity into the individual glycosaminoglycans. Studies on cultured skin fibroblasts gave evidence that the metabolic heterogeneity of glycosaminoglycans is related not only to different rates of biosynthesis but rather reflects the existence of topographically distinct metabolic pools of glycosaminoglycans; an extracellular, a pericellular (cell-membraneassociated, which can be solubilized by trypsin) and an intracellular pool which is destined for immediate degradation may be distinguished. (1) Synthesis and distribution of glycosaminoglycans into diferent metobolic pools
For a more thorough analysis of the formation, composition and turnover of the extracellular, pericellular and intracellular glycosaminoglycan pools, cell cultures grown from the intima of bovine aorta were used for the following reasons. The arterial tissue cells exhibit ‘fibroblast-like’ morphology, they retain their state of differentiation with regard to the synthesis of glycosaminoglycans, and the distribution pattern of their extracellular glycosaminoglycans has been shown to resemble closely the glycosaminoglycan composition of bovine aortic tissue, thus giving evidence that arterial fibroblasts express their characteristic phenotype even under culture conditions. When the incorporation of [35S]sulphate and [14C]glucosamine into glycosaminoglycans was investigated in cultured cells the newly synthesized glycosaminoglycans were found to be distributed among an extracellular pool, a cell-membrane-associated or pericellular pool and an intracellular pool. From the kinetics of 35S incorporation into the glycosaminoglycan pools it was evident that the newly synthesized glycosaminoglycans are transferred in rather similar proportions into the intracellular, pericellular
Vol. 5
396
BIOCHEMICAL SOCIETY TRANSACTIONS
and extracellular pools. In separate short-term experiments a linear incorporation of radioactivity into the glycosaminoglycans of all three pools was found for a period up to 4h. The incorporation of [3sS]sulphate into the intracellular and pericellular glycosaminoglycans increases in a hyperbolic manner towards a constant degree of labelling, which reflects an equilibrium between the entry of labelled macromolecules into the pool and their disappearance from it. This metabolic steady state is reached after 8-12h for the intracellular and after about 24h for the pericellular pool. An equilibrium between the secretion of glycosaminoglycans into the culture medium and their pinocytosis and/or degradation was not reached within an incubation period of 6 days. After incubation for 48h, the proportions of radioactivity incorporated into the intracellular, pericellular and extracellular pool was about 1 :3 :10-25. Cultured fibroblasts synthesize chondroitin sulphate, keratan sulphate, heparan sulphate and hyaluronate, all types of glycosaminoglycans being present in all three pools, each pool having a characteristic distribution pattern. Heparan sulphate was localized predominantly in the intracellular and pericellular pools, whereas chondroitin sulphate is mainly found periceIIuIarly and extracellularly. An alkali-soluble glycosaminoglycan pool, tentatively named ‘undercellular’ pool, localized between the cell monolayer and the surface of the culture flask consists mainly of heparan sulphate. (2) Turnover of sulphated glycosaminoglycans The metabolic fate of sulphated glycosaminoglycans localized intracellularly and pericellularly may be followed in prelabelling experiments. After incubation of the cultures in the presence of NaZJSSO4or [l-14C]glucosamine for 3 days, most of the sulphated glycosaminoglycans inside the cells disappear with a half-life of 7h, as computed from the decay of radioactivity over a period of 12h. However, the radioactivity determined after 24h was regularly and significantly higher than expected from the initial kinetics, thus indicating that a smaller portion has an apparent half-life of about 15h. The intracellular glycosaminoglycans are considered to be mainly a part of a degradative pool, as judged from the size distribution of intracellular glycosaminoglycans. Radioautographic studies on the electron-microscope level, however, gave evidence for the existence of a second intracellular glycosaminoglycan pool localized in the cell nuclei. The pericellular glycosaminoglycans leave their pool with a half-life of 12-14h by two different routes: about 60 % disappear as macromolecules into the culture medium and the remainder are pinocytosed and degraded to a large extent.
(3) Uptake and degradation of extracellular 35S-labelledproteoglycans In the course of catabolism the extracellular proteoglycans are taken up by the cells and subjected to (lysosomal) degradation. The uptake of proteoglycans proceeds as adsorptive pinocytosis, which is defined as internalization of proteoglycans adsorbed on to the cell surface. It does not imply quantitative internalization of the adsorbed proteoglycans. The term adsorption is used to describe the integration of extracellular proteoglycans into the pericellular (trypsin-removable) pool. The rate of pinocytosis is defined as the sum of intracellular glycosaminoglycans and their degradation products released into the medium minus the control value. The alcohol-soluble (inorganic sulphate-containing) part of the pinocytosed sulphated proteoglycans represents the amount of degraded proteoglycans. For studies on the pinocytotic uptake and intracellular degradation of proteoglycans, partialIy purified 35S-labelIedproteoglycan fractions were used, rich in heparan sulphate, chondroitin sulphate and dermatan sulphate. When those 3sS-labelled proteoglycans obtained from arterial fibroblast secretions were added to the culture medium
567th MEETING, DURHAM
397
of aortic cells, the pinocytosis proceeded with high efficiency. The uptake remained linear with time for at most 24h and then reached a plateau, probably owing to the competition for uptake by newly secreted unlabelled proteoglycans. A considerable amount of added proteoglycans, however, became associated with the cell surface (pericellular pool of glycosaminoglycans) and are not removed by repeated washing with Hanks solution. The association of 35S-labelled proteoglycans with the pericellular pool is time-dependent and requires about 4 h until the steady state is reached, but only a small part of this pool represents material required for immediate pinocytosis. The incorporation of proteoglycans into the pericellular pool cannot be explained by adsorption, since this process is in part temperature-dependent. From the kinetics of adsorption and uptake processes it has to be concluded that binding for integration into the pericellular pool and for pinocytotic uptake appear as separate processes. This is suggested by the observation that the proteoglycan concentration that is required for a half-maximal saturation of the pericellular pool is lower than that for the half-maximal rate of pinocytosis. Adsorptive pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan sulphate-rich proteoglycan. N o competition of uptake between dermatan sulphate-rich and heparan sulphate-rich proteoglycans was observed. Competition experiments suggest that the individual proteoglycans are recognized by different receptors on the cell surface. Structural integrity is one of the prerequisites for optimal pinocytosis. Proteolytic digestion of the protein core or elimination of the polysaccharide chain by alkali results in marked decrease in uptake by arterial fibroblasts.
Macromolecular Interactions and Connective-TissueMetabolism HELEN MUIR Biochemistry Division, Kennedy Institute of Rheumatology, Bute Gardens, Hammersmith, London W6 7 D W, U.K.
The particular characteristics of each type of connective tissue result from differences in the relative proportions of fibrillar proteins and other constituents, principally in the relative proportions of collagen and proteoglycan. The types of collagen and proteoglycan vary in different connective tissues and interactions between them will also affect the properties of the tissue. The situation in cartilage has perhaps been studied most extensively, where the contribution of collagen and proteoglycan to its mechanical properties has been established. Cartilage is a highly specialized form of connective tissue that can withstand compressive forces to a remarkable degree. Articular cartilage is able to distribute stress under impact loading (Freeman & Kempson, 1973) because it is not rigid but stiff, although it contains about 70-75% water (Muir et al., 1969). The fluid pressure within cartilage rises immediately a load is applied, but water is driven out only slowly because proteoglycans, which are highly hydrated compounds, are entrapped in the collagen network and impede the flow of interstitial water. Proteoglycans remain within the cartilage, however (Linn & Sokoloff, 1965), and when the load is released the water is reimbibed (Maroudas, 1973; Freeman & Kempson, 1973). The resilience or compressive stiffness of cartilage is therefore directly correlated with the proteoglycan content (Kempson et al., 1970), whereas tensile stiffness and tensile strength depend on the collagen content (Kempson et al., 1973). The molecular interactions of collagen and proteoglycan are likely to affect this situation. The decreased tensile stiffness of articular cartilage from areas adjacent to osteoarthritic lesions compared with cartilage from unaffected joints (Kempson et al., 1973) may be due to some
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